PMID:8787733 82 : In all cases , antibodies against LIN-26 protein stained the nucleus , as appropriate for a transcription factor . 88 : All hypodermal nuclei were stained with about the same intensity . 89 : Socket and sheath nuclei ( except the amphid sheath nuclei ) were less intensely stained than were hypodermal nuclei . PAPERDELIMITER PMID:7997265 3 : We report here that unc-30 encodes a homeodomain protein that is present in the nuclei of the D neurons at high levels in young larvae , in which the motor circuitry is formed , and at low levels in older animals . 107 : Immunocytochemical studies revealed that the UNC-30 protein is found in the nuclei of the D neurons ( Fig 3 ) . 114 : In addition , four nuclei in the head of L1 and L2 animals were weakly stained by the anti-UNC-30 antisera ; these cells were identified as the sensory neurons ASGL and ASGR and the interneurons RIH and RID ( see Fig 3 legend ) . PAPERDELIMITER PMID:12498685 7 : In Caenorhabditis elegans , dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells . 38 : Dynamin Localizes to the Newly Ingressed Furrow and Midbody in C elegans Embryos The distribution and enrichment of dynamin in the midbody of mammalian cells suggested a novel function for methanol or 3 paraformaldehyde , triple labeled for dynamin ( MC63 , red ) [ 33 ] , microtubules ( -Tubulin , green ) , and DNA ( DAPI , blue ) , then viewed as 1 microscope . m sections with a Zeiss Axiovert 35 confocal Figure 1 . 65 : Dynamin Localizes to the Newly Formed Furrow Membranes and the Midbody in C elegans All embryos are arranged with anterior toward the left . 67 : ( [ A ] , arrow ) At the metaphase-anaphase transition , dynamin was observed to localize between the asters . 68 : ( B ) During anaphase , dynamin appeared to localize equatorially , and a population of dynamin appeared on the midzone microtubules . 69 : ( [ C ] , arrow ) During late telophase , dynamin appeared to localize only to the newly formed membrane . 70 : ( [ D ] , arrow ) During early cytokinesis , dynamin localized to the newly formed membrane and along the cleavage furrow . 71 : ( [ E ] , arrow ) As cytokinesis progressed , the dynamin staining became more punctate along the newly ingressed furrow , and the midbody staining was more intense . 77 : dynamin was markedly concentrated around the metaphase plate and was also present on several large cytoplasmic vesicles ( Figure 3A ) . 78 : Later in anaphase , dynamin became concentrated equatorially and also localized along spindle microtubules ( Figure 3B ) in a manner similar to the localization of dynamin along spindle microtubules in mammalian cells ( Figure 1B ) . 79 : Further , in C elegans embryos , dynamin localized to the newly formed cleavage furrow ( Figures 3C3E ) and accumulated at the midbody as the embryos progressed through the Brief Communication 2115 Figure 4 . 96 : Cytokinetic Defects in Temperature-Sensitive dyn-1 C elegans Embryos After establishing that dynamin localizes to the midbody in C elegans embryos in a manner similar to that in mammalian cells , we next tested for functional defects in cytokinesis as a result of altered dynamin function . PAPERDELIMITER PMID:8756296 2 : We find that lin-29 protein accumulates in the nuclei of these cells , consistent with its predicted role as a zinc finger transcription factor . 3 : The earliest detectable LIN-29 accumulation in seam cell nuclei is during the last larval stage ( L4 ) , following the final seam cell division , which occurs during the L3-to-L4 molt . 4 : LIN-29 accumulates in all hypodermal nuclei during the L4 stage . 6 : LIN-29 also accumulates stage-specifically in the nuclei of a variety of non-hypodermal cells during development . 40 : Here we show that hypodermal cell nuclei accumulate lin29 protein in a temporally restricted fashion , beginning during the L4 stage , and we describe the effects of mutations in the upstream heterochronic genes on the time of LIN-29 accumulation . 95 : LIN-29 is a nuclear protein . 105 : LIN-29 has accumulated in the seam cell nuclei . 116 : LIN-29 accumulates in seam cell nuclei positioned along the lateral seam ( open circles in A ) . 118 : The row of tightly spaced nuclei that have accumulated LIN-29 are the progeny of the sex myoblasts ( see Fig 6E ) . 123 : LIN-29 accumulates in L4-stage seam cell nuclei ( arrowheads ) . 127 : Many nuclei in the head accumulate LIN-29 during the L4 stage . 129 : All of the nuclei that fuse into hyp7 A V1 - V4 , V6 C D hyp7 L1 hyp7 hyp7 L2 hyp7 hyp7 L3 hyp7 hyp7 L4 E G F H hyp 7 hyp 7 A B hyp 6 hyp 6 tb m hyp 4 hyp 4 I J hyp12 hyp 7 hyp 11 hyp 10 hyp 8 hyp 9 hyp12 hyp 7 hyp 11 hyp 10 hyp 8 hyp 9 m tb vulva hyp7 seam K G ( marked hyp7 in A ) accumulate LIN-29 during the L4 stage . 131 : Nuclei of the pharynx , particularly the metacorpus ( m ) and terminal bulb ( tb ) , accumulate LIN-29 ( see also Fig 6A ) . 134 : All nuclei of the tail hypodermis [ hyp8-hyp12 ( hyp12P12 . pa ; Sulston et al , 1988 ) ; Ka ; see Fig 6C ] accumulate LIN-29 . 150 : LIN-29 accumulated in these hypodermal nuclei during the L4 stage , and remained detectable in the adult animal At approximately the same time , LIN-29 was detected in the hypodermal nuclei of the head ( hyp1-hyp6 ) , tail ( hyp8-hyp12 , Ka ) ( Figs 3G , I , 6C ) , and the large hypodermal syncytium covering most of the animal ( hyp7 ) ( Fig 3G , I , K ) . 158 : LIN-29 protein accumulates stage-specifically in hypodermal nuclei The anti-LIN-29 antisera recognized a nuclear antigen in lateral hypodermal seam cells in wild-type C elegans ( Fig 2B ) , consistent with the predicted role of LIN-29 as a transcription factor . 168 : The earliest LIN-29 accumulation in lateral seam cell nuclei that we detected was shortly after their final L1 L1 L1 hyp7 hyp7 hyp7 hyp7 hyp7 hyp7 hyp7 hyp7 hyp7 L2 L2 L2 hyp7 hyp7 hyp7 hyp7 hyp7 L3 L3 L3 L4 L4 L4 A A A D E F G H I Fig 4 . 192 : In summary , LIN-29 accumulates stage-specifically , beginning during the L4 stage and persisting into the adult stage , in all hypodermal cell nuclei of the worm . 236 : ( A-B ) Mid-section focal plane through the head of a wild-type L4 molt animal Many non-hypodermal nuclei in the head accumulate LIN29 , particularly in the metacorpus ( m ) and the terminal bulb ( tb ) of the pharynx . 239 : Several tail hypodermal nuclei , including hyp12 and Ka , have accumulated LIN-29 and are also visible in this focal plane . 241 : The eight left sex muscle cell nuclei accumulate LIN-29 and are indicated by vertical lines . 245 : Vulval cell nuclei present on the ventral side of the worm also accumulate LIN-29 and are indicated by a bracket . 248 : A short time later the two distal tip cell nuclei ( DTC ) , present at the tips of the developing gonad , accumulate LIN-29 . 249 : The cell nuclei of the developing vulva also accumulate LIN-29 and are visible in this focal plane ( bracket ) . 251 : LIN-29 accumulation is mainly in pharyngeal cell nuclei . 262 : LIN-29 accumulates in the anchor cell nucleus ( AC ) and in the distal tip cell nuclei ( DTC ) , the posterior one of which is shown in this view . 292 : LIN-29 accumulates stage-specifically in hypodermal cells In this study we have shown that lin-29 protein accumulation in wild-type hypodermal seam cell nuclei is temporally restricted to the L4 and adult stages . PAPERDELIMITER PMID:9875852 74 : In wild-type animals , the nuclei of most if not all cells in embryos and newly hatched L1 larvae stained ( Figures 3B and 3C ) . 75 : In older larvae and adults , staining appeared diminished and became restricted to the nuclei of certain cells in the head and tail regions ( data not shown ) and of the P ( 38 ) . p cells and their descendants ( Figures 3D and 3E ) . 99 : ( D ) LIN-35 staining in nuclei of P ( 38 ) . p cells ( arrows ) in an L3 animal ( E ) LIN-35 staining in nuclei of the developing L4 vulva . 148 : A GFP : : LIN-53 Transgene Is Expressed in Many Nuclei ( A and B ) GFP is expressed in most nuclei in embryos and newly hatched L1s . 149 : ( C ) GFP is expressed in the nuclei of P ( 38 ) . p cells ( arrows ) in an L3 animal ( D ) GFP is expressed in the nuclei of the developing L4 vulva . 157 : A GFP : : LIN-53 Transgene Is Expressed in the Vulval Cell Nuclei A transgene containing the lin-53 cDNA tagged with GFP ( Chalfie et al , 1994 ) at its N terminus and under the control of the endogenous lin-53 promoter was capable of partially rescuing the Muv phenotype of lin53 ( n833 ) ; lin-15A animals . PAPERDELIMITER PMID:10629219 7 : This protein localizes to the spindle apparatus in a cell cycle and microtubule-dependent manner . The LIN-5 protein is located at the centrosomes throughout mitosis , at the kinetochore microtubules in metaphase cells , and at the spindle during meiosis . 9 : show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements , cytoplasmic cleavage , and correct alternation of the S and M phases of the cell cycle . 224 : LIN-5 Protein Colocalizes with Components of the Meiotic and Mitotic Spindle To better understand the function of lin-5 , we determined the subcellular localization of its product . 254 : In immunostaining experiments with antiLIN-5 antibodies , we observed the following localization pattern : LIN-5 was present at the meiotic spindles in the fertilized egg during meiosis I and II ( Figs 5 A and 6 C ) . 255 : Staining associated with the maternal pronucleus disappeared after completion of meiosis and LIN-5 became localized at the duplicated centrosomes that adjoined the paternal pronucleus ( Fig 5 B ) . 256 : LIN-5 remained localized at the centrosomes until completion of the first mitotic division . 257 : In all subsequent mitoses , LIN-5 was associated with the centrosomes and detectable after separation of the centrosomes in prophase until decondensation of chromosomes in telophase ( Fig 5 , CE ) . 258 : LIN-5 was also diffusely present in the cytoplasm and at two other locations ; LIN-5 appeared microtubule-associated in cells with metaphase-aligned chromosomes ( Figs 5 F and 6 I ) . 259 : This localization was limited to the region between the poles and did not overlap with DNA staining . 260 : Therefore , it appeared to reflect specific association of LIN-5 with kinetochore microtubules . 261 : Finally , LIN-5 was diffusely present at the cell cortex in most embryonic cells from the two-cell stage onward ( Fig 5 , DF ) . 262 : During the larval stages , LIN-5 localized to the centrosomes from prophase to telophase and to the cell periphery in late mitosis . 264 : LIN-5 was detected at the centrosomes of such cells and , in addition , diffuse cytoplasmic LIN-5 staining was detected in oocytes ( data not shown ) . 265 : Finally , strong membrane-associated staining was detected in the gonad at all stages of development ( data not shown ) . 266 : LIN-5 colocalized with several components of the spindle apparatus . 267 : Its predominant localization to the centrosomes could reflect association with the centrioles or pericentrosomal matrix or , alternatively , could indicate that LIN-5 associates with microtubules that radiate from the centrosomes . 275 : LIN-5 localizes in a cell cycle-dependent manner to the meiotic spindle , mitotic centrosomes , and kinetochore microtubules . 276 : In addition , LIN-5 is present in the cytoplasm and at the cell periphery . 279 : LIN-5 was localized to the meiotic spindle ( arrow ) . 283 : LIN-5 was located at the duplicated centrosomes of the decondensed sperm pronucleus ( arrows ) and was no longer detected at the oocyte pronucleus ( arrowhead ) . 284 : C , LIN-5 remained associated with the centrosomes ( arrows ) during nuclear migration , rotation , and formation of the first mitotic spindle . 285 : D , LIN-5 is detected at the centrosomes ( arrows ) and kinetochore microtubules ( arrowhead ) of this two-cell embryo . 286 : E , LIN-5 is detected at the centrosomes , metaphase spindle ( arrowhead ) , and cell cortex ( arrow ) of mitotic cells throughout embryogenesis . 287 : F , Magnification of a multicellular embryo to illustrate the localization of LIN-5 at the kinetochore microtubules ( arrowhead ) in metaphase cells . 290 : In conclusion , consistent with its role in chromosome segregation , LIN-5 is localized at the spindle apparatus in meiosis and mitosis . 291 : Its localization at the meiotic spindle , mitotic centrosomes , and metaphase microtubules are all dependent on the presence of microtubules . 353 : Finally , we showed that LIN-5 localization to the spindle depends on microtubules . 354 : Together , our data establish that lin-5 encodes a novel component of the spindle apparatus essential for multiple cell division events . 367 : Such functions are consistent with the prominent presence of LIN-5 at the meiotic spindle and mitotic centrosomes . 409 : LIN-5 is not an integral constituent of the centrosomes , but rather locates to the microtubules that emanate from the spindle poles . 411 : First , LIN-5 was associated with the meiotic spindle , which is Lorson et al LIN-5 Is a Novel Spindle Component 83 formed in the absence of centrosomes ( Albertson and Thomson , 1993 ) . 413 : Third , LIN-5 was detected at the kinetochore microtubules in metaphase cells . 446 : However , LIN-5 is present at the spindle , as well as the cell periphery . PAPERDELIMITER PMID:2922060 1 : The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch Gary Ruvkun & John Giusto DepartmentofMolecularBiology , MassachusettsGeneralHospitalandDepartmentofGenetics , HarvardMedicalSchool , Wellman8 , MassachusettsGeneral Hospital , Boston , MA02114 , USA During wild-type development , a protein product of the Caenorhabditis elegans heterochronic gene lin-14 is localized to nuclei of specific somatic cells in embryos and early larvae , but is absent in late larvae and adult soma . 3 : The normal downregulation of the lin-14 nuclear protein level encodes a temporal switch between early and late cell fates . 12 : We find that the lin-14 protein is localized in the nucleus and that its progressive elimination during C elegans development controls the normal temporal sequence of cell fates mediated by the lin-14 gene . 13 : Lin 14 protein is nuclear and temporally regulated To overproduce the lin-14 gene product in Escherichia coli , we fused the IacZ gene of plasmid pUR290 ( ref . 21 : We used the affinity-purified anti-lin-14 antibodies to detect the lin-14 protein in whole-mount fixed specimens of wild-type C elegans by indirect immunofluorescence staining , and found that the anti-lin-14 antibodies bound antigen in specific somatic nuclei of late embryos and early larvae ( Fig 2 ) . 49 : At this stage , the most intense staining was in intestinal and hypodermal nuclei ( Fig 2a ) . 51 : No lineage defects before larval stage one ( L1 ) have been observed in lin-14 mutant strains , and the temperature-sensitive period for lin-14 lineage defects occurs after hatching , indicating that the accumulation of the lin-14 protein detected in these embryos is not yet functional The most intense staining that we observed at any stage was in nuclei of late embryos just before hatching , and in newly hatched L1 animals . 52 : Significantly , only nuclear staining was observed at these stages ( Fig 2b , c ) . 53 : In the newly hatched L1 animal , the lin-14 protein was present in the nuclei of most of the post-embryonic blast cells ; intense nuclear staining was observed in the hypodermal blast cells Hl , H2 , VI-V6 , and T ( Figs 2c and 3 ) and in all of the intestinal ( E ) cells ( Fig 2b ) , and weaker staining was observed in both neuroblasts Q1 and Q2 , in the mesoblast M cell , and in the P cells . 56 : The nuclei of these progeny cells also stain with the anti-lin-14 antibody during the mid-L1 stage . 58 : The lin-14 staining in the P-cell nuclei fades before their migration into the ventral cord but reappears later in some of their progeny cells ( see below ) . 63 : The nuclei of many cells that do not divide post-embryonically also accumulate the lin-14 protein during the L1 stage . 64 : The embryo-derived nuclei in the hypodermal syncytial cell hyp7 , ABarpppapa , ABplaapppp , Cpaaaa , Cpaapa , Cpaapp , Cpapaa , all accumulate levels of the lin-14 protein similar to those of the hypodermal blast cells ( Figs 2c and 3 ) . 65 : Terminally differentiated nuclei from embryonic body muscle also accumulate the lin-14 protein . 66 : Nuclei of many but not all neuronal cells stain with the anti-lin-14 antibodies . 68 : All of the embryonically generated ventral-cord neurons , and some but not all of the neurons of the nerve ring and posterior ganglion accumulate the lin-14 protein in their nuclei during the Li stage ( Fig 2c , d , e ) . 71 : It is not known , therefore , whether the lin-14 expression that we observe in these differentiated cells is in fact functional Late in the L1 stage , the lin-14 protein staining of all nuclei except the neuronal nuclei is much weaker . 82 : In occasional L2 and L3 stage animals , weak lin-14 protein staining in hypodermal , neuronal , and intestinal cells was observed in nuclei and cytoplasm , as if it was being slowly degraded or not efficiently transported to the nucleus . 83 : Patches of lin-14 staining in hypodermal or intestinal nuclei was only rarely observed in very old adults ( data not shown ) . 88 : The oocyte lin-14 nuclear staining disappears after fertilization ; presumably it is either degraded or greatly diluted during nuclear division . 102 : Bright nuclear lin-14 protein staining can be seen in hypodermal nuclei . b , FITC , wild-type larval stage one ( L1 ) animal with the plane of focus at the midline , showing intense lin-14 protein staining in the large intestinal nuclei . 104 : Below the intestinal nuclei , the smaller nuclei of the ventral cord neurons stain with the antibody . 105 : Towards the anterior the lin-14 protein staining nuclei of the nerve ring can be seen . c , FITC , wild-type early L1 animal with the plane of focus at the lateral hypoderm , showing protein staining in the lateral hypodermal blast cells V1 , V2 , and V3 , the ventral blast cells , P12 , P34 , and P56 , the neurons CAN and ALM , the embryonic hypodermal nuclei Cpaapa , Cpaap and Cpaaaa , and in the neuronal nuclei of the nerve ring . d , FITC , wild-type mid-larval stage-one animal with the plane of focus at the midline , posterior towards the inside of the spiral On the inner edge of the spiral , pairs of lin-14 protein staining nuclei from ventral cord neurons can be seen . 107 : The larger lin-14 protein-staining nuclei are intestinal nuclei before the L1 division . e , DAPI , same animal as d . 112 : Shown are six mature oocyte nuclei that stain with the anti-fin-14 antibody . 117 : Shown are hypodermal nuclei that contain the fin-14 protein . 119 : lin-14 protein staining can be seen in hypodermal nuclei ( large ) and neuronal nuclei ( small ) of the postdereid , V5 . pax . l , DAPI , same animal as in k . 120 : Note that in k only two of the five neuronal nuclei shown in this animal have accumulated lin-14 protein . 140 : Molecular basis of lin-14 cell fate specification The nuclear localization of the lin-14 protein suggests that it regulates the pattern of gene expression of the cells that accumulate it either transcriptionally , by binding to DNA adjacent to downstream genes downstream genes whose expression may be controlled by lin-14 include the lin-14 gene itself , other heterochronic genes , such as lin-28 and lin-296 and cell-type and temporally regulated genes such as the vitellogenin genes5 . 141 : The lin-14 nuclear protein could either activate early larval stage-specific genes or repress late larval and adult stagespecific genes . PAPERDELIMITER PMID:9604932 195 : Green indicates nuclei expressing LIN-31 located in the sagital ventral midline ( same plane as MH27 staining ) , while blue indicates nuclei expressing LIN-31 located laterally left of the midline . 206 : LIN-31 is first expressed in the nuclei of P1 . pP11 . p ( including the vulval precursor cells ) , from the middle of the first larval ( L1 ) stage until the early L3 stage ( Figure 5A ) . PAPERDELIMITER PMID:9165118 217 : UNC-37 is ubiquitously expressed and nuclearlocalized To identify the cells in which UNC-37 is expressed , we raised rabbit polyclonal antibodies to a GST-UNC-37 fusion protein ( Fig 5 ) . 218 : Immunofluorescence staining with the affinity-purified antibody detected UNC-37 in the nuclei of apparently all somatic cells throughout development ( Fig 5 and data not shown ) . 221 : We also observed strong UNC-37 expression in germ line nuclei . 222 : As shown in Fig 5B , UNC-37 is expressed in an apparent gradient in the proximal arm of the gonad with the highest levels of UNC37 in the enlarged nuclei of oocytes adjacent to the Fig 5 . 237 : A lower level of UNC-37 staining was observed in germ line nuclei in the distal arm of the gonad ( Fig 5B , D ) . PAPERDELIMITER PMID:9187144 102 : At all developmental stages , CeEDA antibody staining is nuclear ( except in the germline , see below ) , consistent with the protein functioning as a transcription factor . 112 : CeEDA can first be detected in both nuclei of 2-cell embryos ; the fragility of single-cell embryos in our fixation method precludes us from making definitive conclusions about its presence or absence in the nucleus of singlecell embryos . 113 : Staining persists apparently in all nuclei of the early embryo for the first 150200 minutes of development ( 100-200 cells ) . 127 : Ubiquitous , nuclear-localized CeEDA is observed in early embryogenesis . 128 : Equal levels of CeEDA are detected in all metaphase nuclei ; a diminution in the signal is observed in nuclei entering and exiting mitosis . 147 : CeEDA antibody-positive pharyngeal nuclei . 307 : We assume that the two ABderived nuclei of the cells accumulated CeEDA prior to , and during , pharyngeal organogenesis , and that after cell fusion the nuclear CeEDA must be stable and unable to exchange with its co-syncytial MS-derived nucleus . PAPERDELIMITER PMID:8612272 3 : We have found that LIN-7 is a cell junctionassociated protein that binds to the LET-23 receptor tyrosine kinase . 4 : LET-23 is also localized to the cell junctions , and both LIN-2 and LIN-7 are required for this localization . 97 : LIN-7 Is Localized at Epithelial Cell Junctions The LIN-7 PDZDHR domain is most similar to the third PDZDHR domain in DlgA , which is a protein found at septate junctions in Drosophila ( Woods and Bryant , 1991 ) . 103 : In the Pn . p cells , the majority of LIN-7 staining was closely associated with the cellular junctions following heat shock ( Figures 3D3F ) . 106 : of the mosaic analyses discussed above indicate that LIN-7 acts in the Pn . p cells , and the LIN-7 expression studies suggest that LIN-7 may be associated with cellular junctions when expressed in epithelial cells , such as intestinal cells or the Pn . p cells . 113 : LIN-7 and MH27 colocalize at the intestinal cell junctions ( arrowheads ) and the rectal cell junctions ( arrows ) . 121 : LIN-7GFP was found to be closely associated with the intestinal cell junctions , which were visualized by staining with the cell junctionspecific monoclonal antibody MH27 ( Francis and Waterston , 1991 ) ( Figures 3A3C ) . 122 : To determine the subcellular localization of LIN-7 within Pn . p cells , we used affinity-purified anti-LIN-7 LET-23 Is Associated with the Pn . p Cell Junctions Immunocytochemistry experiments indicate that LIN-7 can be a cell junction protein , and the similarity of LIN-2 to other MAGUKs suggests that LIN-2 might also be associated with cell junctions . 132 : From this perspective , the lateral ring of LET-23GFP expression appears as a thin line that is partly basal and partly overlapping with the MH27-stained cell junctions , indicating that LET-23 is not evenly distributed on the surface of Pn . p cells , but rather is specifically associated with the cell junction . 137 : Figures 4D4F are an example of a ventrallateral view of LET-23GFP expression in P6 . p ( 44 . 5 hr after the L2 molt ) , showing that LET-23GFP is expressed in a lateral ring in the plasma membrane . 172 : cell junctions and that LET-23 localization requires LIN-7 suggest that LET-23 is localized to the cell junctions of Pn . p cells by a physical interaction with LIN-7 . 222 : In this work , lin-7 is shown to encode a protein with a single PDZDHR domain , a proteinbinding domain found in cell junctionassociated MAGUK proteins . 223 : Mosaic analyses indicate that lin-7 acts cell autonomously in the Pn . p cells , and expression studies indicate that LIN-7 can be associated with Pn . p cell junctions . 227 : LET-23 normally colocalizes with LIN-7 at the basal side of the cell junctions in Pn . p cells , and LET-23 is mislocalized in lin-7 and lin-2 mutants . 228 : LET-23 may be localized to the cell junction by binding to LIN-7 , since the intracellular portion of LET-23 binds to LIN-7 . PAPERDELIMITER PMID:9806929 173 : Throughout all stages LIN-25 protein is detected in the nuclei of positive cells but appreciable cytoplasmic staining is also observed . 291 : At all stages , LIN-25 is most clearly visible in the nuclei of positively stained cells but appreciable cytoplasmic staining is also present . PAPERDELIMITER PMID:10583406 5 : CeRhoGDI is a 23-kDa protein that is localized predominantly in the cytosol . 38 : CeRhoGDI is a 23-kDa protein that is located predominantly in the cytosol . 187 : CeRhoGDI is expressed throughout development and is localized predominantly in cytosol C elegans Rho-GTPases have been suggested to play active roles in embryogenesis because of their expression at early stages [ 1921 ] . 195 : In the cell fractionation assay , CeRhoGDI was detected in both the cytosolic and membrane fraction , although it is predominantly cytosolic ( Fig 7C ) . 209 : ( C ) CeRhoGDI is mainly cytosolic . 268 : CeRhoGDI was detected predominantly in cytosol in the cell fractionation assay ( Fig 7C ) . PAPERDELIMITER PMID:7983175 114 : The cell body and neuronal process contain mec-7 antigen , but the nucleus does not . PAPERDELIMITER PMID:11139502 193 : Animals were co-immunostained with antibodies to the common domain of the nuclear LIN-14 proteins ( shown ) as well as the monoclonal antibody MH27 ( data not shown ) to outline hypodermal cells for developmental stage determination . 243 : The LIN-14 nuclear protein is novel , but it may , for example , function as a transcription factor or an activator of transcription factors that in turn interpret the gradient . PAPERDELIMITER PMID:9486792 183 : Staining in the neurons of the ventral cord can also be seen ( small nuclei between the VPCs ) . PAPERDELIMITER PMID:2175254 290 : We observe staining with the CeMyoD antibody in the nuclei of very early embryonic blastomeres at about 120 min postfertilization. 291 : Nuclear staining with the CeMyoD antibody is observed in all descendents of the immunopositive early blastomeres as they continue to divide and ultimately differentiate into body wall muscles. 292 : (A) Adult body wall muscle cells viewed under polarized light microscopy showing nuclear localization of Beta-galactosidase activity (arrows). PAPERDELIMITER PMID:11748140 6 : Antibodies against UNC-83 co-localized to the nuclear envelope with lamin and UNC-84 . 7 : Unlike UNC84 , UNC-83 localized to only specific nuclei , many of which were migratory . 37 : We cloned the unc-83 gene and found that it encodes a novel protein associated with the nuclear envelope and is expressed in cells undergoing nuclear migration . 38 : The localization of UNC-83 to the envelope is dependent on unc-84 ( ) function . 256 : UNC-83 is localized to the nuclear envelope . 276 : UNC-83 localizes to the nuclear membranes of a subset of nuclei To determine the subcellular localization of UNC-83 , we raised monoclonal antibodies against a maltose-binding protein fusion protein containing most of UNC-83 ( from the initiator methionine of UNC-83C to just before the carboxyl predicted transmembrane domain ) . 278 : Antibodies against UNC-83 stained the nuclear envelopes of migratory nuclei and several additional nuclei ( Fig 5 , Fig 6 ) . 279 : UNC-83 co-localized with two other proteins of the nuclear envelope , UNC-84 and lamin ( Fig 6 ) ( Liu et al , 2000 ; Malone et al , 1999 ) . 287 : We first detected UNC-83 at the nuclear envelopes of migrating embryonic hyp7 nuclei ( Fig 5B ) , consistent with the onset of the first observable unc83 mutant defect . 292 : Scale bar : 2 m . development , UNC-83 was localized to the nuclear envelopes of hyp7 cells , P cells and intestinal cells ( Fig 5D-F ) . 295 : In late stages of wild-type embryos , and in larvae and adults , UNC-83 was detected on nuclei in a wide variety of cells , including several cells around the pharynx and in the uterus . 325 : UNC-83 is a novel component of the nuclear envelope and is associated with migrating nuclei Using a monoclonal antibody , we found that UNC-83 localized to the nuclear envelope of a number of different cell types . 329 : UNC-83 first localized to the nuclear envelope of migrating hyp7 precursor nuclei . 351 : The cell specificity of UNC-83 localization to the nuclear envelope could be defined by the complex transcriptional regulation of unc-83 . 353 : Consistent with this hypothesis , unc-84 ; unc-83 double mutant animals have the same nuclear migration defect as animals with either mutation alone ( Malone et al , 1999 ) , and UNC-83 and UNC-84 colocalize at the nuclear envelope ( see Fig 6 ) . PAPERDELIMITER PMID:11514616 51 : We have identified two primary sequences in SNG-1 that are necessary for its synaptic localization , a C-terminal region containing 38 amino acids and an arginine in the loop facing the cytoplasm . 95 : SNG-1 : : GFP is localized to synaptic regions in C elegans . 109 : Immunoreactivity was concentrated in nervous system regions rich in synapses , including the nerve ring ( Figure 1A ) , the dorsal nerve cord ( Figure 1B ) and the ventral nerve cord ( not shown ) . 110 : Discrete puncta were found in the ventral and dorsal sublateral process bundles ( Figure 1B ; our unpublished results 112 : The SNG-1 protein also accumulated in the presynaptic varicosities of SAB motor neurons innervating the head muscle ( Figure 1A ) . 117 : suggest that endogenous SNG-1 is localized to SVs in C elegans , although localization to another membrane such as synaptic endosomes can not be excluded . 311 : Synaptic localization of SNG-1 depends upon two sequence motifs located in the cytoplasmic loop separating the second and third transmembrane domains and in the C-terminal domain . PAPERDELIMITER PMID:11412996 90 : In wild-type one-cell embryos , PAR-3 is at the anterior cortex In two-cell embryos , PAR-3 is found around the entire periphery of the anterior cell , which divides transversely , while PAR-3 is restricted to the anterior cortex of the posterior cell , which divides longitudinally ( Figures 1b and 4a ) . 103 : ( a ) In wild type , cortical PAR-3 localizes to the anterior cell and to the anterior region of the posterior cell , while ( b ) PAR-2 : : GFP is detected in the posterior of the P1 cell . 128 : PAR-2 : : GFP was uniformly associated with the cortex after fertilization , but then became asymmetrically distributed from the posterior of the embryo to the cytokinetic furrow [ 4 , 21 ] . 147 : Our analyses indicate that cdc42 is required for the asymmetric cortical localization of the anterior complex proteins PAR-3 and PAR-6 and the posterior cortical protein PAR-2 . PAPERDELIMITER PMID:12686594 101 : Localization of DPY-7 within Cuticle The DPY-7 protein is detected in circumferential bands within the cuticle of each larval stage and the adult ( Figure 4 ) . 102 : The DPY-7 bands locate within the furrows that delineate the annuli ( Figure 4 , C and D ) . 110 : We conclude that the DPY-7 collagen is assembled into tight band or thread-like structures that run circumferentially around the body of the animal , located within the furrows that delineate the annuli . 138 : DPY-7 location within the cells is detected as a halo surrounding the nucleus ( Figure 3 , A and G ) . 139 : This perinuclear pattern is consistent with the location in a secretary pathway organelle, probably the endoplasmic reticulum. 144 : In B , some extracellular localized DPY-7 ( red arrow ) and some remaining perinuclear-localized DPY-7 ( white arrow ) are both seen . 148 : LIN-26 is present in all hypodermal cell nuclei at this stage . 152 : Arrow in J , the nucleus of one of the seam cells ; the halo of DPY-7 localization is visible around it . 217 : Conversely , we found that the matrix localization of DPY-7 was drastically affected by mutant alleles of dpy-2 , dpy-3 , dpy-8 , or dpy-10 ( Figure 6 , DH , and Table 1 ) , and correspondingly , that the furrows delineating the annuli were either absent or severely altered in an allele-dependent manner ( Figure 5 , EK ) . 272 : As hypothesized , we found that the DPY-10 : : Ty-tagged collagen was located in circumferential bands that correspond to the annular furrows ( Figure 6K ) . 273 : In contrast , DPY-13 : : Ty localized to a distinct matrix structure ( Figure 6N ) . 280 : However , the pattern of matrix substructure formed by DPY-13 : : Ty was dramatically altered when DPY-7 was reduced by RNAi : instead of the normal periodic striped pattern , DPY-13 : : Ty was assembled into a relatively amorphous layer within the cuticle ( Figure 6P ) . 310 : Consistent with the no annuli phenotype of the dpy-2 group of mutants , the DPY-7 and DPY-10 collagens are assembled into narrow band-like substructures in the furrows that delineate the annuli . 339 : We have shown this to be true in the embryo for DPY-7 and DPY-10 , because these collagens are detected by immunofluorescence in a perinuclear location from the comma stage of embryogenesis . PAPERDELIMITER PMID:11914278 7 : SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles . 92 : SMC-4 and MIX-1 localize to the poleward face of chromosomes at times of the cell cycle when chromosomes are condensed SMC-4 antibody staining showed that SMC-4 localized to mitotic chromosomes in a dynamic pattern with two striking features . 93 : First , SMC-4 localized to chromosomes only at times of the cell cycle when they were condensed . 94 : Little or no SMC-4 was observed on decondensed chromosomes during interphase ( Fig 2A , F ) . 95 : SMC-4 localized to chromosomes at prometaphase ( Fig 2B , G ) , when the six C elegans chromosomes , but not their sister chromatids , become visibly distinct . 97 : Second , SMC-4 associated only with certain regions of the chromosomes . 98 : SMC-4 appeared in a faint speckled pattern during prophase , then appeared in two stripes outlining each chromosome during prometaphase ( Fig 2G ) . 99 : When chromosomes were aligned at metaphase , SMC-4 localized to distinct foci on the poleward sides of the metaphase plate ( Fig 2H ) . 101 : SMC-4 colocalizes with MIX-1 at the poleward face of condensed mitotic chromosomes but not on X chromosomes . 104 : ( FJ ) Merged confocal images of wild-type embryonic nuclei costained with SMC-4 antibody ( green ) and DNA dye ( red ) ; overlap appears yellow . 106 : MIX-1 and SMC-4 colocalize in nuclei with condensed chromosomes ( yellow when merged , N ) . 108 : ( OQ ) Metaphase chromosomes costained with DNA dye ( blue ) , MIX-1 antibody ( red , O ) , and SMC-4 antibody ( green , P ) show colocalization of MIX-1 and SMC-4 at the poleward face of condensed chromosomes ( yellow when merged , Q ) . 110 : MIX-1 and SMC-4 both stain nuclei of two mitotic cells ( mit ) , but only MIX-1 shows the punctate subnuclear staining characteristic of dosage compensation proteins bound to hermaphrodite X chromosomes ( X ) . 119 : The wild-type larva of Figure 2 ( RT ) contains two mitotic cells brightly stained by both MIX-1 and SMC-4 , but only MIX-1 shows the punctate nuclear staining characteristic of dosage compensation proteins bound to X chromosomes . 159 : ( A ) MIX-1 associates with mitotic chromosomes in wild-type embryos ( yellow , MIX-1 and DNA overlap ) . 178 : Consistent with this finding , both SMC-4 and MIX-1 localize to condensed mitotic chromosomes in the gonad ( data not shown ) . 180 : pendent localization of SMC-4 and MIX-1 to chromosomes . 193 : Both SMC-4 and HCP-3 outlined prometaphase chromosomes ( Fig 6AC ) and were restricted to poleward-facing foci on cross sections of the metaphase plate ( Fig 6DF ) . 194 : SMC-4 and the conserved kinetochore-associated protein CeMCAK also overlapped in a punctate , poleward-facing pattern on metaphase cross-sections , with CeMCAK staining extending further toward the poles ( Fig 6GI ) . 198 : SMC-4 associates with condensed mitotic chromosomes within mitotic spindles , but not with interphase chromosomes . 224 : In immunofluorescence experiments , both SMC-4 and MIX-1 localized to meiotic chromosomes in metaphase of meiosis I and of meiosis II ( Fig 7AD ) . 225 : In confocal sections , SMC-4 appeared somewhat more concentrated around the periphery of condensed chromosomes in meiosis I ( Fig 7B ) and meiosis II , in a pattern that resembled centromere proteins . 232 : In the merged image and magnified inset ( I ) , CeMCAK partially overlaps ( yellow ) SMC-4 and extends more poleward ( red ) than SMC-4 on metaphase chromosomes . 239 : In costaining experiments with HCP-3 and SMC-4 antibodies , both proteins were coincident at metaphase of meiosis I ( Fig 7AD ) and meiosis II ( data not shown ) , consistent with localization of SMC-4 to the meiotic centromere . 249 : SMC-4 and MIX-1 co-localize with centromere proteins on meiotic chromosomes , and their depletion impairs chromosome segregation in meiosis II but not meiosis I ( AD ) Meiosis I bivalents stained with a DNA dye ( blue , A ) , SMC-4 antibody ( green , B ) , and an antibody to centromere protein HCP-3 ( red , C ) . PAPERDELIMITER PMID:12036960 152 : B , in the elongated embryos the hypodermal location is maintained for the P4H subunits PHY-1 , PHY-2 and PDI-2 , but DPY-7 was incorporated into the developing cuticle . 209 : The nuclear-excluded pattern observed probably represents an endoplasmic reticulum ( ER ) location , as PDIs are known to be located in the ER ( 5 ) , and as double labeling with a 29194 Novel ECM-specific Prolyl 4-Hydroxylase in C elegans monoclonal antibody to the cuticle collagen , DPY-7 , gave an identical staining pattern ( Fig 4A , merge ) . 211 : At this stage , the hypodermal ER location was maintained for all three P4H subunits ( Fig 4B ) , whereas the cuticle collagen DPY-7 has already been secreted from the ER and has been fully incorporated into the developing cuticle2 ( Fig 4B ) . PAPERDELIMITER PMID:14660541 13 : Remarkably , the dosage compensation complex is similar to the evolutionarily conserved 13S condensin complex required for mitotic and meiotic chromosome resolution and compaction , implying the recruitment of ancient chromosome segregation proteins to the new task of regulating gene extensive XX-specific lethality or disrupt the stability or targeting of the dosage compensation complex to X Nonetheless , DPY-21 is a member of the dosage compensation complex and localizes to X chromosomes in a hermaphrodite-specific manner . 36 : We show that DPY-21 is a member of the dosage compensation complex and localizes to X chromosomes in a sex-specific manner . 175 : DPY-21 localizes specifically to both X chromosomes of hermaphrodites If DPY-21 functions as a member of the dosage compensation complex in vivo , as predicted by the biochemical experiments , 6524 Development 130 ( 26 ) Research article Fig 3 . 176 : DPY-21 associates physically with components of the dosage compensation complex ( A ) Western blot of extracts from wild-type or dpy-21 ( e428 ) mutant gravid hermaphrodites serially diluted by 1 . 3-fold and probed with N-terminal antibodies to DPY-21 and antibodies to the loading control SMC-1 , a protein involved in chromosome cohesion . 199 : In embryos with greater than 40 cells , DPY-21 formed punctate , subnuclear foci that coincided with the X-localized SDC-3 foci ( Fig 4B ) . 200 : We showed directly that the DPY-21 foci co-localized with X chromosomes by probing simultaneously with DPY-21 antibodies and X-chromosome-specific DNA probes that were visualized by fluorescence in situ hybridization ( FISH ) ( Fig 5A ) . 201 : DPY-21 maintains its localization to X chromosomes throughout C elegans development , like other dosage compensation proteins , as indicated by its presence on X chromosomes of adult gut nuclei ( Fig 4D ) . 204 : These results establish that DPY-21 is recruited to the X chromosomes of hermaphrodites with other members of the dosage compensation complex at the onset of dosage compensation . 232 : In both embryos ( Fig 6A ) and adult gut nuclei ( Fig 6B ) co-stained with SDC-3 and DPY-21 antibodies , SDC-3 co-localized with her-1 arrays and X chromosomes , but DPY-21 only colocalized with X chromosomes . 252 : DPY-21 localizes to the X chromosomes of XX but not XO embryos , as expected for a component of the dosage compensation complex ( A-F ) False-color confocal images of wild-type XX embryos ( A , B ) , dpy-21 ( e428 ) mutant XX embryos ( C ) , wild-type hermaphrodite gut nuclei ( D ) , him-8 ( e1489 ) XO embryos ( E ) and him-8 ( e1489 ) ; xol-1 ( y9 ) mutant XO embryos ( F ) stained with DPY21 antibodies ( green ) , the DNA-intercalating dye 4 , 6 diamidino-2phenylindole ( DAPI ) ( blue ) and an X-chromosome-specific marker ( red ) ( either an X-chromosome-specific FISH probe or antibodies to SDC-3 or GFP , which identifies the DPY-27 : : GFP fusion protein used in D ) . 254 : The third column shows a merged image of the first two columns , and yellow indicates overlap in staining of DPY-21 and the X-chromosome marker . 258 : ( B ) In 40-cell stage embryos , DPY21 exhibits a punctate pattern that is coincident with the X-localized SDC-3 protein . 260 : SDC-3 localized to the X chromosomes of a dpy-21 mutant , indicating that DPY-21 is not essential for the recruitment of the dosage compensation complex to X ( D ) The X-chromosome localization of DPY-21 is maintained throughout hermaphrodite development , as shown by the X-chromosome localization of DPY-21 in adult gut nuclei , which carry a DPY-27 : : GFP fusion protein . 265 : By contrast , we found that SDC-3 , like SDC-2 ( Chu et al , 2002 ) , localizes to her-1 arrays in dpy-27 ( null ) mutant embryos ( Fig Gene-specific versus chromosome-wide repression 6527 7E , Table 1B ) , providing another example that SDC-3 has different requirements for its recruitment to her-1 versus X Together these results 270 : DPY-21 associates physically with other components of the dosage compensation complex and localizes to X chromosomes of XX embryos to repress gene expression . 292 : Although DPY-21 localizes to X chromosomes as a component of the dosage compensation complex , it does not localize to her-1 , unlike the other dosage compensation proteins . 296 : ( A ) In wild-type embryos , foci of DPY-21 staining co-localize with X chromosomes identified by FISH . 313 : ( A ) In embryos that have activated dosage compensation , both DPY-21 and SDC-3 co-localize with the X chromosome , which is denoted by an asterisk in the inset . 319 : SDC-3 localization to her-1 regulatory regions does not require DPY-27 or SDC-2 , unlike its localization to X chromosomes . 325 : In this single nucleus of a 10-cell embryo , SDC-3 localizes to her-1 regulatory regions at a time prior to its recruitment to X chromosomes . 357 : SDC-2 can localize to X independently of SDC3 , but requires SDC-3 for its localization to her-1 . PAPERDELIMITER PMID:8939869 24 : ( A to C ) DPY-26 is associated with all condensed mitotic chromosomes in young wild-type XX embryos before the onset of dosage compensation . 28 : ( D to F ) DPY-26 is localized to the X chromosomes of XX animals that have activated dosage compensation . 33 : In this optical section both X chromosomes are visible , and the coincident positions of DPY-26 and DPY-27 on the X chromosomes are apparent . 43 : In wild-type XX embryos at a stage ( 30 cells ) before the activation of dosage compensation , DPY-26 was distributed diffusely in interphase nuclei ; DPY-26 was also associated with all chromosomes as they condensed and underwent mitosis ( Fig 1 , A to C ) . 45 : Costaining of XX embryos with antiDPY-26 and anti-DPY-27 antibodies revealed that DPY-26 colocalizes with DPY27 on the X chromosome throughout the cell cycle ( Fig 1 , D to I ) . 58 : We found that DPY-26 was localized in the nucleus of XO animals but was not exclusively associated with the male X chromosome at any time during development ( Fig 2 , A to C ) . 66 : Dosage compensation genes required for the X-chromosome localization or stability of DPY-26 . 91 : DPY26 then becomes selectively localized to X in hermaphrodites . 114 : DPY-26 colocalizes with all chromosomes in the germ cell nuclei ( yellow arrow ) but is localized only to the X chromosomes ( white arrows ) in somatic cells . 117 : ( B to D ) Germ cell nuclei in pachytene were stained with anti-DPY-26 ( green ) ( B ) and propidium iodide ( red ) ( C ) . PAPERDELIMITER PMID:10359617 1 : Molecular Biology of the Cell Vol 10 , 20872100 , June 1999 Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10 Charles W Whitfield , Claire Benard , Tom Barnes , S Hekimi , and Stuart K Kim Department of Developmental Biology , Stanford University Medical Center , Stanford , California 94305 ; and Department of Biology , McGill University , Montreal , Quebec , Canada Submitted February 26 , 1999 ; Accepted March 16 , 1999 Monitoring Editor : Peter Walter In Caenorhabditis elegans , the EGF receptor ( encoded by let-23 ) is localized to the basolateral membrane domain of the epithelial vulval precursor cells , where it acts through a conserved RasMAP kinase signaling pathway to induce vulval differentiation . 2 : lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization , because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective ( vulvaless ) phenotype . 7 : LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi . 82 : In wild-type larvae , LET-23 RTK is first expressed in the vulval precursor cells in the early L2 stage and appears on both the basolateral and apical membrane domains , with higher levels in the apical membrane domain ( Figure 1B ) . 87 : LET-23 staining is observed throughout the basolateral membrane of all six vulval precursor cells ( Figure 1 , CE ) . 88 : In five of the vulval precursor cells ( P3 . p , P4 . p , P5 . p , P7 . p , and P8 . p ) , little or no LET-23 is present in the apical membrane domain . 89 : In the remaining vulval precursor cell ( P6 . p ) , LET-23 is present in both the basolateral and apical membrane domains ( Figure 1 , D and E ) . 90 : As P6 . p divides in early L3 , LET-23 staining continues to be present in both the basolateral and apical membrane domains ( Figure 1F ) . 113 : Strong LET-23 staining is observed in the apical membrane domain , which appears as a thin line along the ventral surface . 114 : Weak LET-23 staining is observed in the basolateral membrane domain , located dorsal and anterior to the cell junctions . 117 : LET-23 staining appears in the basolateral membrane domains of the vulval precursor cells . 120 : LET-23 appears predominantly basolateral in P8 . p and both basolateral and apical in P6 . p . 140 : Basolateral localization of LET-23 requires lin-10 . 226 : In these cells , LIN-10 staining is concentrated in punctate spots around the nucleus , and staining is diffuse in the cytoplasm and at the plasma membrane . 231 : LIN-10 appears in neuronal processes and cell bodies . 232 : In neural cell bodies , a small amount of LIN-10 appears diffusely throughout the cytoplasm , whereas the majority of LIN-10 is concentrated in discrete perinuclear structures ( Figure 7 , D and E ) , similar to perinuclear structures observed in vulval epithelial cells . 238 : These results indicate that LIN-10 is localized in the transcisterna of the Golgi or is localized in a compartment closely associated with the trans-cisterna , such as the trans-Golgi network . 251 : LIN-10 is present in the cytoplasm and at the plasma membrane . 255 : Punctate , perinuclear LIN-10 staining can be seen in some vulval epithelial cells ( D , inset , arrows ) . 266 : ( E ) Lateral view of an adult hermaphrodite tail showing punctate , perinuclear LIN-10 staining around neural nuclei ( arrows ) . 267 : Some LIN-10 staining may also be associated with nonneural nuclei ( arrowheads ) . 271 : These experiments indicate that the relative distribution of LET-23 RTK on the apical and basolateral membrane domains is dynamic . 272 : LET-23 is initially expressed in early L2 on both the apical and basolateral membrane domains of the vulval precursor cells , with higher expression on the apical surface . 273 : In late L2 to early L3 , at the time of vulval cell fate determination ( Kimble , 1981 ) , LET-23 is expressed predominantly on the basolateral membrane domain of the vulval precursor cells ( with the exception of P6 . p ) . 302 : LIN-10 is at the plasma membrane in vulval epithelia and in neural processes , and this localization is consistent with a role in regulation of exocytosis of receptor-containing secretory vesicles at the target membrane domain or tethering receptors at the target membrane domain once they are secreted . 303 : However , the majority of LIN-10 protein appears to be intracellular . 304 : In neurons this pool of LIN-10 is in close association with a marker for the trans-cisterna of the Golgi , suggesting that LIN-10 may be concentrated in the Golgi cisterna or the trans-Golgi network . PAPERDELIMITER PMID:15557118 55 : HCP-6 and MIX-1 colocalize on centromeres of mitotic chromosomes and are required for mitotic chromosome segregation HCP-6 and MIX-1 colocalize with the centromeric histone variant CENP-A on the poleward faces of metaphase chromosomes during mitotic divisions in embryos and in the germline ( Fig 1 E and Fig 2 A ; Hagstrom et al , 2002 ; Stear and Roth , 2002 ) . 56 : This pattern of localization and the biochemistry above indicate that HCP-6 and MIX-1 form a complex that associates with centromeres of mitotic chromosomes. 57 : HCP-6 associates exclusively with the mitotic condensin II complex and colocalizes with MIX-1 on mitotic chromosomes . 65 : ( E ) HCP-6 and MIX-1 colocalized on metaphase chromosomes in embryos and the premeiotic germline . 77 : HCP-6 and MIX-1 associate independently with mitotic chromosomes and are required for chromosome condensation and segregation . 143 : HCP-6 and MIX-1 localize to sister chromatids during diplotenediakinesis of meiotic prophase I The localization of HCP-6 and MIX-1 was examined in wildtype gonads to assess when condensin first associates with meiotic chromosomes to initiate the structural changes essential for chromosome segregation . 144 : In both sexes , HCP-6 was detected in nuclei throughout meiosis , but HCP-6 was excluded from the chromosomes of transition zone and pachytene nuclei ( Fig 5 , A and B ) . 145 : In males , HCP-6 was first detected on chromosomes as they compacted after pachytene and congressed to the metaphase plate , and HCP-6 staining persisted on DNA through meiosis II ( Fig S1 A , available at http : www . jcb . org cgicontentfulljcb . 200408061DC1 ) . 146 : In hermaphrodites , HCP-6 first accumulated on DNA in diplotene ( Fig S1 B ) . 147 : By diakinesis , each bivalent contained four discrete HCP-6 foci ( Fig 5 C ) , which marked the four sister chromatids as demonstrated by two experiments . 151 : Condensin subunit MIX-1 was also detected on chromosomes in diplotene and was present in four foci on diakinesis bivalents ( Fig 5 C ; Fig S1 B ) . 154 : Although HCP-6 and MIX-1 associate independently with mitotic chromosomes ( Fig 2 A ) , MIX-1 depends on HCP-6 for its association with meiotic chromosomes ( Fig 5 E ) . 157 : Condensin is required in diplotene diakinesis for chromosome compaction and formation of discrete diakinesis bivalents The timing of HCP-6 and MIX-1 accumulation on chromosomes suggested that meiotic condensin is first required in diplotene diakinesis , when chromosomes compact to form highly condensed , cruciform bivalents ( Albertson et al , 1997 ) . 160 : HCP-6 and MIX-1 first associate with meiotic chromosomes at diplotene-diakinesis . 162 : ( C ) HCP-6 and MIX-1 accumulate in four quadrants on wild-type diakinesis bivalents . 174 : Moreover , SC central element SYP-1 and cohesin subunits SMC-1 and SMC-3 localized between homologues , as in wild type ( Fig 6 A , Fig 7 A ) . 181 : Additionally , a short stretch of SYP-1 staining persisted on each homologue pair until late diakinesis , as in wild type ( Fig 6 B , arrowhead ; MacQueen et al , 2002 ) . 205 : Moreover , the Aurora B kinase homologue AIR-2 localized to the mid-bivalent in the oldest oocyte of hcp-6 ( mr17 ) gonads ( 81 of oocytes , n 70 ) , as in wild-type gonads ( 78 of oocytes , n 58 ; Fig 7 A ; Fig S1 C ) . 210 : ( A ) AIR-2 localized at the midbivalent in wild-type and hcp-6 ( mr17 ) animals . 213 : ( B ) AIR-2 and SMC-3 colocalized along the length of synapsed pachytene homologues in wild-type and hcp-6 ( mr17 ) animals . 228 : In pachytene nuclei , AIR-2 colocalized with SMC-3 and the SC along the length of synapsed homologues ( Fig 7 B ; Nabeshima , K , M Colaicovo , and A Villeneuve , personal communication ) . 232 : Because both the localization of AIR-2 between homologues in pachytene and the asymmetric localization to synapsed segments of homologues during pachytene exit are specified before the time of condensin function , AIR-2 localization at the midbivalent ( short arm ) might also be specified in a condensin-independent manner . PAPERDELIMITER PMID:12827206 86 : Our biochemical experiments defined the C elegans cohesin complex and revealed its physical association with TIM-1 . 109 : C elegans TIM-1 , a paralogue of the circadian clock protein TIMELESS , associates with the cohesin complex. 115 : Using TIM-1-specific antibodies ( Fig 1d and Methods ) we confirmed the association of TIM-1 with cohesin . 119 : Thus , TIM-1 associates specifically with cohesin but is less tightly connected than other subunits . 128 : At interphase , TIM-1 and cohesin subunits accumulate most intensely and have a diffuse nuclear appearance . 190 : Consistent with this premise is the observation that SMC-1 , SMC-3 and SCC-3 recapitulate the meiotic localization pattern of REC-8 . 191 : All four proteins associate with the chromatin of transitionzone nuclei ( Fig 4a , b , and data not shown ) , which is consistent with the loading of meiotic cohesin before or during pre-meiotic S phase . 192 : SMC-1 co-localizes with REC-8 in pachytene nuclei , and all four proteins assemble along the longitudinal axes of synapsed chromosomes ( Fig 4c ) . 193 : Moreover , two findings show that SMC-1 localization to pachytene chromosomes does not require chromosomes to have a pairing partner and SC . 197 : In contrast , SCC-3 localized throughout the chromatin , indicating a possible additional role for SCC-3 in diakinesis not played by other cohesins ( Fig 4f ) . 199 : TIM-1 was diffusely distributed throughout pre-meiotic nuclei and disappeared abruptly as nuclei entered meiotic prophase ( Fig 4a , b ) . 226 : Figure 4 TIM-1 is restricted to pre-meiotic nuclei , unlike meiotic cohesin , which localizes between sister chromatids throughout meiotic prophase 228 : SMC-1 , SMC-3 and SCC-3 proteins co-localize with REC-8 along the length of pachytene chromosomes . d , Anti-SMC antibody staining along the unpaired male X chromosome shows that SMC-1 localization is not dependent on homologous chromosome pairing and SC formation. SCC-3 localizes to the interface of sister chromatids and homologous chromosomes and also throughout the bivalent , indicative of additional functions. PAPERDELIMITER PMID:15653635 4 : Here, we show that the MUT-7 protein resides incomplexes of 250 kDa in the nucleus and in the cytosol. 94 : This demonstrates that the immune serum specifically recognizes the MUT-7 protein and that MUT-7 is expressed in adult worms , and localizes to both the nucleus and the cytosol . 96 : As is shown in Figure 2A , both the nuclear and the cytosolic MUT-7 protein peak around the catalase marker protein of 232 kDa . 97 : These data show that the MUT-7 protein either oligomerizes or associates with other components to form complexes of 250 kDa in the cytosol and nucleus . 143 : Both RDE-2 ( Figure 4A ) and MUT-7 ( data not shown ) are predominantly present in the cytosol . 150 : However , this was not observed for the nuclear extract ( data not shown ) , indicating that MUT-7 and RDE-2 are associated with each other in the cytosol , but not in the nucleus . 171 : Together these data show that the MUT-7 and RDE-2 proteins are associated with each other in the cytosol , but not in the nucleus . 191 : We show that the RDE-2 protein interacts with MUT-7 in the cytosol . 198 : However , considering the size of the MUT-7RDE-2 complex in the cytosol ( 250 kDa ) and the size of the MUT-7 complex in the cytosol of rde2 ( pk1657 ) animals ( 200 kDa ) , it seems likely that MUT-7 associates with an RDE-2 monomer , rather than a dimer . 214 : Dual role for MUT-7 From our studies , it appears that MUT-7 is present in both cytosol and nucleus , but that the MUT-7 protein in the nucleus is embedded in a different complex compared with the cytosol . PAPERDELIMITER PMID:15798199 265 : ( B ) Representative images of RAD-51 staining in midpachytene nuclei ( panels 1 to 5 ) and quantitation of RAD-51 foci in the six zones of the germ line ( 6 to 10 ) for the indicated genotypes . 283 : In wild-type meiosis , RAD-51 foci appear in the transition zone ( which contains nuclei in the leptotene and zygotene stages ) , become abundant from the late zygotene to the midpachytene stages , and are lost from the late pachytene stage onward ( Fig 5B , panels 1 and 6 ) ( 2 , 12 ) . 288 : In the wild type , RPA-1 is largely diffuse and weakly nuclear in the early prophase , increasing in intensity in the diplotene stage and diakinesis with 2.8 +- 1.4 RPA-1 foci per nucleus. 318 : In nonirradiated wildtype ( N2 ) animals , CeBRC-2 is diffuse and detected at very low levels in nuclei ( Fig 7 , panel 1 ) . 322 : These results imply that CeBRC-2 localizes to presumptive sites of DNA damage after radiation treatment . 383 : ( A ) In wild-type ( N2 ) animals , CeBRC-2 functions in DSBs through HR by transporting RAD-51 into the nucleus and targeting RAD-51 to processed DBSs . PAPERDELIMITER PMID:15916946 2 : Immunostaining of wild-type C. elegans embryos with the purified antibody revealed nuclear staining from early prophase through metaphase. 3 : In wild-type embryos, strong mitotic centrosome staining was seen in addition to nuclear staining 4 : Nuclear and/or chromatin-associated TLK-1P634 staining was present from early prophase through metaphase. 5 : Interphase, anaphase, and telopahse embryos had too much lower levels of nuclear/chromatin-associated TLK-1P634 staining. 6 : Nuclear chromatin-associated TLK-1P634 staining was dependent on TLK-1, AIR-2, and ICP-1 expression and was not reduced in the absence of the AIR-1 kinase. In contrast, TLK-1P634 sentrosome staining was dependent on AIR-2 expression, but not TLK-1. AIR-2. or ICP-1. 7 : TLK-1-, AIR-2-, and ICP-1-dependent nuclear immunostaning was strongest at prometaphase and persisted as a nuclear halo around the metaphase plate despite nuclear-envelope breakdown (Figure 3A). 8 : Phospho-TLK-1 is first detectable in prophase ncleai and persists as a halo around the chromosomes after neclear-envelope breakdown. PAPERDELIMITER PMID:16107639 2 : We show here that the long isoform , PTP-3A , localizes specifically at synapses and that the short isoform , PTP-3B , is extrasynaptic . 39 : PTP-3A specifically localizes to synapses , overlapping the presynaptic proteins SYD-2 and UNC-10 . 141 : These data indicate that PTP-3 is predominantly associated with the presynaptic density . 145 : PTP-3A is synaptic , and PTP-3B is extrasynaptic To address the subcellular localization of the PTP-3A and PTP-3B isoforms , we generated isoform-specific GFP fusion transgenes . 148 : PTP-3A localizes to presynaptic regions . 171 : These results suggest that PTP-3A is the major isoform that is localized to presynaptic domains . 183 : ptp-3A contributes to synapse development Because PTP-3A is localized to presynaptic regions , we asked whether mutations in ptp-3 caused any defects in synaptic development . PAPERDELIMITER PMID:16033794 245 : VAB-1 and SAX-3 co-localized on neuroblasts , consistent with SAX-3 and VAB-1 functioning together during this stage of development . 249 : SAX-3 is expressed in a subset of neuroblasts and neurons that express VAB1 . 262 : ( C ) VAB-1 and SAX-3 are co-expressed in some neuroblasts and neurons during development . PAPERDELIMITER PMID:15986473 3 : During oogenesis CGH-1 associates with P granules , but also accumulates to high levels in additional cytoplasmic particles . 52 : At the distal tip of each hermaphrodite gonad arm , in proliferating germ cells that have not entered meiosis , CGH-1 is present at low levels and is associated with P granules ( Fig 2ac , legend ) ( Navarro et al , 2001 ) . 54 : In oocytes at the proximal gonad end CGH-1 continues to be readily detectable in association with P granules and in these additional cytoplasmic particles ( Fig 2a ) . 64 : Longer exposures consistently reveal that in mitotic cells CGH-1 is detectable primarily in association with P granules ( not shown ) , but in meiotic cells CGH-1 is also apparent in particles within the central gonad cytoplasmic core ( inset ) . 84 : Like CGH-1 , GLD-2 associates with P granules in the germline and early embryo and is required for normal gametogenesis ( Fig 1c ) ( Kadyk and Kimble , 1998 ) . 92 : CGH-1 and P Granule Components CGH-1 associates with P granules throughout the gonad and in the embryo , as is revealed by its partial colocalization with the P granule component PGL-1 ( Fig 2a ) ( Navarro et al , 2001 ) . 106 : Despite the importance of PGL-1 for P granule function , in these pgl-1 ( bn102 ) animals CGH-1 was expressed at normal levels and appeared to be localized to P granules , although its perinuclear staining was slightly more diffuse than normal ( Fig 4b ) . PAPERDELIMITER PMID:16054030 93 : Furthermore , depletion of TPXL-1 prevents AIR-1 localization on spindle and astral microtubules and leads to AIR-1 collapse into the same domain of the centrosome as -tubulin ( Figure 3A ) . 95 : Thus , the interaction between TPXL-1 and AIR-1 targets AIR-1 to the microtubules . 101 : AIR-1 and TPXL-1 colocalize on the outer centrosomal layer and extend along centrosomal microtubules . 176 : Aurora A is localized at spindle poles , suggesting that defects in spindle-pole organization could contribute to a lack of kinetochore microtubule stability . PAPERDELIMITER PMID:15615786 4 : Here , we show that NHR-25 , a transcription factor of the nuclear receptor family , is Science expressed in the seam cells and is necessary for these cells to elongate and reach their neighbors after the asymmetric divisions . 169 : ( A , A ) An antibody against NHR-25 stains nuclei of seam cells , marked with scm : : gfp and outlined by MH27 antibody staining of adherens junctions in the same L1 larva . 176 : To test this possibility , we used an antibody against the NHR-25 protein . 177 : This antibody stained the same nuclei lit by the scm : : gfp seam-cell-specific marker at L1 ( Fig 6A ) and later stages ( data not shown ) . 216 : Consistently with its transcriptional function , we have detected NHR-25 in the nuclei of seam and , at a lower level , in hyp7 epidermal cells . PAPERDELIMITER PMID:15659483 89 : Expression was observed in the nuclei of elopmentv hyp5 , H0-H2 , V1-V6 and T These cells constitute a bilateral row of cells called seam cells ( Fig 1G , H ) . 95 : This additionally confirms the expression of ceh16 in the nuclei of these cells . 111 : Expression was observed in the nuclei of hyp5 , H0-H2 , V1-V6 , T. 112 : All nuclei that expressed ceh-16 : : gfp were also stained with the monoclonal antibody 4D9 ( Patel et al , 1989 ) . PAPERDELIMITER PMID:15790968 228 : Consistent with ALR-1 being a transcription factor , expression was exclusively nuclear in all observed cell types . PAPERDELIMITER PMID:16000383 231 : In wild-type animals , FOG-1 protein was observed in the germline and was predominantly cytoplasmic ( Fig 6 ) . PAPERDELIMITER PMID:16054029 40 : Location of Endogenous C. elegans EBP-2 and EBP-2::GFP at the Growing Plus Ends of Microtubules. 41 : By using spinning disk confocal microscopy to visualize the EBP-2::GFP fusion protein, we observed small dots of fluorescence moving away from centrosomes, consistent with EBP-2 decoration of growing MT ends. 45 : EBP-2 : : GFP Movement in a Wild-Type Embryo EBP-2 : : GFP marks the growing plus ends of microtubules in vivo , providing a visual record of microtubule growth rate and direction , as well as an estimate of the numbers of microtubules emanating from the centrosome . 63 : Microtubule plus ends ( marked by EBP-2 : : GFP fluorescence ) move in random directions throughout the cytoplasm in these early one-cell embryos ( prior to significant MT nucleation by centrosomes ) . 74 : Stream-Lapse Movie of Mitosis Reveals a Brief Accumulation of EBP-2 : : GFP Fluorescence at the KinetochoreMicrotubule Interface A stream-lapse movie of an embryo expressing EBP-2 : : GFP throughout metaphase and anaphase is shown ( ) . 76 : Note the accumulation of EBP-2 : : GFP fluorescence at the kinetochoreMT interface that occurs at the same time as pole separation prior to anaphase . 77 : However, we consistently observed an accumulation of EBP-1::GFP fluorescence at the kinetochore region later in metaphase, coincident with spindle pole separation. 78 : Stream-Lapse Movie of Mitosis Reveals a Brief Accumulation of EBP-2 : : GFP Fluorescence at the KinetochoreMicrotubule Interface A stream-lapse movie of an embryo expressing EBP-2 : : GFP throughout metaphase and anaphase is shown ( ) . 79 : Based on pervious examination of the timing of anaphase relative to NEBD, as well as the inspection of the DIC component of steam-lapse movies, we infer that EBP-2::GFP fluorescence is most prominent at the kinetochore region during early sindle elongation, preceding sister chromatid separation. 80 : Note the accumulation of EBP-2 : : GFP fluorescence at the kinetochoreMT interface that occurs at the same time as the separation of centrosomes prior to anaphase . PAPERDELIMITER PMID:15987734 8 : Gal-T2 : : GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern , consistent with the localization of the Golgi apparatus . 201 : Wild-type embryos showed PIE-1 : : GFP concentrated in the posterior nucleus in the P1 cell ( Figure 2C , top ) , and , in later-stage embryos , PIE-1 accumulation was retained in the P-lineage . 236 : The anti-Gal-T2 scFv showed a strong immunofluorescence signal in all cells of early and late embryo specimens , revealing numerous punctate organelles surrounding the nucleus ( Figure 4 , AC , and Movie M3 ) . 241 : The perinuclear staining pattern detected by the antibody was similar to the detection of a Gal-T2 : : GFP transgene reporter , in which the first 60 amino acids of Gal-T2 ( including the N-terminal transmembrane domain and stem region ) was fused to the N terminus of GFP . 250 : The combined antibody and GFP data support the interpretation that Gal-T2 is a Golgi protein , localized in a perinuclear pattern in early stage embryos . 271 : ( B ) High magnification of Golgi apparatus staining and nuclei from embryo in A ( C ) Anti-Gal-T2 scFv antibody staining in a 28-cell embryo shows perinuclear staining pattern . PAPERDELIMITER PMID:15958510 17 : Localization of FRK-1 to the plasma membrane requires beta-catenin , but not cadherin or catenin , and muscle-expressed -integrin is nonautonomously required for this localization ; in the absence of these components FRK-1 becomes nuclear . 190 : FRK-1 shows dynamic localization to the cell surface and nucleus 193 : Using two independent antibodies elicited to two different FRK-1 peptides we found that immunoreactive FRK-1 was initially present both in nuclei and at cell-cell contact points of all cells in early embryos ( Fig 6A , B ) . 198 : Localization of FRK-1 to nuclei correlates with phases of active cell division : nuclear localization was prominent in early embryos and the adult germline ( Fig 6A , B and not shown ) . 199 : The protein remained in the nucleus and at the cell surface until shortly before embryonic enclosure , at which stage most cells became mitotically inactive and nuclear FRK-1 became undetectable ( Fig 6C , D ) . 200 : In elongated embryos , FRK-1 was present in the cytoplasm and at the membrane of epidermal cells ; cell surface staining was especially prominent in the seam cells ( Fig 6E , F ) . 203 : FRK-1 is localized to the nucleus and at regions of cell-cell contacts elopmentv inearlyembryos , asevidentin4-cell ( A ) and40-cell ( B ) embryos . 204 : ( C ) . The protein remains nuclear-excluded throughout elongation ( eg 1 . 25-fold stage ; D ) , and cell surface localization is especially pronounced in the seam cells of elongated embryos ( eg 3-fold embryo , E ; overlay with MH27 apical junction antigen in F ) . 209 : Later , during larval development , FRK-1 reappeared in the nuclei of seam cells and in the nuclei of the developing germline , correlated with their active post-embryonic division . 210 : In the adult soma , FRK-1 stably localized to apical junctions of the intestine and somatic cells of the gonad ( not shown ) . 211 : Cell adhesion components are required for membrane localization and nuclear exclusion of FRK-1 Our findings that FRK-1 was nuclear-excluded and localized primarily to cell boundaries during embryonic enclosure and elongation suggest that it may perform its essential function in morphogenesis at the plasma membrane , perhaps by coordinating or regulating cell adhesion complexes . 214 : In wild-type embryos , FRK-1 localizes to the plasma membrane of all cells immediately before enclosure ( A ) , and remains nuclear-excluded throughout elongation ( eg 1 . 5-fold embryo ; B ) . 266 : These studies also suggest an earlier role for FRK-1 , correlated with its localization to nuclei . 269 : FRK-1 localized primarily to the cell surface during epidermal morphogenesis , where it probably interacts with cell adhesion components ; indeed , the HMP-2 beta-catenin was required for cell surface localization ( and nuclear exclusion ) of FRK-1 , and FRK-1 interacted with HMP-2 in vitro . 281 : It is during early embryogenesis that FRK-1 was nuclear-localized and it is interesting to note that Fer has been shown to phosphorylate nuclear localized factors such as Stat3 ( Priel-Halachmi et al , 2000 ) and the TATA element modulatory factor ( Schwartz et al , 1998 ) . PAPERDELIMITER PMID:15848803 112 : TBH-1 is expressed in the ( C ) RIC interneurons , where it is mainly localized to synaptic specializations and ( D ) gonadal sheath cells . 123 : Bright staining was observed in RIC synaptic regions , as indicated by a punctate immunofluorescence pattern in the nerve ring , whereas weaker staining was observed in the RIC neuronal processes and cell bodies . 131 : Perhaps TBH-1 is associated with actomyosin filaments in the gonadal sheath cells . 133 : TDC-1 staining was observed in the cell bodies and axonal processes of TDC-1-expressing neurons . PAPERDELIMITER PMID:16107479 149 : MLS-2 protein is localized in the nuclei of early M lineage cells and a subset of head neurons 154 : Expression of MLS-2 was first detectable in one or two cells in embryos at the 50-cell stage ( data not shown ) and is localized in the nucleus . 188 : ( D , H ) Corresponding DIC images for panels C and G Nuclear localized MLS-2 protein was seen in a subset of cells during embryogenesis ( A , C ) , in six head neurons ( arrowhead in E ; the neuronal signal was out of focus in G ) , in the M mesoblast ( arrows in E and G ) and in the eight M descendants ( M , N ; only four cells visible in this focal plane ) . PAPERDELIMITER PMID:16139225 168 : ( A ) SEA-1 accumulates in nuclei of 20 to 100-cell wild-type embryos , the correct time to regulate xol-1 . 215 : SEA-1 Accumulates in Nuclei of Young Embryos Consistent with the molecular identity of SEA-1 as a T-box transcription factor , SEA-1 accumulates in nuclei of young embryos , as indicated by immunofluorescence experiments with anti-SEA-1 antibodies ( Figure 5A ) . PAPERDELIMITER PMID:15539489 3 : Maternal mau-2 gene expression is sufficient for normal development until the fourth larval stage , and a MAU-2 : : GFP fusion protein localizes to the cytoplasm of neurones . 206 : In addition , the MAU-2 : : GFP fusion appears to entirely and uniformly fill the cytoplasm of all neurones , including along their axonal projections , and but excluding the nucleus . 247 : Given that mau-2 functions cell autonomously in AVM and that MAU-2 : : GFP is cytoplasmic , it is possible that mau-2 may function as a cytoplasmic effector of the netrin andor slit cues . 308 : Although the biochemical role of the protein MAU-2 remains unknown , MAU-2 appears to function within the migrating cell and to localize to the cytoplasm , where it may participate in the intracellular interpretation of guidance cues encountered by a cell or axon during its migration . PAPERDELIMITER PMID:15866168 6 : FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion , whereas it is virtually undetectable in nonfusing seam cells . 121 : The localization of FUS-1 supports a role for its action at the plasma membrane in cells that are potentiated to fuse . 123 : FUS-1 is also detected in the excretory cell ( Figure 4K ) and the apical membrane of gut cells ( not shown ) starting later in embryogenesis . 126 : As the embryo develops , FUS-1 levels increase in these epidermal cells , where it concentrates almost exclusively on the apical membrane ( Figures 4H and 4K ) and some colocalizes with apical junctions ( Figures 4L4N ) . 131 : The localization of FUS-1 at the plasma membrane of epidermal cells raises the possibility that it might do so by directly acting on EFF-1 , also an apparent cell surface protein ( W Mohler , personal communication ) . 148 : FUS-1 Localizes to the Apical Membrane of Epidermal Cells in Late-Stage Embryos 153 : Magnified images of the area around the apical junctions between hyp6 and hyp7 ( M ) and between the P1 and P2 cells ( N ) show that FUS-1 is localized to apical membranes of the epidermal cells and partially colocalizes with AJM-1 . PAPERDELIMITER PMID:9819355 4 : Specific antisera were used to show that SPE-4 resides within the fibrous body-membranous organelles membranes during wild-type spermatogenesis . 131 : This cellular compartment is labeled specifically by the monoclonal antibody 1CB4 ( Okamoto and Thomson , 1985 ) , so a coincident immunofluorescent signal from 1CB4 and SPE-4 specific antibodies would indicate that SPE-4 also localizes to the FB-MOs . 192 : SPE-4 localizes within the GolgiER derived FB-MOs and segregates to spermatids as they bud from the residual body during C elegans spermatogenesis . 209 : Staining with 1CB4 ( B3 ) reveals that the punctate signal is mostly found in the cell body and a similar , overlapping signal ( B4 ) is observed after staining with the 9910 SPE-4 antiserum . 256 : The localization of SPE-4 to the FB-MOs is intriguing because two human presenilin family members ( PS-1 and PS-2 ) are localized to the Golgi apparatus and endoplasmic reticulum in transfected COS cells , H4 human neuroglioma cells and primate brain ( Kovacs et al , 1996 ; Doan et al , 1996 ; DeStrooper et al , 1997 ; Lah et al , 1997 ) . 258 : Two affinity purified fusion antisera ( EU20 and EU21 ) that stain MOs and only recognized male gonadal epitopes also recognized epitope ( s ) in spe-4 ( q347 ) mutant spermatocytes . PAPERDELIMITER PMID:15572125 128 : SAS-6 colocalized with SAS-4 to small focal structures in the center of the centrosome throughout the embryonic cell cycle ( Figure 3A ) . 132 : We also performed immuno-EM for SAS-5 and found that it too localizes to the centriolar cylinders ( Supplemental Figure S2D ) . 133 : The combination of light microscopy and EM analysis indicates that SAS-6 , like SAS-4 and SAS-5 , is a bona fide centriolar protein . 136 : Using our antibodies , we could detect ZYG-1 staining at centrioles throughout the cell cycle ( Figure 3A ) . 139 : In summary , ZYG-1 , like SAS-4 , SAS-5 , and SAS-6 , localizes to centrioles at a time consistent with its role in centriole assembly . 151 : SAS-6 and ZYG-1 Colocalize with SAS-4 at Centrioles throughout the Cell Cycle ( A ) Fixed embryos were stained for DNA , microtubules , SAS-4 , and either SAS-6 or ZYG-1 . 157 : ( C ) SAS-6 localizes to centrioles by immunoelectron microscopy . 211 : SPD-2 localizes to centrioles and weakly to the PCM . 212 : In contrast , AIR-1 localizes to the periphery of the centrosomes and extends out along astral microtubules . 227 : SPD-5 colocalizes with -tubulin to the PCM in wild-type embryos . 283 : SAS-5 and SAS-6 colocalize with SAS-4 throughout the embryonic cell cycle and are associated with centrioles by immuno-EM ( Figure 3 and Supplemental Figure S2 ) . 285 : In contrast , we found ZYG-1 to be localized to centrioles throughout the embryonic cell cycle ( Figure 3 ) . 300 : We show that two proteins that localize to the PCM , SPD-5 ( required for the formation of the PCM ; Hamill et al , 2002 ) and -tubulin ( required for centrosomal microtubule nucleation ; Hannak et al , 2002 ; Strome et al , 2001 ) , contribute to the formation of new centrioles . PAPERDELIMITER PMID:15545320 6 : SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern . 55 : SUP-12 localizes to the nuclei in body wall muscle and its RNA-binding domain directly binds to the unc-60 premRNA in vitro . 102 : SUP-12 is expressed in body wall muscle and localizes to the nuclei We next investigated expression and subcellular localization of SUP-12 . 105 : GFP-tagged SUP-12 , which was expressed in the body wall muscle under the control of the myo-3 promoter ( Okkema et al , 1993 ) , predominantly localized to the nuclei ( Fig 5 , BD ) . 129 : ( BD ) Nuclear localization of the GFP-SUP-12 fusion protein driven by the body wall muscle-specific myo-3 promoter . 130 : Staining of nuclei by DAPI ( C ) revealed colocalization of some of the muscle nuclei ( arrows ) with GFP-SUP-12 ( D ) . 154 : SUP-12 localized to the muscle nuclei and bound directly to exon 1 and the first and second introns of the unc-60 premRNA in vitro , suggesting that SUP-12 is directly involved in muscle-specific splicing of the unc-60 pre-mRNA . PAPERDELIMITER PMID:15498497 31 : RIC-8 is localized at the embryo cortex , and its activity is essential for the asymmetric localization of GPR-12 . 92 : We found that the cellular localization of RIC-8 is very similar to the localization of GOA-1 , for example at the cortex and on the asters of the mitotic spindle ( Figure 4 ) . 113 : These results suggest that the cortical localization of RIC-8 is either intrinsic to the protein or depends on another protein that has yet to be identified . 139 : Staining is observed at the cortex , on the asters , at the nuclear envelope , and around the chromatin . PAPERDELIMITER PMID:15313609 93 : The conclusion is that two UNC-89 isoforms containing the interkinase region ( either UNC89-B that is expressed in both body-wall and pharynx , or UNC-89-C that are expressed in body-wall ) , are localized to the middle of A-bands . 192 : Furthermore , these isoforms localize to the middle of A-bands in both muscle types . 257 : Antibody localization of the isoforms in body-wall muscle has not revealed any interesting differences : anti-IK , which should recognize UNC-89-B , -C and -D , shows the same localization ( to the middle of A-bands in both body wall and pharyngeal muscle ) as the previously described antibody , anti-Ig3-6 ( Figure 6 ). PAPERDELIMITER PMID:15314158 10 : SMG-2 is located diffusely through the cytoplasm , and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation . 236 : SMG-2 is localized to the cytoplasm . 253 : SMG-2 is located diffusely throughout the cytoplasm in the wild type ( Fig 6A ) and is eliminated in smg-2 ( r908 ) ( Fig 6B ; r908 is a null allele ) . PAPERDELIMITER PMID:15380065 16 : We demonstrate here that the MES complex has nucleosomal histone H3-specific methyltransferase activity . 18 : Western blot analysis revealed that anti-MES-6 antibodies specifically precipitated MES-6 and coprecipitated MES-2 and MES-3 ( Figure 1 ) . PAPERDELIMITER PMID:15364921 33 : We have now observed that AAT-9 ( WormBase gene number Y53H1C . 1 ) expressed alone in Xenopus oocyte localizes to the cell surface . 90 : AAT-9 Transports Amino Acids When Expressed Alone in Xenopus Oocytes--Unlike the mammalian light chain subunits of HATs and the C elegans light chain subunits AAT-1 and AAT-3 , which must associate with an exogenous heavy chain subunit to reach the membrane surface , AAT-9 localizes to the plasma membrane of X laevis oocytes when expressed alone . 96 : C elegans AAT-9 transporter expressed alone in Xenopus oocytes is localized at the cell membrane . 97 : Immunofluorescence with anti-AAT-9 antibody on cryosections of Xenopus oocytes injected with AAT-9 cRNA ( left ) or H2O ( control ; right ) shows AAT-9 at the cell surface . PAPERDELIMITER PMID:15456850 147 : In oocytes and early stage wildtype and cpl-1 ( ok360 ) mutant embryos , YP170 antibody localised to small electron-dense vesicles which have been identified as yolk platelets in previous studies ( Hall et al , 1999 ) . 152 : Double IEM labelling with antibodies to yolk protein and CPL-1 showed colocalisation to yolk platelets in wild-type embryos ( Fig 5E ) and in the adult pseudocoelom ( Fig 5F ) . 195 : ( E , F ) Double labelling of yolk platelets with anti-pro-CPL-1 antibody ( detected with 10 nm gold ; arrowheads ) and antiYP170 antibody ( detected with 15 nm gold ; arrows ) in a wild-type embryo ( E ) and in the hermaphrodite pseudocoelom ( F ) . 199 : However , instead of rapidly disappearing , RME-2 could still be detected in later stage mutant embryos , in which it localised to the periphery of the large yolk vesicles ( Fig 7E , F ) . 200 : Localisation of RME-2 to the peripheral region and yolk to the lumen , indicated that although present in the same compartment , yolk and RME-2 are separated from one another . 206 : In wildtype embryos , anti-RME-2 antibody localised to small cytoplasmic vesicles , which are possibly sorting vesicles or early endosomes ( Fig 7G ) . 227 : ( G , H ) IEM localisation of RME-2 antibody to small vesicles in wild-type embryo ( G ) and to electron-light regions at the periphery of enlarged yolk vesicles in cpl-1 ( ok360 ) embryo ( H ) . 280 : Using IEM we showed that cathepsin L and yolk proteins colocalise within the lumen of yolk vesicles in embryos and during yolk transport in the pseudocoelom . PAPERDELIMITER PMID:15100407 2 : We show that matefin is a nuclear membrane protein that colocalizes in vivo with Ce-lamin , the single nuclear lamin protein in C elegans , and binds Ce-lamin in vitro but does not require Ce-lamin for its localization . 4 : Embryonic matefin is maternally deposited , and matefin is the first nuclear membrane protein known to have germ line-restricted expression . 8 : We report that this gene ( mtf-1 ) encodes matefin , a nuclear membrane protein that binds Ce-lamin and has essential embryonic and germ line-specific functions . 54 : Matefin Is a Previously Uncharacterized C elegans Nuclear Envelope Protein with a SUN Domain and Two Putative Transmembrane Domains . 68 : By indirect immunofluorescence , both antisera specifically stained the nuclear envelope of C elegans embryos ( Fig 1C ) . 81 : Please note that although both embryonic and adult nuclei are stained with Ce-lamin , only the embryonic nuclei are stained with matefin ( Upper ) . 92 : We concluded that matefin is a lamin-binding protein of the inner nuclear membrane . 96 : Matefin was detected at the nuclear envelope of all early embryonic cells ( Fig 1C ) . 124 : Nuclear-envelope localization of matefin does not require Ce-lamin or UNC-84 , and matefin is not required to localize other known nuclearenvelope proteins . 129 : To our surprise , RNAi-mediated down-regulation of Ce-lamin had no obvious affect on the nuclear-envelope localization of matefin ( Fig 2A ) . 133 : Immunofluorescence studies of early embryos also showed that matefin remained nuclear-envelope-localized through early anaphase and then completely disassembled during mid late anaphase , similar to Ce-lamin and other known nuclear membrane proteins ( refs . 7 and 12 and data not shown ) . 135 : Matefin Is Present in all Early Embryonic Nuclei but Is Restricted to Germ Cells from Midembryogenesis to Adulthood . 175 : The only other gene in C elegans with a SUN domain is UNC-84 , which like matefin is localized to the nuclear envelope ( 7 , 8 ) . 183 : Our findings suggest that matefin is anchored at the nuclear inner membrane by two putative transmembrane domains , with exposed N and C-terminal domains that both bind Ce-lamin in vitro . 184 : These findings predicted lamin-dependent localization of matefin at the nuclear envelope . 189 : Matefin and UNC-84 are both SUN-domain proteins at the nuclear envelope , but their specific localizations and functions appear to differ significantly . 190 : Much evidence localizes matefin to the nuclear inner membrane : its direct binding to Ce-lamin , colocalization with Ce-lamin in embryos , and association with peripheral chromatin in detergent-extracted nuclei ( shown in this work ) . 204 : We conclude that matefin is an essential component of the nuclear envelope in early embryos . 269 : In addition to its presence in the germ line , matefin was present in all nuclei of early embryos . PAPERDELIMITER PMID:15044551 115 : Pida-1CeIA-2 : : GFP was observed in neuronal cell bodies and in puncta along the length of axons of neurons in which it was expressed ( Fig 1 ) . 117 : As shown in Figure 1B , the punctate GFP reporter pattern in axons colocalized with synaptotagmin staining , suggesting clustering of CeIA-2 : : GFP to synaptic regions . 131 : These puncta are visualized by CeIA-2 : : GFP expression ( far left ) and are revealed as synapses by anti-synaptotagmin antibody staining ( far right ) . 216 : In wild-type animals ( Fig 4A ) and ida-1 mutants ( Fig 4B ) , anti-SNT-1 antiserum stained only the synapse-rich axons and did not stain cell bodies , suggesting that SNT-1 is correctly localized to synaptic terminals in the ida-1 mutants . PAPERDELIMITER PMID:14872060 3 : Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts , whereas zygotic RBD-1 rst appears in late embryos and is localized in the nucleolus in most cells , although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage . 23 : We also found that RBD-1 is localized in the nucleolus , like Mrd1p and Ct-RBD-1 and that its zygotic expression starts in late embryos , although transcription of pre-rRNA by RNA polymerase I is known to start as early as the one-cell stage . 80 : As evidenced by its colocalization with a nucleolar marker protein FIB-1 , RBD-1 is localized in the nucleolus . 82 : These results show that RBD-1 is a nucleolar protein , like its counterparts in yeast and Ctentans , and is expressed ubiquitously after the late embryonic stage . 169 : In this study , we have claried for the rst time the outline of the pre-rRNA processing pathway in Celegans and shown that a nucleolar RNA-binding protein , RBD-1 , is required for 18S rRNA processing in Celegans . PAPERDELIMITER PMID:14668347 105 : The heterodimer AAT-1ATG-1 that was non-functional ( Fig 2A ) , was localized intracellularly ( see below ) and contained the faster-migrating , presumably core-glycosylated form of ATG-1 ( empty arrowhead , Fig3B , lanes 5 and 6 ) . 106 : In contrast , when expressed alone , ATG-1 was localized at the cell surface ( see below ) and , correspondinglv , mainly a slower-migrating band , presumably the terminally-glycosylated form , was observed ( black arrowhead , Fig 3A , B , C and D , lane 4 ) . PAPERDELIMITER PMID:14565992 43 : Immunofluorescence studies using CLH-3b-specific antiserum demonstrated that the channel is expressed in the oocyte plasma membrane . 213 : As shown in Figures 12A-B , CLH-3b was detected in the plasma membrane at all stages of oocyte development , including newly formed oocytes in the loop region of the gonad . PAPERDELIMITER PMID:14702387 171 : We next investigated to what degree mutant TBB-2 proteins can assemble into spindle microtubules by double staining fixed tbb-2 mutant embryos with rabbit antibodies that recognize the C terminus of TBB-2 ( Lu et al , 2003 ) , and a mouse antibody that recognizes -tubulin ( Fig 3C , see Materials and Methods). 173 : During mitosis in wild-type one-cell stage embryos , astral microtubules radiated out from both centrosomes , contacting the cell cortex at many points , with the different antibodies exhibiting full overlap in their staining of spindle microtubules . 201 : We also stained tbb-2 ( or362 ) and tbb2 ( gk129 ) with the TBB-1 antibodies and found that TBB-1 was still incorporated into spindle microtubules in or362 and gk129 mutant embryos ( Fig 4C ) . PAPERDELIMITER PMID:14630920 254 : In larval and adult stages , the CPZ-1 native protein was localized in the cuticular regions of the worms ( Fig 8 , a and e ) . 255 : In molting larvae ( Fig 8 , bd ) , the protein was localized in both the new and old cuticles , in particular in the interface where the old cuticle is being degraded before ecdysis ( Fig 8c ) . 289 : The immunoelectron microscopy data clearly indicate the presence of CPZ-1 native protein in both the new and the old cuticles during molting , specifically at the time when the separation between the old and the new cuticles occurs . PAPERDELIMITER PMID:14534135 182 : Asymmetric localization of GPR-12 at the cortex depends on PAR-3 and LET-99 GPR-12 were recently shown to be asymmetrically localized at the cell cortex in response to PAR proteins ( Colombo et al , 2003 ; Gotta et al , 2003 ) . 183 : Because LET-99 is also asymmetrically localized in a PAR-dependent manner , we sought to determine the relationship between PAR-3 , LET-99 and GPR-12 localization . 185 : In early wild-type embryos , GPR-12 were localized both on the asters and the cell cortex ( Fig 3A-I ) . 187 : The cortical localization of GPR12 changed with the cell cycle . 188 : In one-cell embryos , GPR-12 were uniformly present at a low level on the cortex from early prophase to metaphase . 189 : The level of cortical localization of GPR-12 increased and became weakly enriched at the posterior pole of most embryos during anaphase ( 78 , n42 ; Fig 3C ) . 192 : In the P1 blastomere during interphase ( Fig 3E , F ) , GPR-12 were highly enriched around the posterior pole of the cell ( 100 , n53 ) , and were present at low levels uniformly around the cortex of AB . 193 : As the cell cycle progressed , GPR-12 asymmetry in P1 disappeared ; GPR-12 were uniformly localized around the cortex through out prophase , metaphase and early anaphase ( Fig 3G , H ) . 203 : At later stages , when anaphase LET-99 and GGPR in spindle positioning 5723 Fig 3 . 216 : At later stages , when anaphase spindle pole oscillations occur in both G mutant and wildtype embryos , the cortical GPR-12 staining levels in G mutants resembled that in wild-type embryos ( Fig 3I , S ) . 248 : However , the cortical localizations of LET-99 and GPR-12 in cells do not overlap but instead are somewhat reciprocal at anaphase . 275 : However , we observed a cell cycle dependent change in LET-99 localization at cell contacts . 293 : Thus , the asymmetric patterns of both LET-99 and GPR-12 at the EMSP2 cell boundary are MES-1SRC-1 signaling dependent , which suggests that LET-99 and G signaling act downstream of MES-1SRC-1 to promote spindle orientation in EMS . 330 : The G proteins and GPR-12 are localized at the cortex and the microtubule asters . 365 : Based on these results and the pattern of cortical LET-99 localization , we propose that LET-99 antagonizes GGPR-12 signaling , thus downregulating cortical forces asymmetrically during both rotation and anaphase spindle elongation . PAPERDELIMITER PMID:14522947 3 : Using RNA interference-based genomics in Caenorhabditis elegans , we identified KNL-1 , a novel kinetochore protein whose depletion , like that of CeCENP-A or CeCENP-C , leads to a kinetochore-null phenotype . 33 : Using the same assays to rescreen gene products implicated in chromosome segregation by RNAi-based functional genomics , we have identified a novel kinetochore component , KNL-1 , whose depletion results in a kinetochorenull phenotype . 58 : KNL-1 colocalized with CeCENP-C , a bona fide kinetochore marker , throughout mitosis ( Fig 2C ) . 77 : KNL-1 localizes to kinetochores throughout mitosis . 89 : Between prometaphase and early anaphase , KNL-1 also localizes to the spindle region but does not coalign with spindle microtubules ( Fig 2C ) . 94 : Cumulatively , the phenotypic and localization analysis indicate that KNL-1 is present at kinetochores throughout mitosis and is essential for mitotic chromosome segregation . 227 : KNL-1 targeted normally to kinetochores in all cases . 250 : Using high-resolution fluorescence assays to rescreen gene products identified by functional genomic analysis of Chromosome III , we identified KNL-1 , a kinetochore protein that translates the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a microtubulebinding interface on mitotic chromosomes . PAPERDELIMITER PMID:14517331 1 : Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex ( NPC ) . 6 : The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs , confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans . 197 : Ce-Gp210 localized at the nuclear rim in C elegans gonads in a punctate pattern characteristic of NPCs , and colocalized with the family of FG-repeat nucleoporins recognized by mAb mAb414 ( Davis and Blobel , 1986 ; Figure 4C ) . 198 : We concluded that our antibodies specifically recognized endogenous Ce-Gp210 proteins at the NPC and could be used to analyze the downregulation phenotype of Gp210 in C elegans . PAPERDELIMITER PMID:12921736 152 : As new cuticle is synthesized in the stage preceding the one in which it is required , the expression of M142 . 2cut-6 in late embryos and L2d indicated that the protein would be present in the L1 and dauer cuticles . 165 : Immunostaining with anti-M142 . 2CUT-6 was also performed on the C elegans strain SU93 , which contains an integrated array expressing ajm-1 : : GFP : ajm-1 localizes to the apical borders of all C elegans epithelia and thus defines the epitheliumseam cell border . 225 : However , M142 . 2 / cut-6 is novel in that our observation extends this regulatory pattern to a noncollagenous cuticle component . PAPERDELIMITER PMID:15525531 73 : As reported previously ( Crittenden et al , 2002 ) , FBF-1specific antibodies stain weakly in the most distal mitotic nuclei of wild-type germlines and more strongly in the proximal three-fourths of the mitotic region . PAPERDELIMITER PMID:16401427 18 : STA-1 Accumulates in the Nuclei of a Subset of Amphid Neurons 19 : Immunohistochemical localization using a STA-1-specific antibody readily detected STA-1 protein in pharynx , head ganglia , tail ganglia ( Figures 1A and 1B ) , ventral nerve cord ( Figure 1C ) , embryos ( Figure 1E ) , and body muscles ( data not shown ) , with a diffuse staining pattern indicative of a largely cytoplasmic localization . 21 : In contrast , detailed analysis of immunostaining patterns in the head ganglia revealed a distinct pattern of nuclear STA-1 accumulation in a few neuronal cells ( Figure 1B ) , indicative of activation . 22 : Confocal immunostaining analysis of head ganglia identified precise nuclear accumulation of STA-1 in select subsets of neurons ( Figures 1F1H ) . 24 : Therefore , it was possible to determine the identities of cell bodies within the head ganglia that displayed selective STA-1 nuclear staining by comparison to the head ganglion cell map [ 7 ] . 26 : Based on their relative positions ( Figures 1F1H ) , cell bodies displaying nuclear accumulation of STA-1 were identified as five pairs of neurons : ADL , ASK , ASJ , ASH , and AIZ , four of which are amphid neurons . 27 : This pattern of STA-1 nuclear staining was essentially invariant in all worms examined . 30 : Because nuclear accumulation of STAT proteins is indicative of activation [ 3 ] , STA-1 protein nuclear accumulation in these five pairs of neurons under standard laboratory culture conditions suggests involvement in a constitutive neuronal sensory function . 33 : Ubiquitous STA-1 Expression and Constitutive Nuclear Accumulation in Some Amphid Neurons in N2 Worms Revealed by Immunostaining with Affinity-Purified antiSTA-1 Antibody ( A ) Overall view of stained L1 larvae ( ventral view , anterior left ) . 43 : Nuclear accumulation of STA-1 in neurons involved in TGF-b signaling prompted us to investigate crosstalk between these pathways . 46 : N2 wild-type worms and Daf-defective daf6 ( e1377 ) mutants showed the same nuclear staining pattern in the limited set of amphid neurons described previously ( Figure 2A and data not shown ) . 47 : In contrast , Daf-constitutive mutant daf-7 ( e1372 ) and daf-8 ( m85 ) worms displayed much brighter STA-1 staining , as well as nuclear staining in an expanded set of cells ( Figures 2B and 2C ) . 169 : In addition to the cooperative action of TGF-b and STA-1 to inhibit dauer formation , active TGF-b signaling also restricted STA-1 nuclear accumulation to a small subset of posterior ganglion sensory neurons . PAPERDELIMITER PMID:13679921 201 : Likewise , in contrast to rfl-1 ( or198ts ) mutants , NED-8 localized to the nucleus and the cytoplasm in wild-type and mel-26 ( or543 ) mutant embryos ( Fig 2g ) . PAPERDELIMITER PMID:14504223 260 : SPE-39 staining shows a diffuse distribution in the cytoplasm in both wild-type spermatocytes and spermatids ( Figure 7 , D and P ) . 275 : In wild- type spermatocyes at the budding stage , SPE-30 staining is observed in both residual bodies and budding spermatids , but staining is stronger in the residual bodies than in the spermatids ( Figure 7D ) . 276 : In wild-type spermatids , staining of SPE-39 is concentrated in the center of the cytoplasm and diminished in the cytoplasmic cortex ( Figure 7P ) , where the MOs are located . 320 : SPE-4 is an integral membrane protein that resides in the MOs ( Arduengo et al 1998 ) , while SPE-39 is highly hydrophilic and has a diffuse cytoplasmic distribution in both spermatocytes and spermatids . PAPERDELIMITER PMID:12937278 6 : During prometaphase , metaphase , and anaphase , KLP-18 concentrates toward the poles in both meiotic and mitotic spindles . 128 : KLP-18 Localization in Mitotic Spindles 132 : KLP-18 staining was most concentrated in mitotic spindles toward the poles . 133 : During anaphase , KLP-18 staining also concentrated between the separating groups of chromosomes , with a dim zone at the spindle equator ( Figure 2A ) . 135 : After cytokinesis , KLP-18 accumulated somewhat around the nucleus and at the cortex near sites of cell-cell contact ( Figure 2B ) . 140 : KLP-18 Localization in Meiotic Spindles 142 : Immunostaining showed KLP-18 colocalized with the disorganized MTs during prometaphase ( Figure 3A ) . 172 : ( A ) During mitotic anaphase KLP-18 is concentrated on the centrosomes ( arrowheads ) and on the MTs between the separating masses of chromatin , with a dim zone at the spindle equator ( arrow ) . 173 : ( B ) KLP-18 becomes concentrated near plasma membranes at sites of cell-cell contact . 194 : ( A ) After nuclear envelope breakdown , KLP-18 colocalizes with MTs around the condensed chromosomes . 195 : ( BD ) During prometaphase , metaphase , and anaphase KLP-18 is highly enriched at the spindle poles . 196 : ( E ) During telophase , KLP-18 and MTs are concentrated between separating chromosomes . 334 : Like Klp2 homologs in other organisms , C elegans KLP-18 concentrates in spindles , especially at spindle poles during prometaphase , metaphase , and early anaphase , and then in the interzone during late anaphase / telophase . PAPERDELIMITER PMID:12853134 94 : Sub-cellular localization of vha-8 by immunogold electron microscopy using anti-VHA-8 antibodies detected signals in the cytoplasm of the excretory canals ( Fig 2L ) and stacked sheets of the plasma membranes of the syncytial hypodermal cells ( Fig 2M ) . 131 : VHA-8 signals were also observed in stacked sheets of the apical plasma membrane of syncytial hypodermal cells ( M , arrowheads ) . 195 : Our result of immunogold electron microscopy revealed that VHA-8 is also expressed in apical surfaces of syncytial hypodermal cells where the plasma membranes are folded into stacked sheets ( Fig 2M ) . PAPERDELIMITER PMID:12783803 36 : We report that both laminin subunits are secreted between the primary it issue layers and become localized in different patterns to exposed cell surfaces , consistent with a receptor-facilitated process . 133 : Laminin A is first detected between tissue layers near the end of gastrulation ( Fig 2A ) and then becomes localized along the muscle cells as the embryo begins to elongate ( Fig 2B ) . 153 : ( F ) Laminin A is also detected in the basement membranes associated with the excretory canal ( c ) . 169 : Laminin B antiserum stains all the major basement membranes during the remainder of embryogenesis and throughout larval development and in the adult ( Fig 4BE ) . 170 : Although laminin B staining is weak in the basement membranes surrounding pharynx , intestine , body wall muscle and epidermis , the staining is strong in the basement membranes surrounding gonad , distal tip cell , vulval muscle , intestinal muscle , anal depressor muscle and coelomocytes ( Fig 4F-H ) . 174 : ( B ) Laminin B is localized to the muscle and epidermis basement membranes in late stage embryos , larvae and adults . 179 : ( C ) In L3 and L4 larvae , Laminin B is localized to the gonad basement membrane and is associated with the distal tip cell ( dtc ) , a migratory cell that helps form the gonad ( g ) . 180 : ( D ) In late stage embryos , larvae and adults , Laminin B is localized to the basement membranes that separate the intestine ( i ) , rectal epithelium ( a ; anus ) , epidermis and pseudocoelom . 181 : ( E ) Laminin B is localized to the basement membrane that encloses the gonad ( g ) primordium cells in the L1 and L2 larval stages . 182 : ( F ) Laminin B is detected in the basement membranes associated with intestinal muscle ( im ) and anal depressor muscle ( am ) . 269 : These results indicate that the correct distribution of the laminin subunits does not initially require collagen type IV and is consistent with the notion that laminin is localized to cell surfaces before a prototypical basement membrane assembles . 431 : Each subunit is distributed to specific cell surfaces that are exposed between it issue layers near the end of gastrulation . 457 : The laminin subunits associate with cell surfaces before the reported expression of other basement membrane components and they are required to assemble stable basement membranes . 532 : We show that laminin associates with cell surfaces in a process that is probably receptor mediated and occurs before the assembly of a prototypical basement membrane . PAPERDELIMITER PMID:14732404 160 : RFP-1 is a nuclear protein in C elegans embryos , larvae , and adults 180 : RFP-1-specific staining is observed in nuclei , but not in nucleoli , of C elegans embryos , represented by the threefold stage embryo shown ( Fig 5B ) . 181 : In L1 larvae , specific RFP-1 staining was observed in the nuclei of pharyngeal , intestinal , and nerve ring cells , but not in nucleoli ( Fig 5C ) . 182 : The nuclear staining of RFP-1 in most it issues was observed in L2 , L3 , and L4 animals ( data not shown ) . 183 : In adult animals , RFP-1 was localized to the nuclei of oocytes and germ cells in the syncytial gonad . 187 : RFP-1 is localized to the nucleus in C elegans embryos , larvae , and adults . 198 : RFP-1 nuclear staining was also observed in pharyngeal and intestinal cell nuclei in the head region of the adult animal ( data not shown ) . 286 : Indirect immunohistochemical staining shows that RFP-1 is a nuclear protein in embryos , larvae , and adults ( Fig 5 ) . PAPERDELIMITER PMID:14623111 11 : HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells , but not in the hatched larvae or adults . 12 : HMG-5 mainly localized to the chromosomal ends , indicating that HMG-5 also binds to telomeres in vivo . 43 : We also show that HMG-5 is expressed exclusively in the nuclei , and is mainly localized to the chromosomal ends in vivo . 96 : HMG-5 in C elegans also turned out to be exclusively localized in the nuclei ( see below ) . 146 : HMG-5 is expressed in the nuclei of oocytes and all embryonic cells . 152 : Supporting this , we observed that HMG-5 localizes primarily at the chromosomal ends ( see below ) . 157 : The localization of HMG-5 in the cells was limited to the nuclei . 159 : The timing and localization of HMG-5 expression indicates that it may play roles within the nuclei of oocytes and developing embryos , but not in other organelles such as mitochondria . 161 : HMG-5 is localized to the chromosome ends in vivo 162 : We found that HMG-5 mainly localizes at the chromosomal ends in mature oocyte and embryonic cells ( Fig 4 , and data not shown ) . 163 : This indicates that HMG-5 , despite its low binding specicity , indeed localizes to the telomeres in vivo . 170 : HMG-5 is mainly localized at the chromosomal ends in vivo . 174 : HMG-5 was mainly localized to the chromosomal ends . 175 : However , HMG-5 was also localized at other places on the chromosomes . 177 : GI : A close-up image of the HMG-5 localization in a single chromosome . 178 : HMG-5 is clearly localized to the ends of the chromosomes . PAPERDELIMITER PMID:14605367 103 : SAN-1 was detected in the nucleus during prophase ( 17 ) and localized to the poleward faces of chromosomes in metaphase , overlapping with the kinetochore marker HCP-3 ( 18 ) ( Fig 2 ) . 118 : SAN-1 protein localizes to the poleward faces of the metaphase plate . PAPERDELIMITER PMID:14630941 5 : Ce-DAB-1 contains signals that confer subcellular localization to Golgi-proximal vesicles . 162 : As shown in Figure 5E , Ce-DAB-1 localized to vesicular structures , including AP-1-containing , wheat germ agglutinin-positive structures near the Golgi . 164 : In addition , by indirect immunofluorescence , Ce-DAB-1 is also localized to vesicular structures in the P6 . p descendants of animals expressing a Ce-DAB-1 GFP fusion protein ( Fig 5F ) . 168 : Ce-DAB-1 associates with Ce-LRP-1 and CeLRP-2 and with Golgi components . 220 : Ce-DAB-1 also localizes to Golgiproximal subcellular structures when expressed in it issue culture cells , and to vesicles in P6 . p descendants . PAPERDELIMITER PMID:14529613 39 : The previously characterized C elegans synaptotagmin protein , which is expressed in neurons as well as other secretory cells , colocalizes with SNAP-25 at the presumable synapses in the nervous system ( Figure 2 ( E ) ( G ) ) but , unlike synaptotagmin , SNAP-25 is not present in the vas deferens or somatic gonad cells ( Figure 2 ( C ) and ( D ) ) . 40 : SNAP-25 was not mislocalized in unc-104 mutant animals in which synaptic vesicle transport is impaired , supporting the notion that it is associated with the plasma membrane ( Figure 2 ( H ) and ( I ) ) . 56 : E-G , SNAP-25 and synaptotagmin are colocalized in the wild-type animal. 59 : While synaptotagmin staining is abolished in the dorsal cord ( arrowheads in I ) , SNAP-25 is still normally located on the dorsal cord ( arrowheads in H ) , indicating that SNAP-25 is on the plasma membrane . PAPERDELIMITER PMID:14522875 8 : At the onset of elongation , NMY-1 forms filamentous-like structures similar to actin , and LET-502 is interspersed with these structures , where it may trigger contraction . 9 : MEL-11 , which inhibits elongation , is initially cytoplasmic . 10 : In response to LET-502 activity , MEL-11 becomes sequestered away from the contractile apparatus , to the plasma membrane , when elongation commences . 182 : NMY-1 was also present at the adherens junctions ( AJs ) of the developing pharynx ( data not shown ) . 183 : As elongation proceeded , NMY-1 became more punctate ( Fig 4D ) and organized into filamentous-like structures ( Fig 4F ) . 189 : Embryos of similar stages stained for actin showed a similar pattern , albeit filamentous structures become apparent earlier ( Fig 4A , C , E ; embryos were not co-stained because NMY-1 and actin require incompatible fixation methods ) . 192 : Prior to elongation , LET502 and MEL-11 showed extensive colocalization in the cytoplasm with punctate staining at epidermal cell boundaries ( Fig 5A-C ) . 193 : As elongation proceeded , MEL-11 became enriched and was restricted to cell boundaries while LET-502 remained primarily cytoplasmic ( Fig 5D-F ) . 194 : When elongation was completed , MEL-11 again increased in the cytoplasm , with decreased , punctate localization at cell boundaries , similar to the pre-elongation pattern ( Fig 5J-L ) . 210 : ( D-F ) Twofold embryo is shown at a plane corresponding to the epidermal cells where MEL-11 is restricted to epidermal cell boundaries while LET-502 remains cytoplasmic . 211 : ( G-I ) Twofold embryo at a plane corresponding to pharynx and intestine , where LET-502 and MEL-11 colocalized at adherens junctions . 212 : ( J-L ) Threefold embryo showed cytoplasmic LET-502 , while MEL-11 showed punctate staining at the cell boundaries , with a limited amount reappearing in the cytoplasm ( compare E and K ) . 218 : In elongating embryos , LET-502 ( A ) was interspersed with filamentous NMY-1 ( B ) . 225 : Although only low levels of cytoplasmic anti-MEL-11 staining was present in the lateral epidermal cells of wild type ( A ) , higher cytoplasmic levels were present in let-502 ( ca201 ) ( B ) , which arrests at the onset of elongation . 264 : Similar to MYPT during smooth muscle contraction ( Shin et al , 2002 ) , MEL-11 moves from the cytoplasm to the membrane , away from the contractile apparatus , when contraction begins ( Figs 5 and 6 ) . 265 : After elongation is complete , low levels of cytoplasmic MEL11 reappear . 277 : In contrast to MEL-11 membrane sequesterization during elongation , LET-502 is interspersed with the myosin filaments ( Fig 6 ) . PAPERDELIMITER PMID:12950278 8 : In addition , the DAF-21 protein seemed to be localized in the perinuclear region of somatic cells . 80 : Interestingly , as the oocytes matured , the signal strength of the antisense probe seemed to decrease , while the 608F antigen existed ubiquitously in the cytoplasm of the oogonium and all oocytes ( Fig 7A ) . 82 : In contrast , the 608F antigen was distributed ubiquitously in the cytoplasm of spermatogonium and spermatocytes ( Fig 6C , D ) . 87 : DAF21 was apparently expressed in the somatic cells , particularly localized in the perinuclear region , in addition to being constitutively expressed in germ cells ( Fig 7 ) . 89 : This suggests that DAF-21 in C elegans can be inducible in somatic cells in addition to being constitutive in germline cells , and that synthesized DAF-21 is accumulated in the perinuclear region of the somatic cells . PAPERDELIMITER PMID:12967565 67 : SYP-2 Immunolocalization Supports a Structural Role for SYP-2 in SC Assembly Evidence that SYP-2 is a structural component of the SC comes from immunolocalization studies using -SYP-2 antibodies ( Figures 2D , 2E , and 2G ) . 68 : The timing of SYP-2 localization with chromosomes corresponds well with the timing of assembly and disassembly of the SC . 79 : The first nuclei in which SYP-2 is detected contain a single bright SYP-2 focus ; these are followed by nuclei with several chromosomal foci of SYP-2 , then by nuclei with extended chromosome-associated patches of SYP-2 by mid-transition zone . 80 : ( E ) Pachytene nuclei , exhibiting continuous SYP-2 localization at the interface between aligned homologous chromosomes ; images are projections halfway through data stacks encompassing whole nuclei . 87 : Upon entrance into and throughout the pachytene stage , SYP-2 localizes in a continuous fashion at the interface between paired and aligned homologous chromosomes . 114 : In rec-8RNAi worms , chromosomal localization of SYP-2 was severely reduced but not eliminated ; overall staining was faint and many chromatin stretches lacked detectable SYP-2 . 129 : Only nuclei with extensive chromosomal localization of SYP-1 carried 3 or more RAD-51 foci . 141 : High-magnification images of nuclei from the mid-pachytene region of the germline ( zone 5 from Figure 4 ) , showing RAD-51 foci localized on chromosomes . PAPERDELIMITER PMID:15457263 187 : Double labelling experiments with an antibody against the vesicular acetylcholine transporter UNC-17 ( ref .15 ) demonstrated that 93 3 of LEV-10 puncta were associated with cholinergic varicosities ( mean SEM , 104 puncta counted in seven worms ) . 217 : Because LEV-10 is expressed but fails to accumulate at synapses in the absence of levamisole-sensitive AChRs , we can not exclude the possibility that LEV-10 requires AChRs to reach the plasma membrane , although no intracellular staining of LEV-10 is seen by immunofluorescence in unc-29 and unc-38 mutants . 248 : LEV-10 is a synaptic protein that requires levamisole-sensitive AChRs for proper localization but not for expression . PAPERDELIMITER PMID:15363415 6 : PAR-2 , which localizes to the posterior cortex , inhibits NMY-2 from accumulating at the posterior cortex during flow , thus maintaining asymmetry by preventing inappropriate , posterior-directed flows . 14 : During the first 30 min after fertilization of the C elegans egg , PAR-3 , PAR-6 , and PKC-3 are distributed uniformly throughout the cortex of the embryo . 57 : We also examined fixed wild-type embryos costained for NMY-2 and filamentous actin ( F-actin ) , and found a close spatial correspondence between NMY-2 foci and previously described F-actin foci ( Figure 1G ) ( Strome , 1986 ) . 58 : Neighboring F-actin foci were typically joined by prominent F-actin containing bundles whose positions often corresponded to those of NMY-2 interfoci ( Figures 1G and 1H ) . 81 : Cortical PAR-6 and PAR-3 Form Anterior Caps during the Period of Cortical Flow PAPERDELIMITER PMID:15530389 7 : Here , we show that MEC-2 colocalizes with the degenerin MEC-4 in regular puncta along touch receptor neuron processes . 42 : ( A ) MEC-2 puncta in PLM ( lateral , l ) and PVM ( ventral , v ) processes . 58 : Thus , MEC-2 appears to be recruited to the degenerin channel complex by the other components . 66 : Taken together this correlation , the presence of MEC-2 puncta in animals with mec-2 mutations outside the stomatin-like region , and the presence of MEC-4 puncta in the absence of MEC-2 suggest that association of MEC-2 with the degenerin channel complex occurs through the stomatin-like region . 130 : The distribution of MEC-2 along neuronal processes does not require this region [ 16 ] or the other members of the degenerin complex ; instead the MEC-2-specific N-terminal region is sufficient for the dispersal of MEC-2 [ 16 ] . 138 : UNC-24 Localizes with MEC-2 and Is Needed for Touch Sensitivity ( A ) MEC-2 and UNC-24 : : GFP puncta colocalize in a PLM process . PAPERDELIMITER PMID:15558032 67 : Expression in post-embryonic stages was prominent in the myoepithelial cells of the pharynx ( Fig 1d , e ) and in the intestine ( Fig 1f , g ) , where EPS-8 localized in the brush border , as it was more apical than the intermediate filaments of the terminal web ( Fig 1hj ) . PAPERDELIMITER PMID:15907376 156 : DSH-2 staining in the gonad is punctate , and higher levels of DSH-2 are often evident at the cell cortex ( Fig 3D , F ) . 157 : Localization of Dsh to the cell cortex in some situations depends on Wnt signaling ( Axelrod et al , 1998 ; Rothbacher et al , 2000 ; Yang-Snyder et al , 1996 ) . 194 : In both D and F a higher level of DSH-2 antibody staining is observed at the cell cortex organization ( Boutros and Mlodzik , 1999 ; Wharton , 2003 ) . 243 : DSH-2 is present at the cortex in Z1 and Z4 , where it can potentially respond to signaling ligands . PAPERDELIMITER PMID:15572146 46 : In this paper , we report that DAZ-1 protein is enriched in the cytoplasm of the mitotic and early meiotic germ cells in hermaphrodites , in a manner independent of the GLP-1 signaling pathway . 88 : DAZ-1 is a germline-specific cytoplasmic protein expressed in the mitotic and early meiotic regions of the adult gonad 108 : An observation by confocal microscopy under high magnification revealed that DAZ-1 localized to the cytoplasm of syncytial germ cells and to the cytoplasmic core ( rachis ) ( Fig 1D ) . PAPERDELIMITER PMID:15452142 66 : KLP-19 concentrates around chromosomes and in the spindle 71 : In the gonad , staining was bright in germline nuclei of the distal mitotic zone , dim in early meiotic prophase nuclei , and then bright again in late prophase nucleoplasm . 72 : Just before fertilization , KLP-19 concentrated on prophase chromosomes . 73 : During metaphase of meiosis I , staining concentrated slightly in the body of the spindle , more around the periphery of chromosomes , and most between homologous chromosomes ( Fig 1 D ) . 75 : In embryos , during most of mitotic prophase , KLP-19 concentrated in nucleoplasm ( Fig 2 A ) . 76 : In prometaphase , it concentrated slightly in the body of the spindle and more strongly around the periphery of chromosomes ( Fig 2 , B and C ) . 77 : In anaphase , it left chromosomes and concentrated in the spindle interzone ( Fig 2 , D and E ) . 79 : The concentration of KLP-19 at the edges of condensed chromosomes was similar to that of MCAK ( Fig 3 ) , a protein known to associate with the holocentric C elegans kinetochore as well as with spindle poles ( Oegema et al , 2001 ) . 80 : To determine if KLP-19 chromosome localization depends on kinetochores , the effects of kinetochore disruption were studied . 86 : These results indicate that KLP-19 spindle localization is independent of kinetochores and suggest that its association with chromosomes is via nonkinetochore chromatin . 91 : KLP-19 accumulates in distal and late prophase nuclei . 92 : ( B ) High magnification image of the nucleus of a single immature oocyte , showing KLP-19 in the nucleoplasm . 94 : KLP-19 became concentrated on chromosomes just before fertilization . 122 : ( D ) KLP-19 localization around chromosomes was not prevented by HCP-3 depletion . PAPERDELIMITER PMID:15458647 6 : SPD-1 is a conserved microtubule-bundling protein that localizes to the midzone and also to microtubule bundles in the cytoplasm . 7 : The midzone localization of SPD-1 is perturbed in embryos depleted of other midzone components , yet the cytoplasmic bundles are not affected . 47 : To determine whether these structures corresponded to microtubules , we fixed and stained embryos with GFP and tubulin antibodies and found that SPD-1 : : GFP colocalized to microtubules along a part of their length ( Figures 2M2O and insets 2M 2O ) . 51 : Rabbit polyclonal antibodies raised and purified against SPD-1 also localized to nuclei and the spindle midzone ( Figures 2P2R ; Figure S2 ) . 54 : In both cases , there was no effect on SPD-1 localization to and release from the nucleus or on localization to the spindle prior to the metaphase-to-anaphase transition ( data not shown ) . 75 : Antibodies specific to SPD-1 also localize to the spindle midzone and nuclei ( PR ) . 120 : ZEN-4 Localizes to the Cleavage Furrow in Wild-Type and spd-1 ( RNAi-f ) Embryos but Not in air-2 ( RNAi ) Embryos 146 : Furthermore , in the absence of SPD-1 and hence a midzone , furrow localization of ZEN-4 , and possibly other midzone components , is generally sufficient for successful cytokineses . PAPERDELIMITER PMID:15331665 4 : Using indirect immunofluorescence and spinning-disk confocal microscopy , we found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations , including the cell cortex , the vicinity of microtubule asters , the nuclear periphery and kinetochores . 40 : We found that LIS-1 localizes throughout the cytoplasm and is enriched in discrete subcellular locations , including the cell cortex , the vicinity of microtubule asters , the nuclear periphery and kinetochores . 155 : We found that LIS-1 is present throughout the cytoplasm , but is also enriched in discrete subcellular locations ( Fig 3A-I ) . 157 : In favourable specimens , LIS-1 can also be detected at the nuclear periphery during late prophase ( Fig 3G , arrowhead ; two-cellstage embryo ) as well as in the vicinity of chromosomes during prometaphase ( Fig 3C , H , arrows ) . 158 : LIS-1 is depleted from the centres of microtubule asters during prometaphase ( Fig 3C , arrowheads ) , whereas it is enriched throughout the spindle during metaphase and early anaphase ( Fig 3E , arrowhead ; two-cell-stage embryo ) . 159 : During late anaphase and telophase , LIS-1 staining diminishes on the spindle but becomes enriched in the vicinity of the microtubule asters ( Fig 3D , arrowheads , compare squares 1 and 2 ) . 160 : Moreover , LIS-1 enrichment becomes apparent at the cell cortex at this stage ( Fig 3D , arrow ) . 171 : ( D ) Telophase ; arrow indicates LIS-1 enrichment at the ingressing central cortex , arrowheads regions of LIS-1 enrichment in the vicinity of microtubule asters . 174 : ( E ) Two-cell stage ; note enrichment of LIS-1 on the P1 spindle ( arrowhead ) and the cell cortex between AB and P1 ( arrow ) . 175 : ( F ) Four-cell stage ; arrowheads point to LIS-1 enrichment in ABa and ABp prophase nuclei . 177 : Arrows point to LIS-1 enrichment at the nuclear periphery ( G ) and in the vicinity of chromosomes ( H ) . 213 : Conversely , we found that the kinetochore localization of ICP-1 , HCP-4 , as well as that of BUB-1 , is not affected in the absence of lis-1 or dhc-1 function ( Fig S1 , in supplementary material ) . 214 : Overall , we conclude that LIS-1 is present at kinetochores in an hcp-4-dependent manner . 233 : LIS-1 targeting to the cell cortex , the nuclear periphery and kinetochores is microtubule-independent . 298 : LIS-1 localizes to microtubules , but not strictly to their plus ends 299 : In C elegans , we found that whereas LIS-1 colocalizes with microtubules , it is not significantly enriched at plus ends as compared with more internal parts of the lattice . 314 : We also found that dhc-1 , dnc-1 and dnc-2 are essential for LIS-1 localization at the nuclear periphery . 315 : By contrast , all three genes appear to be largely dispensable for LIS-1 enrichment at the cell cortex , and entirely dispensable for its enrichment at kinetochores . PAPERDELIMITER PMID:15282160 156 : In spermatids , SPE-9 is located at or near the cell surface and has some incidental overlap with 1CB4 staining that marks sperm membranous organelles ( MOs ) in the cytoplasm ( Figs 2A , B , E , and F ) . 157 : After sperm activation ( spermiogenesis ) , SPE-9 appears concentrated on the pseudopod ( Figs 2C , D , G , and H ) while 1CB4 staining remains restricted to the cell body . PAPERDELIMITER PMID:15342467 40 : We also show that ATX-2 is a widely expressed cytoplasmic protein that forms a complex with PAB-1 , a poly ( A ) -binding protein . 92 : ATX-2 is a cytoplasmic protein that forms a complex with poly ( A ) -binding protein 99 : In whole-mount preparations of wild-type hermaphrodites and males , the anti-ATX-2 Abs stained throughout the cytoplasm in many , if not all , cell types , with slightly stronger staining in the gonad ( Fig 1H ; data not shown ) . PAPERDELIMITER PMID:15342507 5 : GPA-2 , GPA-3 , GPA-5 , and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites . 84 : Anti-GPA-2 antibodies stained many cilia , cell bodies , and axons in the head ( Figure 1C ; results not shown ) . 91 : Anti-GPA-13 antibodies stained the cilia of amphid and phasmid neurons ( Figure 1D ; Table 2 ) . 94 : Anti-GPA-5 antibodies were detected not only in the cilia , but also in the cell bodies and axons of the AWA neurons ( Figure 1 , E and F ) . 96 : A punctate staining pattern of the axon with anti-GPA-5 antibodies suggested that GPA-5 might be located at the synaptic sites of the AWA axons . 102 : However , in animals overexpressing GPA-6 , the dendrites , cell bodies , and axons of four pairs of amphid neurons , but not the cilia , showed staining ( Figure 1G ; Table 2 ) . 109 : We found anti-GPA-3 antibody staining in the cilia , cell bodies , and axons of many amphid cells ( Figure 1H ) and the two phasmid cells PHA and PHB ( Table 2 ) . 111 : Application of anti-GPA-3 antibodies to gpa-6 : : GFP animals showed GPA-3 colocalization with GPA-6 : : GFP in the AWA cilia and cell bodies ( results not shown ) . 120 : GPA-2 ( C ) localizes to cilia ( arrow ) and cell bodies ( arrowheads ) of amphid neurons . 121 : GPA-13 ( D ) localizes to amphid cilia ( arrow ) . 122 : GPA-5 ( red ) can be observed in the cilia ( arrow ) , axons ( open arrow ) , and cell bodies ( arrowhead ) of the AWA cells ( E ) and is localized nearby snb-1 : : GFP ( green ) in the axons ( F , open arrows ) . 124 : GPA-3 localizes to the cilia of many amphid cells ( H ) . 125 : GPA-3 staining in the rod-like cilia is clearly visible ( arrow ) , surrounded by the more fuzzy wing-shaped AWA and AWC cilia staining ( arrowheads ) . PAPERDELIMITER PMID:15265690 7 : HRP-2 was predominantly localised to nuclei and analysis of transgenic lines expressing C-terminal deletions of HRP-2 defined a functional nuclear localisation signal . 45 : HRP-2 was found to be predominantly nuclear localised in all it issues of the worm and using various mutant forms of the protein , we investigated the relative contribution of specific domains in determining this pattern of localisation . 137 : HRP-2 is expressed in nuclei of all life cycle stages of C elegans . 139 : ( A ) Uterus from an adult hermaphrodite showing fluorescence in early embryos ; ( B ) embryos at three-fold stage showing large gut cell nuclei ; ( C ) L2 with fluorescence in most it issues particularly bright around the pharynx ; ( D ) diffuse nuclear staining in gut cell from isolated adult intestine ; ( E ) punctate staining in neuronal cell nuclei . 141 : Panel F shows colocalisation of HRP-2 and DAPI staining in tail region of freeze-cracked and fixed L2 stained with DAPI ( top ) , 1 : 1000 R64 Ab ( middle ) and merge ( bottom ) . 160 : In contrast to the clear nuclear localisation obtained with the tagged protein and the antibody staining , GFP and Lac-Z localisation in these lines was variable but predominantly cytoplasmic with few nuclei staining ( Fig 3 ) . 294 : Although by fluorescence , the predominant localisation of HRP-2 is nuclear , immunoprecipitation experiments indicated that a component is present in the cytoplasm . 304 : While deletion of the RGG-rich domain does not appear to affect the predominant nuclear localisation of HRP-2 , arginine methylation could still modify the function of HRP-2 in other respects by influencing RNA or protein interactions . PAPERDELIMITER PMID:12781129 53 : Indirect immunofluorescence revealed that CSN-5 was in the cytoplasm during mitosis and was prominently in the nucleus during interphase of early embryos ( Figures 1Da , 1Dc , and 1De ) . PAPERDELIMITER PMID:12756232 112 : In embryos , VAB-10A and VAB-10B antibodies first detected a signal at the basal and apical plasma membranes of dorsal and ventral epidermal cells , soon after the onset of differentiation ( Fig 4 , A and F ) . 121 : VAB-10A staining strictly colocalized with myotactin in adults ( unpublished data ) , but only partially in young larvae ( Fig 5 , CC ; notice the alternating green and red bands ) . 143 : ( A and F ) Early wild-type comma embryos ; VAB-10A and VAB-10B staining is detected at the basal ( b ) and apical ( a ) membranes as further shown in optical cross sections along the apicobasal axis at the level of arrows ( 3 insets surrounded by a dotted line ) . 158 : ( EE ) Differential interference contrast picture ( E ) of the animal immunostained with VAB-10B K22 pAbs ( E ) ; the merged image ( E ) shows that VAB-10B is found at the furrows separating annuli ( arrowheads ; due to the permeabilization treatments , their morphology is rather poor ) . 162 : In the epidermis , VAB-10A ( but not VAB-10B ) is enriched in FOs ( arrowheads , gold particles ; white arrows , dense bodies ; F and G are from different sections ) . 166 : Because VAB-10A is associated with FOs , which are envisioned as key structures for muscleepidermiscuticle attachment , loss of VAB-10A function would be expected to cause the epidermis to detach from the ECM , whereas loss of VAB-10B function should have different consequences . 284 : The fact that both VAB-10B and actin filament bundles are localized at furrows separating annuli , and the observation that the actin cytoskeleton is perturbed in VAB-10Bdeficient embryos , suggest that VAB-10B could play a key role in the initial events that organize VAB-10 and actin distribution . PAPERDELIMITER PMID:12661765 5 : Dna2 was localized to the nuclei of C elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging . 67 : In the adult C elegans gonad , Dna2 was localized to the nuclei of mitotic and premeiotic germ cells , and at a higher concentration to the nuclei of oocytes ( Fig 2A ) . 68 : Dna2 was also localized in the nuclei during the early to mid embryonic stage but not past the comma stage ( Fig 2B ) . 75 : A The Dna2 protein was localized to the nuclei of germ cells and oocytes ( arrow heads ) by staining with anti-Dna2 antiserum . 77 : B The Dna2 protein was observed in the nuclei of embryos before the comma stage but disappeared upon RNAi of Cedna2 . 116 : In agreement with a role of Dna2 in DNA replication in yeast ( Bae et al , 2001 ; 2002 ; Fiorentino and Crabtree , 1997 ) , the C elegans Dna2 protein was immunolocalized to the nuclei of early embryos before the comma stage , where active cell divisions take place ( Fig 2B ) . PAPERDELIMITER PMID:12837290 6 : At the embryonic stage , CeGLC-7 is associated with the nuclear membrane and chromosomes . 25 : By means of immunohistochemical analysis , we revealed that CeGLC-7 localizes to chromosomes during mitotic progression . 164 : The counterstaining of embryos with PI and anti-CeGLC-7 antibodies revealed that the protein is enriched at the cytoplasm , especially on the nuclear membrane and the nucleus before the nuclear envelope breaks down ( Figs 6A , d and e ) . 165 : To confirm the localization of CeGLC-7 in nucleus , subcellular fractions of embryonic lysates were subjected to SDS PAGE and then analyzed by Western blotting ( Fig 6B ) . 168 : These results indicate that some fractions of the embryonic CeGLC-7 are in nucleus and tightly associated with the nuclear membrane , although the protein is intrinsically soluble and mainly localized at both nucleus and cytoplasm . 169 : We noticed that CeGLC-7 localized to chromosomes in the embryonic cell cycles. 182 : Particularly , the anti-CeGLC-7 antibodies strongly decorated the condensed chromosomes during prometaphase and metaphase ( Figs 6Adf ) . 198 : CeGLC-7 localized to chromosomes and in the cytoplasm prior to the breakdown of the nuclear envelope . 199 : Together with the accompanying disappearance of the nuclear membrane , CeGLC-7 migrates into the nucleoplasm . 214 : Intrinsically , CeGLC-7 localizes to chromosomes and the cytoplasmic CeGLC-7 changes its localization from nuclear membrane to nucleoplasm . PAPERDELIMITER PMID:12815436 40 : C elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase . 88 : In adults , CDT-1 is present in germ cell nuclei and is enriched in oocyte nuclei ( data not shown ) . 89 : In early embryos , CDT-1 is localized to chromosomes in mitotic cells during anaphase and early telophase ( Fig 4b ; data not shown ) . 90 : CDT-1 nuclear staining is not present in S phase , which directly follows mitosis in the early embryo19 ( Fig 4b ) . 103 : f , Anti-CDT-1 levels in V1V6 seam cell nuclei and cytoplasm relative to anti-histone ( a permeabilization control ) in wild-type ( green ) and cul-4 RNAi ( blue ) larvae at the times indicated . 117 : The CDT-1 protein in re-replicating cells is generally nuclear , but can also be present in both the nucleus and cytoplasm ( Fig 4g ) . 132 : However , on detergent extraction , anti-MCM2-7 staining is only observed in telophase nuclei and smaller S-phase nuclei , consistent with MCM2-7 removal from replicated chromatin ( data not shown ) . PAPERDELIMITER PMID:12814550 7 : CLC-1 was mainly expressed in the epithelial cells in the pharyngeal region of digestive tubes and colocalized with AJM-1 at their intercellular junctions . 42 : These findings suggested that CLC-1 is expressed in the epithelial cells of the pharyngeal region of the digestive tubes and is concentrated at the intercellular junctions located at the most apical portion of the lateral membranes . PAPERDELIMITER PMID:12761549 187 : Anti-SQV-5 antibodies stained several punctate foci in the cytoplasm of the vulva , the uterus and oocytes ( Fig 3ac ) . PAPERDELIMITER PMID:12702662 7 : SPN-4 is present developmentally from the oocyte to the early embryo and its distribution overlaps with that of POS-1 in the cytoplasm and P granules of the posterior blastomeres . 217 : Indeed , the granular patterns matched exactly those observed with a P-granule-specific antibody ( Fig 6A-C ) , indicating that SPN-4 is a component of P granules . 240 : Co-localization of SPN-4 and the P granules . 257 : SPN-4 was uniformly present in the cytoplasm up to the two-cell stage , then began to localize to the posterior blastomeres P2 and EMS . 258 : Co-localization of SPN-4 and POS-1 was mainly observed in the cytoplasm of the posterior blastomeres , including the germline ( D , G , J ; yellow regions ) and P granules ( yellow dots , see Fig 6 ) . 365 : We also show that SPN4 is a temporal component of the P granules . PAPERDELIMITER PMID:12707312 52 : At anaphase I , CSC-1 localizes to the central region of the meiotic spindle ( C ) . 53 : During mitotic metaphase , CSC-1 localizes to the chromosomes ( D ) , and in anaphase , it localizes to the central spindle ( E ) . 69 : CSC-1 localizes to meiotic and mitotic chromosomes and to the central spindle during anaphase 70 : In oocytes , during meiosis I , CSC-1 localizes to a discrete region of meiotic bivalents ( Fig 2 , A and B ) . 71 : During anaphase of meiosis I , CSC-1 localizes to the midzone of the meiotic spindle ( Fig 2 C ) . 72 : In mitosis , CSC-1 localizes to chromosomes during metaphase ( Fig 2 D ) and to the spindle midzone during anaphase ( Fig 2 E ) and telophase . 73 : The localization of CSC-1 on the central spindle is significantly broader than the localization of ZEN-4 , the kinesin-like protein that is part of the centralspindlin complex ( Fig 2 F ) . 74 : The reactivity of antiCSC-1 antibodies on chromatin ( Fig 2 G ) and the central spindle is abolished in csc-1 ( RNAi ) embryos . PAPERDELIMITER PMID:12640035 292 : WSP-1 localizes at the periphery of cells 293 : During early embryogenesis , WSP-1 colocalized with actin at the blastomere boundary ( Fig 8Aa-c ) . 308 : During early embryogenesis , ARX-1 and ARX-7 were localized in the cytoplasm of all of the cells ( Fig 7B , E ) . 315 : WSP-1 localizes at the periphery of cells . 356 : WSP-1 localization at cell boundaries suggests that WSP-1 functions in the formation of cell polarity . PAPERDELIMITER PMID:12424233 125 : DPY-7 antibody was localized solely to the annular furrows of the hypodermally derived cuticle ( Fig 2D ) . 166 : D , DPY-7 is exclusively localized in annular furrows ( af ) of the larval cuticle . 233 : The DPY-7 expression pattern was examined using a specific monoclonal antibody raised to this collagen , which localizes specifically to the annular furrows of the dorso-ventral hypodermally derived cuticle of all larval and adult stages . PAPERDELIMITER PMID:12606285 32 : Using monoclonal antibodies , we show that CYE-1 protein is provided maternally and is uniformly localized to the nucleus of all dividing blastomeres in the embryo . 83 : ( C , D ) Wild type zygote with pronuclei at meeting stage stained with anti-CYE-1 ( C ) and DAPI ( D ) . 93 : Note that , in the L1 larvae ( M , N ) , the descendents of the proliferating blast cells P12 to P0 . a blast cells are visible as a row of 26 cells with nuclear CYE-1 on the ventral side . 132 : Nuclear localized CYE-1 is present in the germline , early embryo , and proliferating larval cells 137 : Mitotic germ cells in the distal region of the gonad have easily detectable levels of nuclear CYE-1 . 139 : Finally , as oocytes cellularize in the loop region of the gonad , CYE-1 levels increase with mature oocytes having the highest levels of nuclear CYE-1 . 145 : In the zygote , CYE-1 is observed in the maternal and paternal pronuclei as soon as they form ( Fig 2C and D ) . 146 : The specificity of antibody staining was confirmed by cye-1 RNAi treatment of adult hermaphrodites that abolishes both oocyte nuclei and embryonic anti-CYE-1 protein staining ( Fig 2E and F ; data not shown ) . 147 : In early embryos , CYE-1 is enriched in nuclei , and levels appear constant with no evidence of cell cycle fluctuations other than during mitosis . 156 : For example , in the L1 stage , proliferating P blast cells that produce ventral nerve cells have relatively high levels of nuclear CYE-1 ( Fig 2M and N ) . 159 : Nuclear CYE-1 becomes detectable in the VPCs during the L3 larval stage when they begin proliferation ( data not shown ) . 280 : The maternal CYE-1 is distributed uniformly in all embryonic blastomeres and , at least through the 200 cell stage of embryogenesis , no cell cycle fluctuation in the levels of nuclear CYE-1 is detectable . PAPERDELIMITER PMID:12397108 3 : We show that MEC-8 is a nuclear protein found in hypodermis at most stages of development and not in most late embryonic or larval body-wall muscle . 32 : We show that MEC-8 is a nuclear protein and is expressed primarily in hypodermal cells when mec-8-dependent UNC-52 isoforms begin to accumulate . 70 : ( A , C ) Early embryos with anti-MEC-8 staining in all nuclei . 71 : ( D , E ) Older embryos with anti-MEC-8 staining in hypodermal nuclei . 87 : MEC-8 is present in embryonic hypodermal nuclei 88 : Anti-MEC-8 serum recognized a nuclear antigen in wild-type C elegans embryos ( Fig 1A-C ) . 89 : The youngest embryos to exhibit immunostaining contained about 50 cells , all of which showed nuclear staining . 90 : All nuclei showed staining in embryos containing up to hundreds of nuclei . 93 : During the late proliferative phase of embryogenesis , prior to the onset of morphogenesis , MEC-8 staining was confined largely to hypodermal nuclei ( Fig 1D , E ) . 94 : Prior to this shift , MEC-8 was found in most nuclei , including nuclei that were also marked with an hlh-1 : : lacZ reporter , which is expressed in early blastomeres that subsequently produce only body wall muscle cells ( Krause et al , 1990 ) ; but MEC-8 was not detectable in body muscles after the onset of morphogenesis ( Fig 1E , F ) . 102 : MEC-8 is expressed in many different it issues in larvae In L1-L4 larvae , MEC-8 was detected by anti-MEC-8 serum in the nuclei of the large hypodermal syncytium , hyp7 , that covers most of the worm ( Fig 1J , K ) . 104 : The nuclei of head hypodermal cells not fused with hyp7 ( hyp4 and hyp5 nuclei in particular ) stained well with anti-MEC-8 in all larval stages and in adults . 105 : AntiMEC-8 also stained the nuclei of many neurons in the head ( probably including chemosensory neurons ) ; a few neurons in the central body region [ including the ALM ( Fig 1L ) and AVM touch neurons , and neurons in the post-deirid ] ; vulval nuclei in L4 and adult stage hermaphrodites ( Fig 1L ) ; anterior and posterior-most intestinal nuclei ; and other unidentified nuclei in the head and tail. 123 : The anterior-most muscle nuclei in the heads of larvae had low but detectable levels of MEC-8 , but none of the muscle cells in the main body appeared to stain with anti-MEC-8 . PAPERDELIMITER PMID:12403719 132 : When expressed in human 293T cells , EFN-4 is localized to the cell surface , as detected with anti-EFN-4 antibodies ( data not shown ) . 155 : In later embryos , larvae and adults , EFN-4-GFP was expressed in several head neurons , pharyngeal cells and a small number of lateral and tail neurons , within which EFN-4 was localized throughout the neuronal cell body and in axonal processes ( Fig 3M-O ) . PAPERDELIMITER PMID:12391314 4 : SQV-1 localizes to punctate cytoplasmic compartments and colocalizes with the SQV-7 nucleotide-sugar transporter , which probably acts in the Golgi apparatus . 142 : SQV-1 Ab Stains Punctate Foci in the Cytoplasm . 144 : These Abs were found to stain punctate foci in the cytoplasm of many cells in WT worms ( Figs 3 A and B and 4A , data not shown ) . 149 : SQV-7 Ab Stains Punctate Foci in the Cytoplasm . 150 : We suspected that the punctate cytoplasmic staining by anti-SQV-1 Abs was caused by the localization of SQV-1 to a specific subcellular compartment , such as the Golgi apparatus . 153 : The anti-SQV-7 Abs stained punctate foci in the cytoplasm of several it issues in WT animals , including the vulva , seam cells , distal tip cells , and oocytes ( Figs 3 CE and 4B , data not shown ). 154 : SQV-1 and SQV-7 localize to punctate cytoplasmic sites . 161 : SQV-7 Abs localize to punctate cytoplasmic foci . 181 : We speculate that significant amounts of SQV-1 and SQV-7 protein are present in the Golgi apparatus of several cells , including oocytes , and that lower concentrations are present in most if not all other cells . 227 : We found that SQV-1 protein is localized in punctate foci , presumably in a subcellular compartment within the cytoplasm . 228 : The SQV-7 transporter appears to localize to the same subcellular compartment . PAPERDELIMITER PMID:12379863 1 : SYD-1 , a presynaptic protein with PDZ , C2 and rhoGAP-like domains , specifies axon identity in C elegans 4 : The SYD-1 protein contains PDZ , C2 and rhoGTPase activating protein ( GAP ) -like domains , and is localized to presynaptic terminals in mature neurons . 27 : The SYD-1 protein has PDZ , C2 and rhoGAP-like domains , and is localized to presynaptic terminals . 171 : SYD-1 is localized in presynaptic terminals 200 : To investigate how the presynaptic localization of SYD-1 might be mediated , we constructed several SYD-1 : GFP fusion constructs and expressed them in wild-type animals . PAPERDELIMITER PMID:12204276 10 : IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes . 36 : Double staining with C2 and desmosomal MH27 antibodies shows that C2 is located in the cytoplasm of intestinal cells and at desmosomes in intestine and pharynx of the larva ( Fig 4A , D ) , late embryo and adults ( data not shown ) . PAPERDELIMITER PMID:12498686 28 : At prometaphase , highly condensed chromosomes were seen , and FZY-1 was localized around condensed chromosomes ( Figures 1A and 1C ) . 29 : At metaphase , FZY-1 localized along chromosomes aligned at the metaphase plate ( Figures 1E and 1G ) . 30 : After the separation of sister chromatids , the FZY-1 signal disappeared from the chromosomes , and at anaphase and interphase , cytoplasmic staining was observed ( Figures 1O and 1Q ) . 31 : FZY-1 was observed around the meiotic chromosomes in cells at metaphase of meiosis I ( Figures 1H , 1J , 1L , and 1N ) , but no signal was detected in cells at anaphase of meiosis I or meiosis II ( our unpublished data ) . PAPERDELIMITER PMID:12213836 70 : CeCDC-14 is a phosphatase and localizes to the central spindle and the midbody 91 : However , in anaphase , striking staining of the central spindle area was observed in anaphase ( Fig 1 B , top , arrowhead ) . 92 : Later in mitosis , during telophase , this staining compacted to a single dot that was positioned between the two daughter cells , highly reminiscent of the midbody ( Fig 1 B , bottom , arrowhead ) . 93 : Older embryos displayed multiple dots in the cytoplasm or between cells , again consistent with midbody staining ( not depicted ; see Fig 7 , bottom , left ) . 95 : CeCDC-14 localizes to the central spindle in anaphase and the midbody in telophase . 108 : As illustrated in Fig 1 C , the two antigens displayed extensive colocalization at both the central spindle and the polar body ( arrow and arrowhead , respectively ) . 125 : The localization of CeCDC-14 to the central spindle and the midbody suggested an involvement of this protein in cytokinesis . 234 : In wild-type embryos at later stages , strong midbody remnant staining was observed with the antiCeCDC-14 antibody , but the extent of antiphospho-histone H3 staining differed between individual cells , depending on which cell cycle state they were in at the time of fixation ( Fig 7 , left , bottom row ) . PAPERDELIMITER PMID:12296824 7 : Although MOE-2 protein is rapidly removed from germ granules after fertilization , we found that MOE-2 associates with the centrosomeperipheral structure in dividing blastomeres . 86 : Immunocytochemical staining using antiMOE-1 and anti-MOE-2-specific antibodies revealed cytoplasmic expression in oocytes in the proximal region of the gonads ( Fig 6A , C ) . 87 : The oocyte-specific expression indicates that the reduction in the number of laid eggs by moe-1moe-2 double RNAi is caused by a defect in the oocyte itself and that the cytoplasmic accumulation of MOE-1 and MOE-2 proteins in maturing oocytes is required for proper oocyte maturation . 98 : MOE-1 and MOE-2 are novel components of P granules and regulate P granules distribution in growing oocytes. Double-staining experiments using anti-MOE1 and anti-P granules ( Strome &Wood 1983 ) antibodies revealed that MOE-1 was also localized on P granules in growing oocytes ( Fig 6Ba , b , c ) , as well as in the cytoplasm . 100 : Similar results were obtained using an antiMOE-2 antibody ( Fig 6Bd , e , f ) . 101 : The MOE-1 and MOE-2 signals on P granules were eliminated by absorption using their corresponding antigens , indicating that there was no cross-reaction of the secondary antibodies ( Fig 6C ) . 105 : Interestingly , MOE-1 and MOE-2 were found to prominently associate with P granules in oocytes in the middle to proximal region of the gonad , although P granules themselves are present from the most primordial stage of female germ cell formation ( Fig 6C ) . 106 : Because MOE-1 and MOE-2 proteins have been shown to be associated with P granules in the developing oocytes , we next investigated the effect of MOE protein removal on the distribution of P granules . 112 : As mentioned previously , MOE-2 protein associates with P granules in maturing oocytes . 122 : MOE-1 and MOE-2 are components of P granules in developing oocytes . 139 : MOE-2 signals on P granules were observed just after fertilization ( Fig 8Ba , b , c ) , but the intensity of the signals decreased to an undetectable level in cleavingstage embryos ( Fig 8Bd , e , f , and our unpublished observations ) . There were essentially no MOE-2 signals on P granules in embryos after the two-cell stage ( Fig 8Aa , b , arrows ) . 144 : The egg just after fertilization ( a , b , c ) shows pronuclei ( c ) , and MOE-2 signals were co-localized with P granules ( a , b ) , while the fertilized egg at the mitotic stage ( d , e , f ) did not show a MOE-2 signal on P granules ( d , e ) . 147 : These observations suggest that the association of MOE-2 with P granules is a transient event , only detectable from diakinesis-stage oocytes to fertilized eggs . 158 : MOE-2 protein associates with the centrosomeperipheral structure in dividing blastomeres Although MOE-2 protein is removed from P granules after fertilization , we found that MOE-2 closely associates with the centrosome-peripheral structure after the twocell stage ( Fig 10Aa , d ) . 172 : MOE-2 protein is localized around the peripheral structure of the centrosome in dividing blastomeres . 194 : MOE-2 protein is a component of P granules that disappears after fertilization . We found that MOE-1 and MOE-2 are associated with P granules in developing oocytes ( Fig 6B ) . 197 : MOE-2 continued to associates with P granules from diakinesis-stage oocytes to pronuclear-stage fertilized eggs ( Fig 8B ) , its expression level thereafter decreased to an undetectable level ( Fig 8B and our unpublished observations ) . 198 : These observations suggest that the association of MOE-2 with P granules is a transient event , and that MOE-2 might have a specialized function for P granules , such as control of proper assembly andor distribution of P granules during the final maturation stage of oocytes . 201 : Although P granule-associated MOE-2 protein is removed after fertilization , we found that MOE-2 closely associates with the centrosome-peripheral structure ( Fig 10Aa , d ) . 205 : MOE-2 protein was found to be accumulated in the cytoplasm of proximal oocytes and was removed after fertilization by the proteasome-mediated degradation pathway , suggesting that it participates in processes in the final step of the meiotic cell cycle , a novel function for CCCH-type zinc-finger family proteins thus far discovered . 206 : Furthermore , MOE-2 protein was found to be associated with P granules in maturing oocytes and with mitotic spindles in dividing embryos . PAPERDELIMITER PMID:12169634 61 : LIP-1 is associated with the plasma membrane of pachytene germ cells 72 : When a lipophilic dye that outlines the germ cell plasma membrane was included in the staining reactions , it became apparent that LIP-1 was localized near or at the junctions where the plasma membranes that separate the different germ cells are connected to each other ( Figure 2E ) . 85 : ( E ) Co-staining of LIP-1 with the hydrophobic dye Dil that labels the plasma membrane surrounding the pachytene germ cells . 198 : In contrast to its uniform subcellular localization in somatic cells ( Berset et al , 2001 ) , LIP-1 is localized at or near the junctions between the plasma membranes that partially enclose the individual germ cells in the distal gonadal syncytium ( Hirsh et al , 1976 ) . PAPERDELIMITER PMID:12239571 90 : By immunocytochemistry , GLD-2 was found to be predominantly cytoplasmic in both germ line ( Fig 3b ) and early embryo ( Fig 3c ) . 93 : In early embryos , GLD-2 was diffuse in the cytoplasm of early P0 embryos , colocalized with P granules in late P0 embryos and remained associated with P granules in germline blastomeres ( Fig 3c , not shown ) . 101 : We conclude that gld-2 activity is required for embryogenesis , and that GLD-2 protein co-localizes with P granules . 127 : Given its sequence similarity to nucleotidyltransferases and its cytoplasmic location , we considered that GLD-2 might be a cytoplasmic PAP , even though its architecture and sequence diverged substantially from classical PAPs . 159 : b , GLD-2 protein is in germline cytoplasm . 162 : c , GLD-2 protein is associated with P granules in early 314 embryos . 164 : Top , late P0 embryo , GLD-2 co-localizes with P granules ; second panel down , 28-cell embryo , P4 , white arrowhead ; third panel , , 100-cell embryo , germline precursor cells , Z2 and Z3 , arrows ; bottom , magnified view of P2 blastomere to show PGL-1 and GLD-2 co-localization ( arrows ) . PAPERDELIMITER PMID:12679112 1 : The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development 2 : Innexins are the proposed structural components of gap junctions in invertebrates . 7 : Through immuno-EM techniques , INX-3 was observed at gap junctions in the adult pharynx , providing supporting evidence that innexins are components of gap junctions . 48 : We document by immuno-EM that INX-3 is localized to gap junctions , providing new evidence that innexins are integral components of gap junctions . 116 : Because INX-3 is localized principally in puncta at plasma membranes , these experiments do not allow us to assign expression unambiguously to individual cells ; however , expression in major cell types or organs is clear . 123 : In early L1 animals , INX-3 accumulates at all hypodermal cell boundaries except along the ventral midline between the paired P1P12 cells ( arrow in C indicates P9P10 interface ) . 128 : Hypodermal expression is strong at the time of hatching , and INX-3 is present in plaques at the intercellular boundaries between most hypodermal cells except at the ventral midline between paired P cells ( Fig 2C and D ) ; however , INX-3 becomes undetectable in the hypodermis shortly after hatching . 132 : Subcellular distribution of INX-3 in both these cases is somewhat unusual , since INX-3 is readily detectable in the cytoplasm of these cells , as well as in cell-surface plaques . 145 : INX-3 is localized to gap junctions 146 : INX-3 , as visualized by light microscopy , is concentrated into plaque-like structures at intercellular boundaries , as expected for gap junctions . 150 : Using antiINX-3 as the primary antibody and a gold-labeled secondary antibody , we were able to detect labeling with gold particles of large gap junctions in the pharynxes of adult animals ( Fig 4B ) . 168 : Immuno-EM localization of INX-3 to gap junctions in the pharynx . 266 : Through immunoelectronmicroscopy we have now shown that anti-INX-3 antibodies bind to large gap junctions in the pharynx of C elegans , providing ultrastructural data that reinforce this conclusion . 282 : INX-3 is initially detected on the plasma membrane at the two-cell stage of embryogenesis , at approximately the time that Bossinger and Schierenberg ( 1992 ) first detected dye coupling between C elegans blastomeres , and continues ubiquitously until the beginning of the morphogenetic phase of development , when embryonic cellular proliferation is almost complete . PAPERDELIMITER PMID:12088146 10 : Like the MOG proteins , MEP-1 is localized to the nucleus and is expressed in both somatic and germline it issues 16 : By immunolocalization , MEP-1 was found in all nuclei in the germline , including oocytes , but not those of mature sperm and spermatocytes ( Fig 4F , G ) 21 : As shown in Figure 7 , FBF-1 is normally expressed and localized in the cytoplasm around the nuclei of both wild-type and mep-1 L4 larvae ( Fig 7A , C ) 24 : Third , similar to the MOG proteins , MEP-1 is a nuclear protein that is broadly expressed in both somatic and germline cells 28 : A : In wild-type worms , FBF-1 is detected in the cytoplasm that surrounds the dark nuclei of all immature germ cells PAPERDELIMITER PMID:12036960 152 : B , in the elongated embryos the hypodermal location is maintained for the P4H subunits PHY-1 , PHY-2 and PDI-2 , but DPY-7 was incorporated into the developing cuticle . 211 : At this stage , the hypodermal ER location was maintained for all three P4H subunits ( Fig 4B ) , whereas the cuticle collagen DPY-7 has already been secreted from the ER and has been fully incorporated into the developing cuticle2 ( Fig 4B ) . 254 : All three polypeptides are coexpressed in the hypodermal cell lineage , as evidenced by the complete overlay with the DPY-7 cuticle collagen expression pattern . PAPERDELIMITER PMID:12091304 327 : We found that REF-2 localized to the nucleus in cells in which it was present , consistent with the idea that it functions as a transcription factor . 359 : We also detected REF-2 protein in the nuclei of the B and Y cells in the tail region during L1 . 427 : However , it is possible that upon cell fusion , REF-2 protein can diffuse into the much larger syncytium , causing an apparent decrease in the concentration of REF-2 in the nuclei of the Pn . p cells . PAPERDELIMITER PMID:12082149 3 : Unlike normal linker histones , H1 . X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes . 4 : H1 . X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein , the tonofilaments . 5 : Additionally H1 . X : : GFP is expressed in the cytoplasm of body and vulva muscle cells , neurons , excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells . 35 : H1 . X is the first linker histone-like protein with a prominent cytoplasmic localization and function . 151 : H1 . X is tightly associated with the tonofilaments in the marginal cells 203 : H1 . X is a nucleolar protein 211 : Antibody labeling with H1 . X-101 revealed a prominent labeling of the nucleoli in the polyploid gut nuclei ( Fig 6B ) of adult C elegans hermaphrodites , which colocalized with fibrillarin ( Fig 6D ) , a structural component of small nucleolar RNPs ( snoRNPs ) implicated in pre-rRNA processing . 212 : A prominent co-immunolabeling was further detected in the nucleoli of the germ nuclei and many other cell types and it issues in C elegans . 214 : Nucleolar localization of H1 . X ( A ) shows a Nomarski micrograph of the nuclear region of a polyploid gut cell in a hermaphrodite C elegans with the nucleolus and its substructures visualized by differntial interference contrast ( B ) H1 . X detected with the antibody H1 . X-101 localizes to the nucleolus ; 296 : H1 . X is a prominent cytoplasmic protein in a limited number of cell types ; these are the marginal cells , a set of nine epithelial cells in the pharynx ( three of which form a syncytium ) , the body-wall muscle cells and the vulva muscles in the hermaphrodite . 298 : H1 . X is tightly associated with these tonofilaments . 300 : Prominent and uniform cytoplasmic expression of H1 . X was detected by indirect immunofluorescence in some head neurons . 345 : In many cells of C elegans H1 . X is associated with the nucleolus , where it co-localizes with fibrillarin , but not with rDNA . PAPERDELIMITER PMID:12810585 3 : CEH-2 protein is restricted to the nuclei of one type of small muscle cell , one type of epithelial cell , and three types of neurons in the anterior pharynx in the head . 110 : Antibody staining is nuclear as expected for a transcription factor ( Fig 2 ) . 111 : We find ceh-2 expression restricted to eleven cells ( fourteen nuclei ) of five types in the anterior pharynx ( corpus ) of larvae and adults : the I3 neuron that lies embedded in the dorsal sector of the pharynx muscle ; the pairs of NSM and M3 motoneurons in the left and right subventral sectors ; the three m2 muscle cells , each possessing two nuclei resulting from cell fusion during development ; and the three e2 epithelial cells with the anterior-most pharynx nuclei ( Fig 2A ) . 123 : ( A , C ) An antibody against a CEH-2 peptide stains 14 nuclei in the anterior pharynx of larvae ( A ) and five or six cells in gastrulating embryos ( C ) . PAPERDELIMITER PMID:12819787 2 : We have analysed VAB-9 , a cell junction protein in Caenorhabditis elegans . 4 : Here , we show that VAB-9 requires HMR1cadherin for localization to the cell membrane , and both HMP-1-catenin and HMP-2-catenin for maintaining its distribution at the cell junction . 16 : In this study , we provide an initial characterization of the cell junction protein VAB-9 . 83 : ( c , d ) VAB-9 is localized to epithelial cell junctions in seam cells , as shown in c , and pharynx ( ph ) and intestine ( it ) , as shown in d . 104 : indicate that HMR-1 is required for localization of VAB-9 to cell junctions and that HMP-1 and HMP-2 are required to maintain the distribution of both VAB-9 and HMR-1 around the periphery of the adherens junction . PAPERDELIMITER PMID:12810918 91 : Staining for each of the antibodies is predominantly nuclear . PAPERDELIMITER PMID:12672828 102 : CED-9 and Cyt-1 Protein Levels Are Abnormal in mev-1 Mitochondria 105 : In contrast , hyperoxia induced CED-9 protein levels in wild-type mitochondria , but caused a statistically insignificant reduction in mev-1 mitochondria . 127 : Protein levels of Cyt-1 and CED-9 in mitochondrial preparations from wild-type and mev-1 young adults . 184 : Fourth , we have demonstrated that mitochondrially localized CED-9 protein levels are altered in a mev-1 genetic background ( Fig 5 ) . 191 : The lower amount of mitochondrially localized CED-9 in mev-1 may in turn be insufficient to protect against the oxidative damage induced by hyperoxia . PAPERDELIMITER PMID:11884032 87 : Cytoplasmic extracts of staged populations were prepared as described above for sucrose gradients and were normalized by protein concentration . PAPERDELIMITER PMID:11707322 173 : Significantly , the antibody staining was associated with the nuclei of embryos . 213 : The ability of Ce-MIF to translocate into the nucleus of developing embryos ( Fig 6 ) suggests that MIF may play a role in gene regulation leading to cellular differentiation and proliferation. PAPERDELIMITER PMID:11714689 7 : OOC-5 protein co-localizes with a marker of the endoplasmic reticulum in all blastomeres of the early C elegans embryo , in a pattern indistinguishable from that of OOC-3 protein . 48 : OOC-5 co-localizes with a marker for the endoplasmic reticulum in all cells of the early embryo , and ooc-3 is required for this localization . 149 : OOC-5 co-localizes with an ER marker in early blastomere 163 : During metaphase in one and two-cell embryos , OOC-5 remained perinuclear and was also associated with the poles of the mitotic spindle ( Fig 3J-L , right cell ; Fig 4D ) . 167 : In addition , OOC-5 was enriched at the regions of cell-cell contact in some two-cell embryos ( 40 , n41 ) and virtually all four-cell embryos examined ( 96 , n24 ; Fig 3M , Fig 4J ) . 175 : These data suggest that OOC-5 protein is localized to the ER or secretory pathway in C elegans embryos . 181 : These results suggest that OOC-5 and OOC-3 localize to the same subcellular structures . 229 : Our immunolocalization results show that OOC-5 localizes to all blastomeres of the early embryo in the same pattern as that seen for ER proteins in other systems . 235 : Our results are thus consistent with localization of OOC-5 to the secretory pathway , in particular the endoplasmic reticulum . 239 : Taken together , these results strongly suggest that OOC-5 is an ER protein , which implies a molecular link between secretion and the reestablishment of polarized domains in the C elegans embryo . PAPERDELIMITER PMID:11729150 11 : In the adult germline , SET-2L protein is localized in mitotic and mid-late-stage meiotic nuclei but is undetectable in early pachytene nuclei . 12 : SET-2L protein is localized in all nuclei of embryos . 52 : The larger protein product , SET2L , is distributed similarly to the MES proteins in the nuclei of germ cells and embryos . 201 : SET-2L protein is localized in the nuclei of embryos and germ cells : To determine the distribution of SET-2L protein , antibodies were raised against the N-terminal peptide of SET-2L . 203 : SET-2L protein is localized in the nuclei of all cells in embryos at all stages of embryogenesis ( Figure 6 ) . 204 : In L1 larvae , SET-2L remains visible in the nuclei of most cell types but is most prominent in Z2 and Z3 , the primordial germ cells . 205 : In adults , SET-2L staining is nuclear and is strongest in the germline ( Figure 7 ) , although it is also detectable in other cell types , such as intestinal , pharyngeal , and neuronal cells ( data not shown ) . 221 : During all stages of embryogenesis , SET-2L is localized in the nuclei of all cells . PAPERDELIMITER PMID:11738032 134 : SLO-1 Functions Presynaptically to Modulate Neurotransmission 151 : revealed immunoreactivity in synaptic regions of the nervous system including in both the nerve ring ( Figure 4A ) and nerve cords , as well as in the body-wall ( Figure 4C ) and vulval muscle . 158 : The presence of SLO-1 in the nerve ring where the density of synapses is high , and its presence in motor neurons is consistent with a rolein consrolling synapic release at both interneuronal synapses and neuromuscular junctions (NMJs). PAPERDELIMITER PMID:11861469 86 : VAB-7 is expressed in nine neuronal nuclei ( seven in the VNC and two nuclei in the head ) , and in the five most posterior epidermal nuclei , which form the hyp8 to hyp11 cells ( Fig 1C-E ) . PAPERDELIMITER PMID:11923211 194 : We detected PAG-3 during embryonic development in many nuclei 280 minutes after fertilization ( data not shown ) . PAPERDELIMITER PMID:12015966 6 : EVL20 is closely associated with both the cell cortex and astral microtubules , suggesting that it may directly interact with microtubule structures at those locations . 7 : Our data indicate that EVL-20 functions in the cytoplasm and at the plasma membrane to regulate cytoskeletal dynamics during cytokinesis and morphogenesis . 224 : ( A and B ) EVL-20 localizes to the cortical plasma membrane of embryonic blastomeres . 225 : ( C ) EVL-20 is associated with astral microtubules . 232 : As shown in Figure 7A , B EVL-20 localizes to the cortical plasma membrane of embryonic blastomeres . 233 : While some diffuse cytoplasmic staining is detectable , the staining intensity is highly concentrated next to centrosomes and is excluded from the mitotic spindle area , suggesting that EVL-20 is associated with astral microtubules during cell divisions ( Figure 7C ) . PAPERDELIMITER PMID:11914278 7 : SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles . 92 : SMC-4 and MIX-1 localize to the poleward face of chromosomes at times of the cell cycle when chromosomes are condensed SMC-4 antibody staining showed that SMC-4 localized to mitotic chromosomes in a dynamic pattern with two striking features . 93 : First , SMC-4 localized to chromosomes only at times of the cell cycle when they were condensed . 95 : SMC-4 localized to chromosomes at prometaphase ( Fig 2B , G ) , when the six C elegans chromosomes , but not their sister chromatids , become visibly distinct . 97 : Second , SMC-4 associated only with certain regions of the chromosomes . 98 : SMC-4 appeared in a faint speckled pattern during prophase , then appeared in two stripes outlining each chromosome during prometaphase ( Fig 2G ) . 101 : SMC-4 colocalizes with MIX-1 at the poleward face of condensed mitotic chromosomes but not on X chromosomes . 106 : MIX-1 and SMC-4 colocalize in nuclei with condensed chromosomes ( yellow when merged , N ) . 108 : ( OQ ) Metaphase chromosomes costained with DNA dye ( blue ) , MIX-1 antibody ( red , O ) , and SMC-4 antibody ( green , P ) show colocalization of MIX-1 and SMC-4 at the poleward face of condensed chromosomes ( yellow when merged , Q ) . 116 : For example , in a wild-type embryo that had not yet implemented dosage compensation , SMC-4 and MIX-1 were both absent from decondensed chromosomes and coincident on condensed chromosomes ( Fig 2KN ) . 117 : During metaphase , MIX-1 and SMC-4 decorated the chromosomes in the same distinctive poleward pattern ( Fig 2O Q ) . 149 : These defects in mitotic chromosome structure and segregation indicate a critical role for SMC-4 in mitosis and support the view that SMC-4 is part of a mitotic condensin complex in C elegans . 178 : Consistent with this finding , both SMC-4 and MIX-1 localize to condensed mitotic chromosomes in the gonad ( data not shown ) . 190 : Unlike condensins of other organisms , which associate with a central chromosome axis ( Hirano and Mitchison 1994 ; Schmiesing et al 1998 ; Steen et al 2000 ; Steffensen et al 2001 ) , SMC-4 and MIX-1 outline condensing prophase chromosomes along their length , then localize to the poleward faces of the metaphase plate . 224 : In immunofluorescence experiments , both SMC-4 and MIX-1 localized to meiotic chromosomes in metaphase of meiosis I and of meiosis II ( Fig 7AD ) . 239 : In costaining experiments with HCP-3 and SMC-4 antibodies , both proteins were coincident at metaphase of meiosis I ( Fig 7AD ) and meiosis II ( data not shown ) , consistent with localization of SMC-4 to the meiotic centromere . 249 : SMC-4 and MIX-1 co-localize with centromere proteins on meiotic chromosomes , and their depletion impairs chromosome segregation in meiosis II but not meiosis I PAPERDELIMITER PMID:11901171 153 : At the comma stage ( 290 350 min ) , both UNC-60B and CeTM are diffusely localized in the cytoplasm and positions of nuclei are devoid of staining ( Fig 5 , ad ; DAPI staining of the nuclei is not shown ) . 154 : After the 1 . 5-fold stage ( 430 min ) , CeTM was gradually localized to a narrow region ( Fig 5 f ) which represented myofibrils as determined by double staining with anti-actin antibody, wheras UNC-60B remained in the diffuse cytoplasm. 170 : show that CeTM is associated with the outer parts of the I-bands , whereas UNC-60B is localized near the barbed ends of the actin filaments . 213 : In CeTM-suppressed cells , UNC-60B was unevenly distributed in the cells and associated with bundles where actin was also localized ( Fig 9 , B , D , and F ) , whereas , in the control cells , UNC-60B was evenly distributed and associated with a part of myofibrils ( Fig 9 , A , C , and E ) . PAPERDELIMITER PMID:11983919 62 : In KAL-1-overexpressing animals , the KAL-1 protein remains associated with the surface of the overexpressing cell ( see Fig 6A ) that is consistent with the reported cell surface-binding activity of vertebrate KAL-1 protein ( 9 , 14 ) and PAPERDELIMITER PMID:11896189 227 : Confocal microscopy of worms stained with FEH-1 antibody and Texas-Red-conjugated phalloidin , which decorates actin-rich muscle cells , indicates , instead , that FEH1 , at least in part , colocalizes with actin in muscle cells in some areas of the two pharyngeal bulbs and in some areas just anterior to the second bulb ( Fig 5C ) . PAPERDELIMITER PMID:12077420 82 : On the basis of immunofluorescence staining ( 10 ) , MES-4 is localized to nuclei and associated with chromosomes . 84 : In embryos , MES-4 is present in both somatic and germline nuclei until the 80 to 100-cell stage ( Fig 1 , B to D ) . 108 : MES-4 associates with autosomes and complex extrachromosomal arrays , but not with X chromosomes and repetitive arrays . PAPERDELIMITER PMID:12062106 150 : Confocal microscopic analyses revealed that the HEN-1 protein was localized in the cell bodies of AIY and ASE and appeared in a punctate pattern in the axons in the nerve ring ( Figure 5C ) , where these neurons form synapses , but was not in the dendrite of ASE . 233 : The localization pattern of the HEN-1 protein was punctate in axons and was dependent on the motor protein KIF1A homolog for synaptic vesicular transport . PAPERDELIMITER PMID:10846178 3 : Immunofluorescence analysis indicated that VHA-11 , the C subunit of Caenorhabditis elegans V-ATPase , was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage . 30 : We observed that VHA-11 , the C subunit of V-ATPase , was predominantly localized in the intracellular organelles of intestinal cells during late embryogenesis and that the generation of acidic compartments in the intestine was dependent on vha-11 expression . 57 : In C and D , VHA-11 was localized in dot-like structures around the nuclei and also detected in the cytoplasm . 91 : In embryonic stages , VHA-11 was detected as dot-like intracellular compartments around the nuclei ( Fig 1C , arrowheads ) . 92 : Diffuse staining in the cytoplasm could be seen at all embryonic stages . 95 : indicate that V-ATPase is localized in intracellular compartments 194 : It may be associated with intracellular organelles such as lysosomes and endosomes , similar to in other eukaryotic cells ( 2 ) . 195 : In comma stage embryos , V-ATPase was highly expressed in intestinal cells being localized in their intracellular compartments. PAPERDELIMITER PMID:10804188 4 : During the GLP-1-mediated cell interactions in the C elegans embryo , APH-2 is associated with the cell surfaces of both the signaling , and the responding , blastomeres . 30 : We show that a novel cell-surface protein , APH-2 , is required for both the 4-cell and 12-cell interactions , and that mutations in the aph-2 gene result in phenotypes that are indistinguishable from those of glp-1 mutant embryos . 262 : APH-2 protein localizes to the cell membranes of embryos 266 : APH-2 is associated with the plasma membranes that partially surround the developing oocyte nuclei and with the surface membranes of mature oocytes ( Fig 7A ) . 267 : In newly fertilized eggs and in early cleavage stage embryos , APH-2 is associated with the peripheral membranes of all blastomeres ( Fig 7B-D ; data not shown ) . 268 : The APH-2 protein can be detected on , or near , plasma membranes throughout the first 5-6 hours of embryogenesis . 278 : APH-2 is localized to the cell surface , and our chimera experiments suggest that it might act at , or through , the cell surface . 291 : By immunostaining , APH-2 appears to be associated with surface membranes , and the carboxyl terminus of APH-2 contains a potential transmembrane domain or a glycosyl phosphatidylinositol ( GPI ) linkage site . 332 : The association of APH-2 with plasma membranes in the embryo suggests that APH-2 may facilitate receptor-ligand interaction . PAPERDELIMITER PMID:11029047 4 : The most abundant protein form is localized to most or all synapses . 41 : The most abundant form localizes to most or all synapses . 262 : UNC-13 Protein Localization Antibodies produced against the UNC-13L region labeled most or all neurons ( Figure 8 ) as well as nuclei in the gut and gonad ( our unpublished results ) . 266 : Antibody staining in the nervous system is punctate and appears to be synaptic , because in cholinergic neurons it colocalized with antibodies to the synaptic vesicle protein UNC-17VAChT ( Figure 8 ) . 271 : We find that UNC-13L staining remains at synapses in unc-104 mutants ( Figure 8 ) , suggesting that it is not transported to synapses by the UNC-104 protein , and therefore is unlikely to be on synaptic vesicles . 279 : Anti-UNC-13L antibody stains a form of UNC-13 protein that is synaptic but is unlikely to be on synaptic vesicles . PAPERDELIMITER PMID:11071916 168 : ( C and D ) A twofold stage embryo shows NID-1 accumulation on the basal face of body wall muscles ( arrowhead ) and on the surfaces of the developing pharynx ( p ) , intestine ( i ) , and gonad ( g ) . 180 : Once the embryo has elongated to the twofold stage , NID-1 has localized to the basal face of the body wall muscles and shows strong accumulation on the surfaces of the pharyngeal , intestinal , and gonad primordia ( Figure 4 , C and D ) . 187 : NID-1 also accumulates strongly at the outer edges of the muscle quadrants and more weakly at the boundaries between muscle cells within each quadrant . PAPERDELIMITER PMID:10983970 57 : BIR-1 Localizes to Chromosomes and to the Spindle Midzone during Mitosis and Meiosis To characterize the subcellular localization of BIR-1 in dividing cells , we focused on the first embryonic division , in which mitotic structures are largest ( Figure 2A ) . 58 : BIR-1 immunoreactivity was first detected on prometaphase chromosomes ( Figure 2A , PROPHASE ) and persisted on these chromosomes at the metaphase plate ( Figure 2A , METAPHASE ) . 60 : During anaphase , BIR-1 was present both on chromosomes and the spindle midzone ( Figure 2A , ANAPHASE ) . 62 : BIR-1 Localizes to Chromosomes and to the Spindle Midzone during Mitosis and Meiosis 70 : BIR-1 immunoreactivity was no longer detectable on decondensing DNA but was still present on the spindle midzone ( Figure 2A , TELOPHASE ) . 89 : In hermaphrodite oocytes , BIR-1 first became visible on the condensed chromosomes of the oocyte most proximal to the spermatheca ( Figure 2B , OOCYTE ) . 90 : BIR-1 localization to chromosomes in this oocyte required sperm in the spermatheca and either preceded or was concomitant with nuclear envelope breakdown ( data not shown ) . 91 : BIR-1 was also associated with meiotic chromosomes in spermatocytes ( Figure 2B , SPERM ) but was not associated with DNA in mature sperm or condensed paternal DNA following fertilization ( data not shown ) . 92 : Following fertilization , BIR-1 remained localized to condensed maternal chromosomes during metaphase I ( Figure 2B , META I ) and was found associated with chromosomes as well as the spindle midzone during anaphase I ( Figure 2B , ANA I ) . 94 : BIR-1 was again associated with both polar body DNA and the spindle midzone during anaphase II ( Figure 2B , ANA II ) . 95 : The localization of BIR-1 to chromosomes and the spindle midzone suggested that BIR-1 may function in chromosomal and spindle processes during meiosis and mitosis . 234 : The presence of BIR-1 on both chromosomes and the spindle midzone is similar to the localization of some mammalian chromosomal passenger proteins ( Choo , 1997 ) . PAPERDELIMITER PMID:11099459 48 : Immunoreactivity is predominantly in synaptic regions along neuronal processes and is sparse in neuronal cell bodies ( compare with diagram in panel B ) . 56 : Immunoreactivity to both proteins is present in synaptic regions of 115 neurons , and additional experiments showed that UNC-17 was associated with synaptic vesicles ( Fig 1 ) ( 1 ) . 99 : Immunolocalization studies indicate that the VMAT protein is primarily localized to synaptic regions of a subset of neurons and is associated with synaptic vesicles ( Fig 1 ) ( 7 ) . PAPERDELIMITER PMID:11001875 44 : A5 , HSP12s localized in cytoplasm of sperm cells ( green ) , with nuclei shown in red ( DAPI ) . 58 : B3 , HSP16s ( green ) are localized in sperm cytoplasm , with nuclei shown in red ( DAPI ) . 89 : The spermathecal labelling seems to consist of cytoplasmic localization of HSP16-2 in sperm ( Figure 1 , B3 ) , and perhaps also in the spermathecal cage cells ( results 91 : Counterstaining with DAPI revealed HSP16 localized in discrete regions within the cytoplasm of intestinal cells , characterized by their large nuclei ( Figure 1 , B4 and B5 ) . PAPERDELIMITER PMID:11311162 3 : All three IMAs are expressed in the germline ; however , only IMA-3 is expressed in the soma . 51 : At various stages of gametogenesis , each IMA protein was associated with germ cell nuclei , but RNA interference ( RNAi ) assays indicated that IMA-3 had a unique role . 172 : Within the adult germline , IMA-2 and IMA-3 were detected in the common cytoplasm of the rachis and in association with the nuclei throughout the germline . 182 : In contrast , IMA-1 was detected only weakly in the distal region , but became increasingly enriched in proximal nuclei at the point when female germ cells exit the pachytene stage and enter diplotene of meiotic prophase I ( Fig 6B , asterisk ) . 186 : In male germlines , IMA-3 was detected at the nuclear envelope and in the rachis similar to hermaphrodite germlines ( data not shown ) . 191 : IMA-1 was associated with nuclei primarily in the proximal region of a male germline ( data not shown ) . 290 : While IMA-3 is distributed between the cytoplasm and nuclear envelope during all stages of germline development , IMA-1 is predominantly seen in the nucleoplasm of germ nuclei that have exited pachytene . 349 : Third , as IMA-3 is the only importin expressed in the soma , it is possible that a somatic signal or factor from the gonadal sheath cells may be lost , preventing progression through meiosis ( McCarter et al , 1997 ) . 375 : IMA3 mainly associates with nuclear envelope during all stages of oocyte development , while importin 3 initially localizes to the nucleoplasm and then within the cytoplasm of developing D melanogaster egg chambers ( Mathe et al , 2000 ) . PAPERDELIMITER PMID:11250150 89 : Staining is observed at the membranes of the neuron cell bodies ( the position of cell bodies is indicated by the bracket ) . 105 : GPB-2-specific staining was also observed in a large number of neural cell bodies located on either side of the nerve ring ( Figure 3b ) . PAPERDELIMITER PMID:11470828 5 : MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments , including the rectum , vulva , mechanosensory neurons , and excretory ductpore . 6 : In addition , it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments ( IFs ) at these sites . 39 : The MUA-3 protein is shown to be a novel transmembrane protein that localizes to hypodermal hemidesmosomes at the sites of skeletal muscle contact and to other epithelial sites where stress-resistant cuticular adhesion is required . 40 : Finally , we show that MUA-3 colocalizes with cytoplasmic IFs in the hypodermis , suggesting that it may physically link IFs to the cuticle . 163 : Localization of MUA-3 to hemidesmosome zone of hypodermis in animals stained with antiMUA-3 antibodies . 188 : MUA-3 localizes to hemidesmosomes where hypodermis contacts body wall muscle , a localization that is shared with several other hypodermally expressed proteins implicated in muscle force transmission . 198 : MUA-3 colocalizes with cytoplasmic IFs at hypodermal hemidesmosomes . PAPERDELIMITER PMID:11532911 291 : Anti-ELT-5 staining is readily detected in the nuclei of seam cells during mid to late-embryogenesis ( Fig 6C , D ) . PAPERDELIMITER PMID:11717359 141 : Our data demonstrate that RBF-1 is localized in the synaptic-rich neuropil of C elegans in concert with other synaptic vesicle proteins and suggest that C elegans rabphilin is associated with synaptic vesicles , as has been shown for vertebrate rabphilin ( Li et al , 1994 ; Mizoguchi et al , 1994 ; Shirataki et al , 1994 ) . 147 : Thus , localization of rabphilin to synaptic vesicles and regulation of the transport of rabphilin were both independent of rab3 . 152 : Rabphilin is localized to synaptic-rich regions of the C elegans nervous system . 154 : RAB-3 ( A ) and rabphilin ( B ) colocalize in wild-type animals doublelabeled with -RAB-3 and -RBF-1 antibodies . 155 : Synaptotagmin ( C ) and rabphilin ( D ) colocalize in wild-type animals double-labeled with -SNT-1 and -RBF-1 antibodies . 291 : Second , our work suggests that C elegans rabphilin associates with synaptic vesicles independently of rab3 . 317 : C elegans RBF-1 localizes with synaptic vesicles independently of rab3 PAPERDELIMITER PMID:11030350 49 : CeTLF Protein Is Nuclear and Present in All Cells throughout Embryogenesis ( A ) Schematic representation of the Caenorhabditis elegans ( Ce ) tlf-1 genomic structure . PAPERDELIMITER PMID:11717360 180 : Axon bundles in the nerve ring and ventral nerve cords of wild-type animals were brightly stained with the anti-egl-3 PC2 antibody ( Fig 4D ) . 181 : Staining of axons with anti-egl-3 PC2 antibodies could correspond to PC2 that has been trafficked into the secretory pathway . 187 : Thus , proper enzymatic activity is required for trafficking of the egl-3 PC2 protein into axons , as would be predicted if axonal staining consists of PC2 that has been transported into post-Golgi secretory organelles . PAPERDELIMITER PMID:11034910 287 : The distribution of UNC-59 and UNC-61 in the cleavage furrow and the midbody region . 288 : ( A-F ) illustrate that UNC-59 co-localizes with the actomyosin contractile ring . 293 : Similar to NMY-II ( G ) , UNC-61 localizes to the leading edge of the cleavage furrow ( H ) . 295 : At the terminal phase of cell division , both NMY-II ( J ) and UNC-61 ( K ) co-localizes to the midbody region . 316 : Localization of actin , non-muscle myosin II , UNC-59 and UNC-61 in the cleavage furrow and the midbody 317 : To determine the localization of UNC-61 relative to other cytoskeletal elements , we labeled UNC-59 and UNC-61 in combination with several proteins that have been shown to localize to the cleavage furrow . 363 : Finally , UNC-59 and UNC-61 antibodies detected the presence of these septins in the cleavage furrows of wild-type embryos but not in the unc-59 ( e1005 ) or unc-61 ( n3169 ) mutant embryos . 380 : Localization of UNC-59 and UNC-61 in early wildtype and mutant embryos Even though we saw no early embryonic defects in septin mutants , we found that UNC-59 and UNC-61 localize to cleavage furrows throughout embryonic development . PAPERDELIMITER PMID:11441002 174 : The compartment was found exclusively around the nucleus and observed during embryogenesis ( 4-cell stage , Fig 5A ; 8-cell stage , Fig 5 , B and C ; and 100-cell stage , Fig 5 , E and F ) . 188 : VHA-6 was clearly detectable on the apical surface of intestinal cells , forming two lines along the worm body ( Fig 6 , G and I , arrowheads ) . 191 : indicate that VHA-6 on the apical surface of intestinal cells may function by taking up nutrients through microvilli . 225 : G I , in adult worms , VHA-5 was strongly expressed in an H-shaped excretory cell and was detectable in its cell body ( G , arrowheads ) and canals ( H , arrow ) . 241 : VHA-6 is distributed specifically on the apical surface of intestinal cells in larval and adult worms . PAPERDELIMITER PMID:11553721 122 : Staining is observed in the cytoplasm of early embryo stage ( F ) , along the intestinal wall of a larva ( G ) , and in the terminal bulb region of the pharynx ( arrows ) and some excretory cells ( arrowhead ) in the head ( H ) . 146 : CRT-1 was detected in the cytoplasm , presumably from the endoplasmic reticulum network , of the early embryo ( 100-cell stage ) ( Figure 2F ) and a strong intestinal staining was observed in both larval and adult worms ( Figure 2G ) . PAPERDELIMITER PMID:10882103 4 : MEX-5 is a novel , cytoplasmic protein that is localized through PAR activities to the anterior pole of the 1-cell stage embryo . 155 : Toward the end of the 2-cell stage , MEX-5 appears on both centrosomes of the nascent spindle in P1 ( Figure 5C , arrows ) . 156 : As the spindle rotates onto the anteriorposterior ( ap ) axis of the egg and P1 begins division , MEX-5 disappears from the anterior centrosome ( Figure 5D ) ; MEX-5 persists on the posterior centrosome for a brief period before also disappearing ( Figure 5E ) . 166 : The orientation of the nascent mitotic spindle is indicated by arrows ; note MEX-5 on both centrosomes . 169 : MEX-5 is present at high levels on the posterior centrosome . PAPERDELIMITER PMID:10811831 176 : -G Spectrin Associates with the Plasma Membrane at all Sites of CellCell Contact throughout C elegans Development 180 : -G spectrin was localized to the plasma membrane at sites of cell cell contact throughout development , starting at the first cell division ( two-cell embryo ; Fig 3 C , i ) . 190 : -G spectrin is associated with the plasma membrane at sites of cellcell contact throughout development in C elegans . 204 : -G spectrin is associated with plasma membranes at sites of cellcell contact ( i and ii , arrows ) , starting at the two-cell stage , with cell membranes in the gut ( iii , arrows ; v , gt ) , in epidermal cells ( iv , arrow indicates row of lateral seam cells ) , and in the developing pharynx ( v , ph ) . PAPERDELIMITER PMID:11029035 12 : are consistent with the interpretation that ICA69RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles . 265 : are consistent with the interpretation that ICA69 is a cytosolic protein participating in the process of neuroendocrine secretion via association with the outside of secretory vesicles . PAPERDELIMITER PMID:11245684 124 : Antibody staining of wild-type animals for UNC-17 and ChAT produces a punctate pattern that is characteristic of presynaptic varicosities at en passant synapses . 133 : As shown in Figure 2 , UNC-17 and ChAT staining of VC neuromuscular synapses is readily detected in the wildtype animal 232 : A , D , UNC-18 antibody ( red ) stains the axonal processes of the VCs in a diffuse pattern ( arrow ) . 235 : AC , In wild type , the UNC-17 and UNC-18 antibodies colocalize to VC axonal projections . PAPERDELIMITER PMID:11525245 5 : Immunochemistry and cell fractionation experiments indicate that , when induced , AIRAP is present in both the nucleus and the cytoplasm , and cross-linking experiments indicate that it associates with RNA in vivo . 103 : AIRAP staining was observed in both the nucleus and the cytoplasm following arsenite treatment of NIH3T3 cells ( Fig 3A ) . 104 : The nuclear staining was diffusely nucleoplasmic and was occluded from the nucleolus . 115 : suggest that most AIRAP is incorporated into an insoluble complex in the cytoplasm . 116 : As AIRAP was detected in both the nucleus and the cytoplasm and formed insoluble complexes , we examined its association with any polynucleotide species that might 139 : Whole cell extracts ( WCE ) , nuclear fraction , cytoplasmic fraction , and 200 000 g ultracentrifugation supernatant and pellet from untreated ( ) and arsenite-treated ( 30 M , 6 . 5 hours ) ( ) cells were resolved by 12 SDS-PAGE and immunoblotted with the antiAIRAP antisera . PAPERDELIMITER PMID:11161560 5 : DLG-1 is restricted to adherens junctions of all embryonic epithelia , which contrasts with the localisation of the Drosophila and vertebrate homologues in septate and tight junctions , respectively . 44 : The protein encoded by the C elegans discs-large ( dlg-1 ) gene is localised in the zonula adherens and is necessary for correct junctional assembly in all embryonic epithelia . 150 : We further tested if the correct localisation of DLG-1 in the zonula adherens depends on the cadherincatenin system ( Costa et al , 1998 ) or the basolateral protein LET-413 , which is required for the assembly of adherens junctions in C elegans ( Legouis et al , 2000 ) . 161 : DLG-1 expression in C elegans is restricted to the zonula adherens in hypodermis , pharynx , and intestine . 243 : of CRB-1 are found immediately apical to the zonula adherens , forming a second narrow band ( Figs 8G8I ) . 253 : In WT embryos ( A , a ) aPKC is distributed over the entire apical surface . 258 : In the wild-type C elegans embryo cytoplasmic CRB-1 is already detectable in the late 8 E-cell stage ( data not shown ) . 263 : This conclusion is based both on the expression of DLG-1 in the zonula adherens of all epithelia as well as on the failure of dlg1 ( RNAi ) embryos to form continuous belts of junctionassociated antigens . 276 : In wild-type embryos ( AI ) at the end of the proliferation phase ( AC ) anti-phosphotyrosine ( PY , green ) epitopes and CRB-1 ( red ) colocalise at the apical membrane domain of the gut epithelium ( as indicated by the orange colour ) . 278 : ( GI ) In the plum stage anti-CRB-1 fluorescence defines a narrow belt in the immediate vicinity of the zonula adherens in which PY epitopes are not found . 306 : In C elegans ( Figs 7 and 8 ) , colocalisation of CRB-1 , PKC-3 , PAR-3 , and PAR-6 is immediately apical to the continuous circumferential zonula adherens . PAPERDELIMITER PMID:11312268 114 : Within the expressing cells , the FL-GFP fusion protein localized efficiently to several structures beyond the cell soma , including axons that form the ventral nerve cord ( Fig 2D ) ; dendrites that extend to the anterior tip of the animal ( Fig 2 , A and B ) ; and the cilia of neurons AWB , AWC , and BAG ( Fig 2C ) . 152 : Immunostaining of wild-type L1 larvae with the anti-CePPEF antibodies demonstrated CePPEF in several cell bodies in the region of the nerve ring , in dendritic processes leading from these cells to the anterior end of the animal , and in sensory cilia at the extreme anterior end of the animal ( Fig 3C ) . 263 : Our observation that CePPEF immunoreactivity in wild-type nematodes and GFP fluorescence in full-length CePPEF-GFP ( FL-GFP ) transgenic animals encompass both the cell soma and membrane-rich subcellular compartments such as dendrites and cilia is consistent with the possibility that there may be populations of CePPEF proteins that are differentially targeted . PAPERDELIMITER PMID:11134055 187 : Furthermore , immunocytochemistry of PMR1-overexpressing and control cells reveals the correct targeting of PMR1 to the Golgi compartment of the COS-1 cells ( Fig 3B ) . 188 : In conclusion , by using our polyclonal anti-PMR1 antiserum it became clear that PMR1 is expressed in the worms , that it can be overexpressed in COS-1 cells and that it contains all the information needed to target the PMR1 protein to the Golgi membranes . 298 : The C elegans PMR1 protein overexpressed in COS-1 cells showed a predominantly Golgi-like distribution as shown by immunocytochemistry . 307 : We have shown that the PMR1 protein overexpressed in COS-1 cells shows a predominantly Golgi-like distribution and that its ion transport activity can be measured following permeabilization of the plasma membrane . PAPERDELIMITER PMID:12054525 82 : Immunocytochemical analysis of the cells expressing nRTN-C showed a typical ER-localization ( Fig 1C ) . 122 : Cytosolic and microsomal fractions were prepared from the cells , and coimmunoprecipitation experiments were carried out . nRTN-C was detected comparably in both fractions ( Fig 4D ) . 123 : Calnexin , an ER marker protein , was not detected in the cytosolic fraction , indicating that detection of nRTN-C in the cytosolic fraction was not due to contamination of microsomes . 144 : The nRTN-C protein transiently expressed in mammalian cells was localized in ER , which is characteristic PAPERDELIMITER PMID:12810918 91 : Staining for each of the antibodies is predominantly nuclear . PAPERDELIMITER PMID:11470827 5 : Here , we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix . 98 : MUP-4 localization in larval and adult stages at cuticular attachment sites and to hemidesmosomes MUP-4 continues to localize to circumferential rings over body wall muscle in larvae and adults ( Fig 7 , AC ) . 157 : In addition to the annular localization , MUP-4 and IF proteins are sometimes seen diffusely throughout the hypodermal syncytial cell cytoplasm , possibly reflecting protein not yet recruited to the regions overlying muscle . 158 : MUP-4 also shows coincident localization with the MH4 IF in other structures such as the touch neuron channels , further supporting that MUP-4 associates with IFs ( Fig 7 , EG ) . 172 : We report that MUP-4 is a novel transmembrane protein with domains consistent with its function as an IFAP at epithelial junctions . 173 : mup-4 is expressed in the hypodermis ( also see Gatewood and Bucher , 1997 ) and localizes to hypodermal cellmatrix boundaries , rather than cellcell boundaries . PAPERDELIMITER PMID:14704165 131 : Moreover , staining with anti-ODR-7 antibodies showed that ODR-7 ( R372A ) was localized to the nuclei and that levels of ODR-7 ( R372A ) were less than twofold different from those of wild-type ODR-7 ( Figure 2 and data not shown ) . 135 : ODR-7 ( K393AR394G ) was localized to the nucleus at levels comparable to those in wild-type animals ( Figure 2 ) , indicating that nuclear localization of ODR-7 is mediated by additional residues or via alternate mechanisms in the AWA neurons . 154 : ODR-7 ( R356E ) was localized to the nucleus and expressed at levels similar to those of transgenic animals misexpressing wild-type ODR-7 in the AWC neurons ( Figure 2 ) . 162 : Consistent with the K393R394 residues in the DBD being required for nuclear localization of ODR-7 in the AWC neurons , deletion of the DBD resulted in mislocalization of the ODR-7 protein to the cytoplasm and failure to repress str-2 expression ( Figure 2 ) . PAPERDELIMITER PMID:11715019 2 : We have characterized the novel coiled-coil protein AJM-1 , which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMRHMP ( cadherincatenin ) complex. 48 : AJM-1 localizes to the apical borders of all C elegans epithelia . 92 : To address the functional role of AJM-1 in apical junctions specifically , we analysed ajm-1 embryos at the ultrastructural level by TEM . 145 : Consistent with another report that was published while this work was being reviewed was our observation , by laser scanning confocal microscopy analysis , of complete co-localization with AJM-1 to the apical junctions in the hypodermis ( Fig 4d ) , pharynx and the intestine ( data not shown ) 27 . 146 : We conclude that AJM-1 and DLG-1 physically interact at apical junctions . 216 : indicate that AJM-1 is a novel coiled-coil protein that localizes to a distinct apical domain of epithelial junctions of C elegans and is required for maintaining the integrity of this domain . 218 : AJM-1 seems to interact directly with DLG-1 at apical junctions , whereas LET-413 mediates the rapid apical localization of both DLG-1 and AJM-1 . 219 : Analysis of AJM-1 localization by immunofluorecence and immunogold TEM reveals that AJM-1 occupies a distinct apical junctional domain in C elegans epithelia that is basal to the HMRHMP ( cadherincatenin ) complex 228 : Apart from the apical junction to which AJM-1 localizes , we observe no morphologically distinct structures in the cell borders between epithelial cells of C elegans that could be candidates for maintaining the paracellular seal 258 : indicate that LET-413 mediates a rapid accumulation of DLG-1 and AJM-1 at apical junctions . 282 : A better understanding of the nature and function of the apical junctional domain to which DLG-1 and AJM-1 localize in C elegans will require the identification of other binding partners for these proteins . PAPERDELIMITER PMID:10862717 6 : LMP-1 was found to be present from early embryonic stages through adulthood and to be predominantly localized at the periphery of a population of large , membrane-bound organelles , called granules , that are seen throughout the early embryo but in later stages are restricted to the cells of the intestine . 200 : The LMP-1 protein is primarily expressed in a subset of intestinal granules 201 : In early embryos , immune serum stained granules broadly distributed throughout the embryo ( Fig 4A and B ) . 204 : Intestinal granules are labeled in all subsequent stages of C elegans development ( Fig 5 ) . 206 : At all stages ( Figs 4D , 5F ) labeling occurs at the periphery of the granules , as would be expected for labeling of a membrane protein . 208 : we were interested in knowing if the granules constaining LMP-1 might be lysosome-like. 220 : LMP-1 is present in C elegans intestinal granules from late embryonic stages through adulthood . 251 : Thus , dsRNA mediated inhibition potently inhibited accumulation of LMP-1 protein , and therefore the gut granule staining pattern described in the previous section was specific for LMP-1 . 311 : By immunofluorescence microscopy LMP-1 was located in subcellular granules distributed throughout the early embryo . 312 : Beginning with the lima bean stage , LMP-1 positive granules accumulated in cells differentiating into gut it issue and progressively disappeared from other cells . 313 : The switch from uniformly distributed LMP-1 positive granules to intestinal concentration of the granules corresponds well with the timing of yolk granule redistribution from the periphery to the intestine , and with the first appearance of granules in the intestinal cells that are autofluorescent , birefringent and can accumulate Acridine Orange . PAPERDELIMITER PMID:11343658 31 : Scale bar : 10 M served a dendritic , axonal , and cell body subcellular distribution with Ce-NCS-1-specific antibodies ( data not shown ) . PAPERDELIMITER PMID:11257122 5 : The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system . 142 : ( D ) Under body wall muscle of an adult , CLE-1 accumulates strongly at the junctions between muscle cells ( arrowheads ) and along muscle-dense body lines ( small arrows ) but is not coincident with the dense bodies . 149 : ( I and J ) Wild-type and cg120 L1 larvae show type IV collagen localization in the basement membranes of the pharynx ( p ) , intestine ( i ) , and gonad primordium ( g ) . 184 : Beginning in the L4 larval stage , CLE-1 is also observed in basement membranes surrounding the gonad , pharynx , and intestine ( Fig 3 C ) , and under body wall muscles where it preferentially accumulates at junctions between muscle cells and along dense body lines ( Fig 3 D ) . 237 : Also , the accumulation of CLE-1 at the junctions between body wall muscle cells is absent from cg120 animals ( Fig 3 H ) , suggesting that the NC1 domain is necessary for this localization . 348 : CLE-1 is broadly distributed in C elegans basement membranes but accumulates to highest levels in association with the nervous system . PAPERDELIMITER PMID:11290289 6 : osm-5 is expressed in ciliated sensory neurons in C elegans and its expression is regulated by DAF-19 , an RFX-type transcription factor that governs the expression of other genes involved in cilia formation in the worm . 7 : Similar to murine polaris , the OSM-5 protein was found to concentrate at the cilium base and within the cilium axoneme as shown by an OSM-5 : : GFP translational fusion and immunofluorescence . 47 : In addition , the OSM-5 protein was found to concentrate at the distal end of the dendrites and within the cilia of the sensory neurons , as determined by immunofluorescence and in transgenic worms expressing an OSM-5 : : GFP translational fusion . 206 : osm-5 is expressed in ciliated sensory neurons 215 : Thus , similar to the expression of Tg737 in the mouse , osm-5 is strongly associated with ciliated cells . 277 : In all of these mutants , OSM-5 protein was still observed at the distal tips of the dendrites at the transition zones where cilia would form ( data not shown ) . 324 : Furthermore , the OSM-5 protein was found to concentrate at the distal end of the dendrites in the transition zone and within cilia . 337 : In agreement with a ciliogenic role , osm-5 expression appears exclusive to ciliated sensory neurons in the Fig 8 . PAPERDELIMITER PMID:11514595 101 : Comparison shows that expression of Ce-Duox1 and MH4 occurs in the same cellular pattern , although the staining intensity differed in different regions of the hypodermal cell layer . 107 : Merged images ( E and H ) show areas of overlapping staining of Ce-Duox1 and MH4 in yellow , confirming the expression of Ce-Duox1 in the hypodermal layer of cells . PAPERDELIMITER PMID:10508609 123 : At all stages of germ-line development , NOS-3 was predominantly cytoplasmic : this was evidenced by the dark , non-staining holes corresponding to nuclei in the germ line at the larval L3 stage ( Figure 3b ) . 124 : The distribution of NOS-3 overlapped with regions of the cytoplasm containing P granules , detected using anti-PGL-1 antibodies ( Figure 3c , d ; [ 20 ] ) ; however , within the cytoplasm , NOS-3 staining was uniform and diffuse throughout . 138 : ( b ) NOS-3 protein was uniformly distributed throughout the cytoplasm in the anterior and posterior germ-line tubes of wild-type animals ( arrowheads ) , and there was faint staining in the somatic gonadal it issue ( arrow ) . 143 : ( h ) NOS-3 protein was found in the germ-line cytoplasm and was most prominent in germ cells in mitosis and meiotic pachytene . 149 : NOS-3 was localized to the cytoplasm of P blastomeres , which generate the germ line . PAPERDELIMITER PMID:10545592 6 : Here we show that CeSMN is transmitted maternally as a predominantly nuclear factor , which remains present in all the blastomeres throughout embryonic development and onwards into adulthood . 89 : The CeSMN protein is expressed in all the nuclei of Celegans embryos from the zygotic stage 92 : A strong homogeneous staining is present in all of the interphasic nuclei , although some diffuse fluorescence is also visible in the cytoplasm . 96 : CeSMN is present in virtually all the cell nuclei of the young nematodes . 203 : Its abundance in the germ cell nuclei and subsequently in both pronuclei and the zygote implies that it is synthesized by the germline and incorporated into the developing gametes . PAPERDELIMITER PMID:10066248 5 : OSM-10 is a novel cytosolic protein expressed in ASH and three other classes of sensory neurons . 144 : The OSM-10 protein was uniformly distributed throughout the cell bodies , sensory processes , and axons of the expressing cells but was excluded from nuclei on the basis of confocal resolution microscopy ( data not shown ) . 148 : demonstrate that OSM-10 is a novel cytoplasmic protein expressed in the osmosensory ASH neurons . 152 : The OSM-10 protein is diffusely distributed throughout the cell body , sensory processes , cilium , and axons of expressing cells . PAPERDELIMITER PMID:9819355 4 : Specific antisera were used to show that SPE-4 resides within the fibrous body-membranous organelles membranes during wild-type spermatogenesis . 135 : These two antisera exhibit an apparently coincident signal , suggesting that SPE-4 resides in the FB-MOs ( Fig 3A5 ) . 192 : SPE-4 localizes within the GolgiER derived FB-MOs and segregates to spermatids as they bud from the residual body during C elegans spermatogenesis . 197 : Both missense mutants synthesize detectable SPE-4 protein that localizes within the FB-MOs . PAPERDELIMITER PMID:9716526 48 : In this report , we show that embryos depleted of PKC-3 die displaying Par-3-like phenotypes , and that PKC-3 can bind to PAR-3 in vitro and is co-localized with PAR-3 at the anterior cortex of the 1-cell embryo . 158 : The antibodies stained the cell periphery of early blastomeres as well as the nuclear envelope . 178 : PKC-3 staining is weak and uniform throughout the cytoplasm . 180 : PKC-3 becomes restricted to the anterior periphery . 182 : PKC-3 can be seen at the anterior periphery . 184 : PKC-3 is present uniformly at the periphery of the AB cell and at the boundary of AB and P1 blastomeres . 186 : PKC-3 can be seen at the periphery of somatic cells ABa , ABp and EMS but peripheral staining of the germ-line cell P2 is restricted to the boundaries with ABp and EMS . 207 : Overall , these observations lead us to propose that PKC-3 and PAR-3 cooperate to achieve a mutually dependent anterior peripheral localization in 1-cell embryos . PAPERDELIMITER PMID:10224255 8 : spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface . 48 : In a continuing molecular characterization of genes that affect this signaling pathway ( Minniti et al 1996 ) , we identify the spe-12 gene and show that it encodes a sperm plasma membrane protein . 243 : These experiments strongly suggest that within spermatids , SPE-12 is predominantly localized to the plasma membrane . PAPERDELIMITER PMID:11514595 101 : Comparison shows that expression of Ce-Duox1 and MH4 occurs in the same cellular pattern , although the staining intensity differed in different regions of the hypodermal cell layer . 107 : Merged images ( E and H ) show areas of overlapping staining of Ce-Duox1 and MH4 in yellow , confirming the expression of Ce-Duox1 in the hypodermal layer of cells . PAPERDELIMITER PMID:9508766 214 : Indirect immunofluorescence using mAb D3C2 showed that in wild-type embryos , NCL-1 is a cytoplasmic protein with no obvious nuclear accumulation . 229 : Immunostaining with mAb D3C2 showed that NCL-1 is present in the cytoplasm of all cells except those of the developing gut ( Fig 7 c ) ; these cells are among the first to differentiate in the embryo , are the largest cells in the embryo , and have very large nucleoli . PAPERDELIMITER PMID:9645949 6 : Consistent with cyk-1 function being required for a late step in embryonic cytokinesis , we show that the CYK-1 protein colocalizes with actin microfilaments as a ring at the leading edge of the cleavage furrow , but only after extensive furrow ingression . 32 : Consistent with this hypothesis , we show that the CYK-1 protein localizes to the leading edge of the cleavage furrow near the end of cytokinesis . 149 : In early stage wild-type embryos , NMY-2 localizes to the cortex of blastomeres ( Guo and Kemphues , 1996 ) , and is enriched in the cleavage furrow during cytokinesis ( Fig 3C ) . 174 : NMY-2 is present in the cleavage furrows of both wildtype and cyk-1 mutant embryos during cytokinesis . 195 : CYK-1 protein localizes to the leading edge of the cleavage furrow late in cytokinesis . 197 : Consistent with it acting at a late step in cytokinesis , CYK-1 protein is detectable only well after the initiation of furrow ingression , when it forms a ring at the leading edge of the cleavage furrow ( Fig 6A ) . 216 : Thus only after the cleavage furrows close down substantially around the midzone of the mitotic spindle do detectable amounts of CYK-1 co-localize with the actin contractile ring . 241 : Consistent with it acting at a late step in cytokinesis , we detect CYK-1 protein at the leading edge of the cleavage furrow only after substantial ingression has occurred . 251 : CYK-1 protein localizes to the leading edge of the cleavage furrow at a late stage in furrow ingression . 258 : ( B ) CYK-1 protein is associated with the plasma membrane at the site of polar body extrusion during meiosis ( arrow ) , with DAPI staining showing the maternal chromosomes to the left ( paternal pronucleus is to right ) . 262 : Consistent with the late defect in cytokinesis in cyk-1 mutant embryos , CYK-1 protein localizes to the leading edge of the cleavage furrow late in cytokinesis . PAPERDELIMITER PMID:9614171 162 : ( C ) A lateral view of the midbody showing UNC-64 immunoreactivity on the basolateral surface of the intestine . 173 : Syntaxin immunoreactivity also was detected in the vast majority of neuronal cell bodies as well as in commissural and dendritic processes ( Figure 2B ) . 183 : In addition , syntaxin expression was high in intestinal nuclei , in the spermatheca , and in the uv1 cells of the vulva ( Figure 2H ) . 191 : In neurons , syntaxin A and C were found ubiquitously on neuronal cell bodies , axons , and dendrites . PAPERDELIMITER PMID:9502737 146 : We first detected anti-CeOE staining in 8 nuclei in the 400 to 550-cell embryo ( Fig 4A ) , the stage during which the embryonic motor neurons are first formed ( Sulston et al , 1983 ) . 173 : Lateral views of a 3-fold embryo ( D ) and L1 larva ( E ) ( 250 m in length ) showing intense staining in 16 nuclei in the ventral cord . PAPERDELIMITER PMID:9950681 5 : Additionally , immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites . 156 : These antibodies react specifically with polypeptides of the appropriate molecular mass on immunoblots of MT proteins prepared from mixed stage C elegans cultures and clearly show that these polypeptides cosediment with MTs with the addition of AMPPNP and elute from MTs with ATP as expected for kinesinrelated motors ( Figure 3B ) . 219 : Antibodies to CeKRP95 , CeKAP , and OSM-3 all stained the cell body cytoplasm , dendrites , and ciliated endings of the amphid , inner labial , and phasmid chemosensory neurons . 220 : Cell body staining was most dramatic in anti-OSM-3 and anti-CeKRP95 stained worms ( Figure 7 , AF ) . 221 : In addition , worms stained with anti-CeKRP95 and anti-OSM-3 antibodies demonstrated a perinuclear , punctate staining in cell bodies and a punctate staining along corresponding dendrites of amphid neurons ( Figure 7 ) 236 : These punctae clearly align along neuronal processes in single confocal optical sections , although this is slightly obscured because of the abundance of overlapping punctae in the confocal projections provided in Figure 7 . 239 : The immunolocalization of CeKRP95 to the cell bodies , dendrites , and ciliated endings of chemosensory neurons in the head of the worm ( Figure 7 , AC ) is consistent with CeKRP95 promoter : : GFP expression . 250 : ( AF ) Perinuclear , punctate staining of the polypeptides is observed in the cell bodies and along dendrites of the amphid chemosensory neurons . 253 : Although all three antibodies stain the cell bodies and dendrites of these neurons in a punctate manner , the most intense staining is seen in the ciliated endings , suggesting a concentration of the CeKinesin-II and CeOsm-3 holoenzymes in these structures . 281 : Our data clearly demonstrate that both CeKinesin-II and CeOsm-3 are localized in the cell bodies , dendrites , and ciliated endings of chemosensory neurons . PAPERDELIMITER PMID:9584117 3 : We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells , of all five cell types . 82 : In six comma-stage embryos , we counted an average of 80 . 5 ( 2 . 5 s . d . ) PHA-4staining pharyngeal nuclei by confocal microscopy ( maximum count 85 ) . 85 : We conclude that , within the error associated with counting closely apposed nuclei , all cells of the pharyngeal primordium ( valve ) contain the nuclear factor PHA-4 . 86 : The same embryos also show nuclear PHA-4 in 6-8 rectal cells , including the two rectal valve cells and the three rectal epithelial cells . 92 : The embryo shown in Fig 2E contains 21 nuclei with detectable levels of PHA-4 protein , as counted by confocal microscopy . 94 : 100 total cells ; 16 pharyngeal precursor cells , of which 11 give rise only to pharyngeal cells ) , we can detect PHA-4 in 3-10 nuclei but at very low levels ( data RESULTS 101 : 250 minutes of development , when there are 52 pharyngeal precursor cells of which 49 give rise to only pharyngeal cells ; 40-50 strongly PHA-4-staining nuclei were counted in embryos at this stage . 105 : pha-4 persists in all stages of the life cycle PHA-4 can be detected immunologically in most but not all nuclei of the larval and adult pharynx . 106 : PHA-4 is detected in nuclei of all epithelial cells , muscle cells , marginal cells , gland cells and pharyngeal intestinal valve cells , but is detected only in about 8 of 19 neuronal nuclei ( Fig 2I , L ) . 109 : As expected from previous transgenic analysis ( Azzaria et al , 1996 ) , PHA-4 can also be detected in nuclei of the developing somatic gonad , including the distal tip cell and ventral uterine cells ( data not shown ) . PAPERDELIMITER PMID:9581759 227 : VAB-8S is punctate and roughly colocalizes with thin filaments in preparations double-stained with phalloidin to detect actin ( Figures 5d and 5e ) . PAPERDELIMITER PMID:11641215 4 : We show that one of the five isoforms of C elegans eIF4E , IFE-1 , is enriched in the germline and is a component of germ granules ( P granules ) . 5 : The association of IFE-1 with P granules requires the P-granule protein PGL-1 . 41 : that IFE-1 protein associates with P granules , and that this association is dependent on PGL-1 protein . 190 : ( D ) The merged image of B , C demonstrates double staining of P granules by anti-GFP and antiGLH-2 antibodies in N2 . 228 : IFE-1 associates with P granules in vivo and requires PGL-1 for this association 239 : IFE-1 may associate with P granules via a direct interaction with PGL-1 . 244 : suggest that PGL-1 is required for IFE-1 localization to P granules in wildtype animals . 378 : Interestingly , IFE-1 is a component of P granules . PAPERDELIMITER PMID:10587644 29 : The highest CUL-2 levels are found in oocytes , where CUL-2 is mainly nuclear with some cytoplasmic staining ( Fig 1jk ) . 30 : Disruption of cul-2 expression by double-stranded RNA-mediated interference ( RNAi ) 24 severely reduces the levels of both nuclear and cytoplasmic anti-CUL-2 staining ( Fig 1l , m ) . 71 : As germ cells enter meiosis , the level of CKI-1 increases , with the highest intensity being observed in oocyte nuclei ( Fig 5e , g ) . PAPERDELIMITER PMID:15454571 1 : Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo. The Caenorhabditis elegans UNC-45 protein contains tetratricopeptide repeats and a domain with similarity to fungal proteins , and it differentially colocalizes with myosin heavy chain B in the body wall muscles of adult worms . 5 : Yeast twohybrid screens show that UNC-45 can directly interact with NMY-2 , a non-muscle type II myosin , and UNC-45 and NMY-2 colocalize at cell boundaries in early embryos . 91 : Arrows indicate concentration of UNC-45 at cell boundaries . 100 : and appears to be concentrated at the cell cortex ( Fig 1C , D ) . 181 : Fig 4A-F shows images of two-cell and 20-cell stages of wild-type embryos , demonstrating that NMY-2 and UNC-45 are indeed concentrated at the cell cortex , and the staining patterns are largely coincident ( although there are slight differences in intensity ) . 182 : Cortex staining for UNC-45 is apparent both where a fluorescent secondary antibody is used , and where UNC-45 antisera is directly labeled with a fluorescent marker ( data not shown ) . 189 : In Fig 4G , H , the UNC-45 staining at the cortex of the cell is disrupted in nmy-2 ( RNAi ) -treated embryos , indicating that the localization of UNC-45 at the cleavage furrow is dependent on NMY-2 . 205 : ( A-F ) Wild-type embryos of increasing age stained with anti-UNC-45 ( green ) and anti-NMY-2 ( red ) to show the concentration of UNC-45 and NMY-2 at the cell boundaries . 208 : Images G and H show embryos treated with RNA-mediated interference ( RNAi ) against NMY-2 , stained with anti-UNC-45 showing that the concentration of UNC-45 at the cell boundaries is disrupted when NMY-2 is knocked down . 225 : The localization of UNC-45 staining to the cell cortex is not unique , as several maternally contributed components show cortical localization in the two-cell embryo ( Rose and Kemphues , 1998 ) . 233 : As mentioned above , both UNC-45 and NMY-2 are expressed in the germline cells of the gonad as well as the premorphogenesis embryos , and the localization of maternal UNC-45 at the cell boundaries is dependent on the presence of NMY-2 . PAPERDELIMITER PMID:12421563 6 : CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining . 129 : 26 Note that the region stained with the R16 antibody is located in the I-band of the thin filament region . 139 : The R16 antibody stained actin-localizing areas in thin filaments ( I-bands ) , but not in paramyosin-containing areas in thick filaments ( Figure 3 ( d ) ) . 232 : with the R16 antibody indicated that CeRyR was located in the I-bands ( Figure 3 ) and , to a much lesser extent , in the A-band ( Figure 3 ( b ) ) . 233 : Thus we suggest that CeRyR is found in areas surrounding dense bodies within both I-bands and A-bands where flattened vesicles are located close to I-bands . PAPERDELIMITER PMID:11641272 164 : The lysate was cleared by spinning 10 min at 12 , 000g . PAPERDELIMITER PMID:14627718 31 : Anti-SMA-9 antibody staining reveals that SMA-9 is present in many it issues , where it localizes to cell nuclei ; this is consistent with its predicted transcriptional cofactor function . 240 : Antibody staining detects SMA-9 protein in most , if not all , somatic nuclei of wild-type animals , but not in sma-9 ( wk55 ) animals ( Fig 4E , G ) . 242 : The localization of SMA-9 to the nucleus is consistent with a function as a transcriptional cofactor . 334 : SMA-9 is similarly detected in nuclei of hyp7 . PAPERDELIMITER PMID:15068795 74 : TRR-1 Is Expressed Broadly and Is Localized to Nuclei 76 : In whole-mount stainings of wild-type animals , TRR-1 was detected in the nuclei of one-cell and subsequent stage embryos ( Figures 1C and 1F ) . 84 : Among the nuclei expressing TRR-1 were those of P ( 38 ) . p ( Figures 1D and 1G ) . 85 : TRR-1 was readily detectable in germ nuclei of larvae and adults . 86 : During the pachytene ( data not shown ) and diakinesis ( Figures 1E and 1H ) stages of meiosis I , as observed in syncytial germ nuclei and cellularized oocytes , respectively , TRR-1 was localized to condensed chromosomes . PAPERDELIMITER PMID:15138888 11 : Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potetial interaction in vivo. 205 : The sub-cellular localization of LFI-1 and LIN-5 overlap at kinetochore microtubules. 209 : LFI-1 remained in the nuclear region, in a pattern reminiscent of proteins associated with the nuclear matrix. 213 : Surprisingly , we found diffuse area of staining surrounding the kinetochore that the LFI-1 staining was not eliminated following microtubules that overlapped with the localization of RNAi treatment for lfi-1 , or RNAi for lfi-1 and LIN-5 ( Fig 4 ) . 214 : Partly different locations were seen in F35D11 . 11 together , which might explain the absence of interphase cells : while LIN-5 remained cytoplasmic , an LFI-1 showed a nuclear localization in late interphase cells. 216 : Even after nuclear envelope breakdown in mitosis, LFI-1 remained in the nuclear region, in a pattern reminiscent of proteins associated with the nuclear matrix. PAPERDELIMITER PMID:15122256 22 : ( e ) Localization of HRP-1 at the chromosome ends , as shown by antibody staining of HRP-1 in a germ cell . 48 : Using antibodies against HRP-1 , we found that HRP-1 was localized at the ends of the chromosomes of the germ cells ( Fig 1e ) . PAPERDELIMITER PMID:15208641 53 : FSN-1 is expressed specifically in the nervous system in the nerve processes ( Fig 2a , b ) . PAPERDELIMITER PMID:15115755 101 : The protein was non-uniformly distributed in nuclei from the early embryonic stage throughout embryogenesis . 105 : In larval stages WRN-1 was present in the nuclei of Research article numerous cells , and the fraction of WRN-1-positive somatic cells was significantly lower in adults ( Fig 2B ) . 107 : When nuclei in the intestine were magnified , an uneven distribution of the protein was observed ( Fig 2C ) . 108 : Both mitotic and meiotic prophase germ cells in the L4 stage gonad contained WRN-1 in the nuclei ( Fig 2C ) , but the level was significantly reduced in the meiotic prophase germ cells of adults ( data not shown ) . 109 : In the oocytes of adult gonads , WRN-1 localized to condensed chromosomes ( Fig 2D ) . 206 : However , the localization of WRN-1 to the poleward periphery of metaphase chromosomes in the early C elegans embryo has not been observed in other organisms . PAPERDELIMITER PMID:15210732 181 : Immunostaining with a pAb that we raised against RNF-5 revealed its expression in the larval muscle dense bodies ( Fig 6 A ) , the apical cell junctions of the spermatheca ( septate junctions ) , and the junctions between the gonadal sheath cells and the gonadal basal lamina ( unpublished data ) . 182 : During embryogenesis , RNF-5 is not expressed specifically in the developing muscle and is localized to the nucleus of most of the embryo cells ( unpublished data ) . PAPERDELIMITER PMID:15242805 6 : ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex ERM-1 depletion results 57 : Depletion of the protein encoded by the erm-1 ( ezrin-radixinmoesin ) gene specifically perturbs the F-actin cytoskeleton in the cell cortex of the apicallumenal membrane domain of the intestine . 151 : In WT embryos , we detect the first anti-ERM-1 staining in the two-cell stage ( see inset in Fig 2A ) highlighting the cell cortex below contacting plasma membrane domains . 153 : At the onset of morphogenesis ( lima bean stage ) , several it issues develop a polarized phenotype and ERM-1 becomes enriched at the apical cell cortex of the hypodermis and the pharyngealintestinal primordium ( Fig 2B ) . 156 : There is little or no overlap between ERM-1 and AJM-1 staining ; however , ERM-1 co-localizes with apically enriched F-actin ( Figs 2GI ) . 160 : ( A ) Beginning at the two-cell stage ( see inset ) , ERM-1 ( n 18 ) predominantly localizes to the cell cortex of contacting membranes . 161 : This localization shows some variability during later rounds of proliferation so that there are cells in which ERM-1 can be found in the whole cortex ( asterisk ) . 294 : In C elegans , we find ERM-1 expression associated with the cell cortex in most cells . PAPERDELIMITER PMID:15930113 200 : ANI-2 localizes to the surface of the rachis in the adult gonad 211 : In control gonads , ANI-2 localized strikingly and exclusively to the surface of the rachis ( Fig 6 ; green in schematics ) . 212 : ANI-2 framed the windows between the pseudocells and the rachis in the distal and middle gonad , and also localized to the surface of the rachis between the windows ( Fig 6A , B ) . 248 : The ANI-2 staining on the surface of the rachis is visible along the edges in cross-section ( arrows ) . PAPERDELIMITER PMID:15728376 72 : Indirect immunofluorescence staining with serum 3779 , which localizes endogenous Ce-BAF in the nucleus of embryos from worms fed empty L4440 vector ( Right ) . 108 : Serum 3779 , which recognizes a different epitope on Ce-BAF , also stained the nucleus , but the envelope signal was much weaker ( Fig 2A ) . PAPERDELIMITER PMID:15616192 96 : Affinity-purified antibodies revealed a distribution of DHC-1 in wild-type embryos similar to that described previously ( Gonczy et al , 1999 ) : punctate in cytoplasm , an elevated concentration on nuclear envelopes during pronuclear migration , enrichment in the central spindle during metaphase , and faint enrichment over the entire anaphase spindle ( Figure 2 , AC ) . 97 : We also noted a transient accumulation of DHC-1 in a narrow cortical zone between the AB and P1 cells during rotation of the P1 centrosomecentrosome axis onto the anterior-posterior ( AP ) axis ( Figure 2D ) . 131 : All embryos displayed punctate DHC-1 staining in the cytoplasm ; other localized concentrations are described below . 134 : ( B ) DHC-1 is lightly concentrated on the central spindle at metaphase ; there is no concentration on spindle poles . PAPERDELIMITER PMID:16207815 6 : In addition we find that Dcp2 is localized to P-granules , showing that Dcp2 is stored andor active in these structures . 50 : Colocalization with GLH-1 and other markers suggests that in addition to these small cytoplasmic foci , Dcp2 is found in P-granules . 75 : C elegans Dcp2 is found in cytoplasmic bodies from early embryogenesis . 114 : C elegans Dcp2 Is Enriched in Cytoplasmic Bodies 119 : Immunostaining of C elegans embryos revealed that Dcp2 protein was found enriched in particles in the embryonic cytoplasm ( Figure 1 ) , and these particles disappear in embryos depleted of Dcp2 using RNAi ( see below and Figure 1B ) . 125 : Dcp2 staining was faint in nuclei . 135 : Such immunostaining indicates that Dcp2 is enriched in P-granules ( Figure 2A ) . 143 : In summary , Dcp2 is found in P-granules , showing that a component of the mRNA degradation machinery localizes to these sites in a manner that is compromised in pgl-1 mutants . 196 : Although we found that Dcp2 is localized to P-granules , the progeny of dcp2 RNAi-treated mothers did not show sterility ( unpublished data ) , showing that depletion of Dcp2 alone from the P-granules does not affect germ line development . 281 : In addition Dcp2 is also found in P-granules , colocalizing with the P-granule markers GLH-1 and the antigens recognized by the K76 and OIC1D4 antisera . PAPERDELIMITER PMID:16197937 181 : The GOB-1 protein appears cytoplasmic throughout all of the stages examined although there are sporadic exceptions : rare embryos ( 1 ) show GOB-1 staining highly concentrated in nuclei ; the significance of these observations could not be determined , and experimental conditions could not be found in which such nuclear staining was reproducible . PAPERDELIMITER PMID:16183052 4 : FAX-1 protein accumulates in the nuclei of 18 neurons , among them the AVA , AVB , and AVE interneuron pairs that coordinate body movements . 111 : We detected FAX-1 protein in the nuclei of wild-type embryonic neurons beginning at mid-embryogenesis ( approximately 350 min ) , the time at which most embryonic neurons differentiate , and through all larval and adult stages ( Figs 2A C ) . 113 : Accumulation of FAX-1 protein in nuclei of wild-type animals . 136 : In addition , FAX-1 protein accumulated to high levels in the nuclei of AVA , AVB , and AVE bilateral interneuron pairs of the head , which have been shown to function in coordinating movement ( Figs 2C , 4 ) . PAPERDELIMITER PMID:16143610 257 : SPE-10 localizes to the FBMO 268 : These data indicate that SPE-10 mostly localizes within FBMOs during spermatid formation . 273 : Overlaid composite image of 1CB4 , anti-SPE-10 sera , and DAPI staining ( merge ) shows that most , but not all , SPE-10 localizes within FBMOs . 334 : Our analysis indicates that SPE-10 is an integral membrane protein mostly located in the MO . PAPERDELIMITER PMID:16360035 5 : HIM-8 bound chromosome sites associate with the nuclear envelope ( NE ) throughout meiotic prophase . 33 : HIM-8 immunostaining reveals that this locus is also associated with the nuclear envelope ( NE ) during meiotic prophase . 129 : Wild-type gonads stained with these antibodies showed conspicuous chromosome-associated foci from premeiotic stages through late pachytene of both males ( data not shown ) and hermaphrodites ( Figure 3A ) . 143 : HIM-8 is a C2H2 Zinc-Finger Protein that Localizes to Distinct Nuclear Foci during Meiosis 144 : Subnuclear HIM-8 foci are present in all germline nuclei throughout the premeiotic , transition-zone , and pachytene region of the gonad . 153 : Hermaphrodites carrying two copies of mnDp66 , a duplication of the left two megabases of the X chromosome that includes the PC region ( Villeneuve , 1994 ; Colaia covo et al , 2003 ; MacQueen et al , 2002 ) , revealed up to four distinct HIM-8 foci in premeiotic nuclei and two foci at pachytene , indicating that each copy of mnDp66 introduces an extra HIM-8 signal ( Figure 4C ) . 158 : ( Figure 4E and data not shown ) We therefore conclude that HIM-8 associates with the X chromosome in a PC-dependent fashion and that its binding site coincides with the genetically defined PC region . 161 : The HIM-8 Protein Localizes to the PC Region of the X Chromosome All images show projections through fields of pachytene-region nuclei from animals of the indicated genotypes . 194 : HIM-8 Associates with the Nuclear Envelope 206 : We conclude that the prominent X chromosome-associated foci of HIM-8 are located at or very close to the nuclear envelope throughout most of meiotic prophase . 233 : HIM-8 Foci Associate with Nuclear-Envelope Components in Both Wild-Type and him-8 ( me4 ) Animals ( A and B ) Pachytene nuclei were stained with antibodies against HIM-8 ( yellow ) and anti-LMN-1 ( red ) , which marks the nuclear lamina , or nuclear envelope . PAPERDELIMITER PMID:16530049 57 : Endogenous SMK-1 could also be detected in the nuclei of intestinal cells , hypodermal cells , and head and tail neurons by staining with affinity-purified SMK-1 antibodies ( Figure S2 ) . 59 : Importantly , these assays indicated that SMK-1 was temporally and spatially colocalized with active DAF-16 , which is active in transcribing genes when expressed in the nuclei of these cells ( Libina et al , 2003 ) . 243 : In this study , we find that smk-1 is expressed in the nuclei of intestinal cells and in subsets of neurons . PAPERDELIMITER PMID:15890334 3 : We show that SMA-1 , an ortholog of hH-spectrin required for normal morphogenesis , localizes to the apical membrane of epithelial cells when these cells are rapidly elongating . 4 : In spc-1 a-spectrin mutants , SMA-1 localizes to the apical membrane but its organization is altered , consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton ( SBMS ) . 51 : SMA-1 localizes to the apical membrane of elongating epithelial cells , where it is an essential part of the SBMS that stabilizes changes in the membrane skeleton . 107 : SMA-1 localizes to the apical membrane in the hypodermis , gut , pharynx , and excretory canal cell during cell elongation . 116 : SMA-1 is localized to the apical membranes of hypodermal cells ( white arrow ) and gut ( arrowhead ) . 118 : SMA-1 localizes to apical cell membranes of hypodermal ( white arrow ) , gut ( arrowhead ) , and pharynx ( black arrow ) cells , but does not co-localize significantly with AJM-1 ( green ) . 119 : ( H , I ) Cross-section of 3-fold stage embryo , SMA-1 is localized to the apical cell membranes of hypodermal ( white arrow ) and pharynx ( black arrow ) cells . 144 : During early morphogenesis , as the hypodermal cells migrate to enclose the embryo , SMA-1 is present at all epithelial cell boundaries , with some protein in the cytoplasm ( Figs 1B , C ) . 148 : One candidate protein for targeting SMA-1 to the apical membrane is a-spectrin . 255 : We conclude that the N-terminus of SMA-1 is sufficient for localization to the apical membrane of epithelial cells . 298 : SMA-1 localizes to the apical membrane of cells of the pharynx , gut , hypodermis , and excretory canal ( Fig 1 ) during the stages of development when these cells are rapidly elongating . 299 : In the hypodermis , just prior to cell elongation , SMA-1 localizes to the apical membrane simultaneously with the reorganization of actin into apically localized bundles that circumscribe the embryo ( Fig 1 , Priess and Hirsh , 1986 ) . PAPERDELIMITER PMID:15137946 6 : We show that this novel protein localizes to basal bodies in mouse and C elegans , is under the regulatory control of daf-19 , and is necessary for the generation of both cilia and flagella . 182 : In agreement with a conserved role for BBS-5 in basal bodies at the base of cilia , GFP fluorescence was detected in all ciliated sensory neurons including the amphids , labial neurons , phasmids , and the sensory rays of the male tail ( Figures 6A6C and data not shown ) . 205 : The localization to basal bodies was confirmed by colocalization of BBS5 with -tubulin , a centrosomal and basal body marker , and the localization of BBS5 just beneath the cilia as detected by antibodies to acetylated -tubulin . 206 : The domain of BBS5 staining was also found to extend beneath that of -tubulin signal , indicating that BBS5 is also localized to regions surrounding the basal bodies . PAPERDELIMITER PMID:15936327 11 : The intracellular domain of the long form was detected at the cell membrane and in the nucleus . 205 : The long cytoplasmic domain of Ten-1 can be detected in nuclei We raised antibodies against both the N ( anti-N ) and the C-terminal peptides ( anti-C ) of the mature long form of the Ten-1 protein . 210 : Anti-C stained all membranes while anti-N stained in addition to membranes also the nuclei of Ten-1-expressing cells . 211 : Within the nucleus , the Ten-1 staining was punctuate ( Fig 6I ) . 212 : The nuclear staining was particularly prevalent in cells lining the gut where anti-C stained the cell membranes and anti-N stained predominantly the nuclei ( Figs 6G and H ) . 218 : Also in this case , the nuclear staining was punctuate , in contrast to that seen following the expression of a GFP containing a standard nuclear localization signal resulting in homogeneous nuclear staining ( Fig 6M ) . 266 : The most interesting finding was the discovery that the intracellular domain of the long form of Ten-1 can be detected in cell nuclei . PAPERDELIMITER PMID:16236031 1 : Regulatory machinery of UNC-33 Ce-CRMP localization in neurites during neuronal development in Caenorhabditis elegans 7 : In this study , to understand the regulatory mechanisms of localization of UNC-33 in neurites , we screened for the mutants that were involved in the localization of UNC-33 , and identified three mutants : unc14 ( RUN domain protein ) , unc-51 ( ULK kinase ) and unc-116 ( kinesin heavy chain ) . 11 : suggest that the UNC-14UNC-51 complex and Kinesin-1 are involved in the localization of UNC-33 in neurites . 39 : In this study , to examine the regulatory mechanism of UNC-33 localization in neurites , we identified the molecules that were essential for the intracellular localization of UNC-33 and examined the interactions between UNC-33 and these molecules . 200 : Although UNC-33LMS was accumulated at the ventral nerve cord and nerve ring ( Figs 4ad , Table 1 ) , UNC-33LMSMut was mislocalized to cell bodies ( Figs 4eh , Table 1 ) , which indicates that the molecular lesion of UNC-33 caused by the e204 mutation contributed to the regulation of UNC-33 localization in neurites . 202 : suggest that the localization of UNC-33 in neurites is important for neuronal morphogenesis and functions . 206 : These observations prompted us to examine the regulatory mechanisms of the localization of UNC-33 in neurites . 271 : This result suggests that the dimer formation between UNC-33 proteins is required for the localization of UNC-33 in neurites and the neuronal development . 275 : Molecular mechanism of UNC-33 accumulation in neurites PAPERDELIMITER PMID:16319925 106 : Under normal growth conditions , germline nuclei from wild-type ( N2 ) animals are devoid of any detectable ATL-1 staining ( Figure 3B1 ) . 107 : In contrast , ATL-1 is detected in multiple nuclear foci in mitotic germline nuclei arrested in S-phase following HU-treatment , but is absent in nuclei at all stages of meiotic prophase ( Figure 3B2 ; data not shown ) and is dramatically reduced in atl-1 ( tm853 ) mutants or in animals subjected to atl-1 RNAi depletion ( Figure 3B3 and 4 ) . 125 : ( A ) ATL-1 staining of fixed germline nuclei from N2 ( Wt ) animals before and after 1h post-IR-treatment ( 75 Gy ) and in animals of the indicated genotypes 1h after IR-treatment ( 75 Gy ) . 126 : ( B ) RPA-1 staining of fixed mitotic nuclei for the indicated genotypes 1 h after IR-treatment ( 75Gy ) . 127 : ( C ) ATL-1 staining of fixed diplotene nuclei during meiotic prophase for the indicated genotypes before and 1h after IR-treatment ( 75 Gy ) . 146 : ATL-1 nuclear foci are elevated in number in the mitotic zone of these mutants following IR ( Supplementary Figure S4A and B ) . PAPERDELIMITER PMID:16501168 32 : SOR1 and SOP-2 colocalize in SOP-2 nuclear bodies and direct interact with each other . 205 : SOR-1 colocalizes to the same nuclear bodies as SOP-2 213 : SOR-1 is nuclear localized 231 : From the two cell-stage onwards , SOR-1 is found to be inhomogenously expressed in the nuclei with obvious accumulations in distinct nuclear speckles ( Fig 4B ) . 234 : The localization of SOR-1 in nuclear bodies lead us to investigate whether it is colocalized with SOP-2 , a C elegans PcG protein that is also localized in nuclear bodies , termed SOP-2 bodies ( Zhang et al , 2003 ) . 237 : SOP-2 is required for the localization of SOR-1 into nuclear bodies 249 : Colocalization of SOR-1 and SOP-2 in nuclear bodies . 254 : SOR-1 is localized in distinct nuclear bodies ( arrow ) . 318 : Both GFP reporters and antibody staining indicate that SOR-1 and SOP-2 are colocalized in nuclear bodies and the localization of SOR-1 depends on SOP-2 . 355 : First , components of the PRC1 complex and the SOP-2 SOR-1 complex are localized into distinct nuclear bodies , although the mechanistic role of the bodies in Hox gene repression remains unknown . PAPERDELIMITER PMID:16401427 3 : Here , we report that the nematode STAT ortholog STA-1 accumulated in the nuclei of five head neuron pairs , three of which are amphid neurons involved in dauer formation [ 1 , 4 ] . 19 : Immunohistochemical localization using a STA-1-specific antibody readily detected STA-1 protein in pharynx , head ganglia , tail ganglia ( Figures 1A and 1B ) , ventral nerve cord ( Figure 1C ) , embryos ( Figure 1E ) , and body muscles ( data not shown ) , with a diffuse staining pattern indicative of a largely cytoplasmic localization . 21 : In contrast , detailed analysis of immunostaining patterns in the head ganglia revealed a distinct pattern of nuclear STA-1 accumulation in a few neuronal cells ( Figure 1B ) , indicative of activation . 22 : Confocal immunostaining analysis of head ganglia identified precise nuclear accumulation of STA-1 in select subsets of neurons ( Figures 1F1H ) . 24 : Therefore , it was possible to determine the identities of cell bodies within the head ganglia that displayed selective STA-1 nuclear staining by comparison to the head ganglion cell map [ 7 ] . 26 : Based on their relative positions ( Figures 1F1H ) , cell bodies displaying nuclear accumulation of STA-1 were identified as five pairs of neurons : ADL , ASK , ASJ , ASH , and AIZ , four of which are amphid neurons . 27 : This pattern of STA-1 nuclear staining was essentially invariant in all worms examined . 30 : Because nuclear accumulation of STAT proteins is indicative of activation [ 3 ] , STA-1 protein nuclear accumulation in these five pairs of neurons under standard laboratory culture conditions suggests involvement in a constitutive neuronal sensory function . 46 : N2 wild-type worms and Daf-defective daf6 ( e1377 ) mutants showed the same nuclear staining pattern in the limited set of amphid neurons described previously ( Figure 2A and data not shown ) . PAPERDELIMITER PMID:11076762 234 : All nine Rn cells have nuclear HLH2 staining . PAPERDELIMITER PMID:16890160 36 : ( D ) The endogenous NHR-25 protein is detected in the nuclei of Z1 and Z4 ( arrows ) but not in the Z2Z3 germline precursors ( arrowheads ) . 58 : The NHR-25 protein can be detected in the nuclei of Z1 and Z4 ( Figure 1D ) as well as in the nuclei of their daughter cells ( Figure 1E ) , but not in the Z2 and Z3 precursors of the germline . PAPERDELIMITER PMID:15282161 219 : In addition , while HLH-2 accumulation in the DTCs is nuclear , as expected for a transcription factor , HLH-2 is detectable in the cytoplasm as well as the nucleus of other gonadal cells ( eg , Figs 5IK ) . 205 : lag-2DlacZ expression ( anti-LacZ : blue ) marks the four daughters of Z1 and Z4 ( and nongonadal cells ) , propidium iodide ( red ) marks all nuclei , and HLH-2 , if present , would be detected using anti-HLH-2 as green and nuclear staining . 212 : In this individual , HLH-2 is evident in the nuclei of Z1 . aa and Z4 . pp ( out of focus ) , and in the cytoplasm as well as the nuclei of Z1 . pp , Z4 . aa ( the parents of Z1 . ppp and Z4 . aaa ) , and Z4 . ap . PAPERDELIMITER PMID:2922060 1 : The Caenorhabditis elegans heterochronic gene 1in-14 encodes a nuclear protein that forms a temporal developmental switch. During wild-type development , a protein product of the Caenorhabditis elegans heterochronic gene lin-14 is localized to nuclei of specific somatic cells in embryos and early larvae , but is absent in late larvae and adult soma. 3 : The normal downregulation of the lin-14 nuclear protein level encodes a temporal switch between early and late cell fates. 11 : We find that the lin-14 protein is localized in the nucleus and that its progressive elimination during C elegans development controls the normal temporal sequence of cell fates mediated by the lin-14 gene . 12 : Lin 14 protein is nuclear and temporally regulated 20 : We used the affinity-purified anti-lin-14 antibodies to detect the lin-14 protein in whole-mount fixed specimens of wild-type C elegans by indirect immunofluorescence staining , and found that the anti-lin-14 antibodies bound antigen in specific somatic nuclei of late embryos and early larvae ( Fig 2 ) . 22 : Because both the 70K protein detected on the immunoblots and the somatic nuclear lin-14 staining disappear in these lin-14 mutant strains , the somatic nuclear staining probably corresponds to this 70K lin-14 protein . 48 : At this stage , the most intense staining was in intestinal and hypodermal nuclei ( Fig 2a ) . 50 : The most intense staining that we observed at any stage was in nuclei of late embryos just before hatching , and in newly hatched L1 animals. 51 : Significantly , only nuclear staining was observed at these stages ( Fig 2b , c ) . 52 : In the newly hatched L1 animal , the lin-14 protein was present in the nuclei of most of the post-embryonic blast cells ; intense nuclear staining was observed in the hypodermal blast cells Hl , H2 , VI-V6 , and T ( Figs 2c and 3 ) and in all of the intestinal ( E ) cells ( Fig 2b ) , and weaker staining was observed in both neuroblasts Q1 and Q2 , in the mesoblast M cell , and in the P cells . 55 : The nuclei of these progeny cells also stain with the anti-lin-14 antibody during the mid-L1 stage . 57 : The lin-14 staining in the P-cell nuclei fades before their migration into the ventral cord but reappears later in some of their progeny cells ( see below ) . 62 : The nuclei of many cells that do not divide post-embryonically also accumulate the lin-14 protein during the L1 stage . 63 : The embryo-derived nuclei in the hypodermal syncytial cell hyp7 , ABarpppapa , ABplaapppp , Cpaaaa , Cpaapa , Cpaapp , Cpapaa , all accumulate levels of the lin-14 protein similar to those of the hypodermal blast cells ( Figs 2c and 3 ) . 64 : Terminally differentiated nuclei from embryonic body muscle also accumulate the lin-14 protein . 65 : Nuclei of many but not all neuronal cells stain with the anti-lin-14 antibodies . 67 : All of the embryonically generated ventral-cord neurons , and some but not all of the neurons of the nerve ring and posterior ganglion accumulate the lin-14 protein in their nuclei during the Li stage ( Fig 2c , d , e ) . 70 : Late in the L1 stage , the lin-14 protein staining of all nuclei except the neuronal nuclei is much weaker . 75 : The disappearance of the lin-14 protein during the L1 stage from the nuclei of the post-embryonic blast cells correlates well with the mid to late L1 temperaturesensitive period for fin-14 mutations . 81 : In occasional L2 and L3 stage animals , weak lin-14 protein staining in hypodermal , neuronal , and intestinal cells was observed in nuclei and cytoplasm , as if it was being slowly degraded or not efficiently transported to the nucleus . 82 : Patches of lin-14 staining in hypodermal or intestinal nuclei was only rarely observed in very old adults ( data not shown ) . 84 : In most adults , lin-14 immunostaining reappears only in the mature oocyte nuclei of hermaphrodites ( Fig 2h ) , at meiotic prophase I when the chromosomes are condensed as shown by DAPI staining ( Fig 2i ) . 87 : The oocyte lin-14 nuclear staining disappears after fertilization ; presumably it is either degraded or greatly diluted during nuclear division . 90 : The embryonic and L1 stage staining in these mutants was equivalent to that seen in the wild-type in its intensity , nuclear localization , and cellular distribution ( data not shown ) . PAPERDELIMITER PMID:8144590 109 : Transfection with this shortened cDNA led to the production of an active enzymesecreted into the culture medium . Five days postinfection approximately half of the AChE activity was found in the medium and half in the cell pellet . PAPERDELIMITER PMID:17084364 30 : In whole-mount indirect immunofluorescence experiments , the affinity-purified antibody detected nuclear-localized antigen in many it issues of adult tra-1 ( + ) hermaphrodites ( Figures 1B1E ; see also the Supplemental Data available with this article online ) . 32 : Wild-type males exhibited far weaker TRA-1 immunofluorescence than their hermaphrodite siblings ( Figures 1H and 1I ) ; however , when TRA-1 was detectable in males , it was primarily nuclear . 33 : Therefore , TRA-1 proteins appear predominantly nuclear in both sexes and are present at much higher levels in hermaphrodites than in males . 87 : They differed in that XX animals expressing N-terminally tagged GFP : : TRA-1 exhibited nuclear fluorescence , whereas those expressing Cterminally tagged TRA-1 : : GFP fluoresced only weakly ( Figures 3C3F ) . PAPERDELIMITER PMID:17218259 175 : Consistent with this idea , TPXL-1 amounts at centrosomes were substantially reduced after depletion of RSA-1 ( Figure 6A ) . 176 : The decrease in TPXL-1 at the centrosome did not result from the reduced microtubule number , as it was also observed when microtubule amounts were restored to wild-type levels by codepletion of KLP-7 and RSA-1 ( Figures 6A and 6B and Movie S10 ) . 181 : Based on these results , we conclude that the RSA complex contributes to mitotic spindle assembly , at least in part by targeting TPXL-1 to centrosomes , likely via a direct physical interaction . 198 : did not alter the targeting of LET-92 or TPXL-1 to centrosomes ( our unpublished observations ) . 218 : Regulation of Spindle Formation by Targeting of the C elegans Ortholog of TPX2 The most direct evidence we have found for how the RSAPP2A complex regulates spindle assembly is through physical interaction with and targeting of TPXL-1 to centrosomes ( Figures 6A6C ) . 239 : RSA-1 Is Required for the Centrosomal Localization of TPXL-1 ( A ) Shown are still images taken from time-lapse series of wild-type , rsa-1 ( RNAi ) , and rsa-1 ( RNAi ) ; klp-7 ( RNAi ) double depletion embryos expressing TPXL-1 : : GFP . 247 : ( B ) Quantification of centrosomal TPXL-1 : : GFP amounts from single frames of time-lapse recordings as shown in ( A ) . 254 : Recruitment of TPXL-1 to centrosomes by the RSA-PP2A complex is required for stabilization of kinetochore microtubules ( gray ) and thereby spindle stability ( right branch ) . PAPERDELIMITER PMID:16774992 225 : pig-1 : : gfp is present throughout the cytoplasm and excluded from the nucleus . 231 : In all cells , the fusion protein was localized to the cytoplasm and excluded from nuclei ( Fig 6G , H ) . PAPERDELIMITER PMID:17576746 63 : SUT-1 is present both in the nuclear and cytoplasmic compartments , with nuclear staining being prominent . 64 : SUT-1 protein exhibited nucleo-cytoplasmic expression from mid embryogenesis to adulthood . 144 : On the other hand , SUT-1 has primarily a nuclear localization though some cytoplasmic staining is also in evidence ( Fig 2E-J ) . 151 : Likewise , SUT-1 may also have both a cytoplasmic function when bound to UNC-34 and an as yet unknown nuclear function ( Fig 7 ) . 155 : The observation that most of SUT-1 is located in the nucleus suggests SUT-1 could shuttle between cytoplasmic UNC-34 and the nucleus to regulate transcription in response to UNC-34-dependent processes . PAPERDELIMITER PMID:15371340 42 : Consistent with the phenotypic analysis , endogenous KNL-3 and a GFPKNL-3 fusion protein expressed in the germ line by stable integration both localized to the diffuse mitotic kinetochores of C elegans ( Fig 1C ; Supplementary Video 10 ) . 43 : KNL-3 staining overlaps with the bona fide kinetochore component CENP-CHCP-4 ( Fig 1D ) . 44 : Like CENP-CHCP-4 and KNL-1 , KNL-3 is first detected on chromosomes during prophase and persists on chromosomes until the end of mitosis . 45 : Kinetochore targeting of KNL-3 requires CENP-CHCP-4 , but not vice versa , as was found previously for KNL-1 ( Fig 1D ; Desai et al 2003 ) . 54 : KNL-3 is a kinetochore protein whose depletion results in a kinetochore-null phenotype . 64 : ( C ) KNL-3 localizes to the kinetochore throughout mitosis . 226 : 2262 GENES & DEVELOPMENT A conserved kinetochore protein network We observed a severe reduction in KNL-3 at kinetochores in KBP-2-depleted embryos relative to similarstage wild-type embryos ( Fig 7A ) . 232 : GFPKNL-3 is clearly visible on kinetochores prior to NEBD in control embryos ( Fig 7C ; Supplemental Video 10 ) . 244 : In contrast , KNL-3 persisted at kinetochores in KNL-1-depleted embryos , albeit at a reduced level relative to wild type ( Fig 8A , B ; Supplementary Videos 10 , 24 ) . PAPERDELIMITER PMID:15814591 170 : As was true for the abts-4 transcriptional fusion , the abts-4 translational fusion was expressed exclusively in the intestine , where it was localized to the basolateral membrane of the gut cells ( Fig 4E ) . 192 : The ABTS-4 : : GFP fusion was expressed on the basolateral membrane of the midgut , most strongly in the posterior intestinal cells ( E and F ) . PAPERDELIMITER PMID:16207815 4 : Here we show that Caenorhabditis elegans Dcp2 is localized to distinct foci during embryogenesis , reminiscent of P-bodies , the sites of mRNA degradation in yeast and mammals . 5 : However the decapping enzyme of the 3 to 5 transcript decay system ( DcpS ) localizes throughout the cytoplasm , suggesting this degradation pathway is not highly organized . 6 : In addition we find that Dcp2 is localized to P-granules , showing that Dcp2 is stored and/or active in these structures . 48 : We find that Dcp2 localizes to foci in the somatic and germ-line precursor cells ( P-cells ) . 49 : Colocalization with GLH-1 and other markers suggests that in addition to these small cytoplasmic foci , Dcp2 is found in P-granules . 51 : In contrast we find that the decapping enzyme of the 3 to 5 mRNA decay pathway , DcpS , is found throughout the cytoplasm , indicating that this mRNA decay pathway is apparently not organized at foci . 73 : C elegans Dcp2 is found in cytoplasmic bodies from early embryogenesis . 83 : Cytoplasmic Dcp2 foci are seen in both the somatic cells and the germ line lineage in the 2-cell ( second row ) , and 8-cell ( third row ) embryo . 84 : The fourth row shows a closer view of punctate Dcp2 staining in an embryonic cell in C The intensity of foci typically appears to increase during the initial divisions in development up to the 8-cell stage , with strong labeling of somatic foci then observable throughout embryogenesis . 86 : ( D ) CGH-1 and Dcp2 completely colocalize in cytoplasmic foci in early embryos , with CGH-1 becoming difficult to detect subsequently in development . 111 : RESULTS C elegans Dcp2 Is Enriched in Cytoplasmic Bodies To test the hypothesis that decapping enzymes are found in cytoplasmic foci during metazoan development , we raised polyclonal antibodies to Dcp2 . 116 : Immunostaining of C elegans embryos revealed that Dcp2 protein was found enriched in particles in the embryonic cytoplasm ( Figure 1 ) , and these particles disappear in embryos depleted of Dcp2 using RNAi ( see below and Figure 1B ) . 119 : Though present around nuclei in the germ cells of the gonad , cytoplasmic ( nonperinuclear ) Dcp2 foci were first obvious after fertilization and were present by the time the two pronuclei meet and fuse ( Figure 1C ) . 120 : By the two-cell stage , Dcp2 bodies were observed throughout the cytoplasm of both the somatic and germ line lineage , though they often appear stronger in the P-cell than the somatic cell at this early point in development ( Figure 1C ) . 121 : From the four-cell stage onward , many Dcp2 foci were observed in the cytoplasm of all cells . 122 : Dcp2 staining was faint Molecular Biology of the Cell 5882 Decapping Proteins and C elegans Development in nuclei . 123 : Because it has previously been noted that CGH-1 ( the C elegans ortholog of S cerevisiae Dhh1 , an enhancer of decapping ) is found in granules throughout the soma during early development , we tested whether CGH-1 and Dcp2 colocalize . 124 : We find that somatic CGH-1 foci are coincident with Dcp2 , indicating that Dcp2 particles also contain a second homolog of a protein found in S cerevisiae P-bodies ( Figure 1D ) . 125 : In contrast to previous observations showing that CGH-1 somatic foci disappear after the first few embryonic divisions , we observed that cytoplasmic Dcp2 foci persist throughout embryonic development . 126 : C elegans Dcp2 Is Found in P-Granules in a pgl-1dependent Manner P-bodies have been previously compared with C elegans P-granules because they both contain proteins associated with RNA regulation . 130 : We observed by immunostaining that the germ line precursor cells contained much larger sites of Dcp2 localization that resembled Pgranules in shape . 132 : Such immunostaining indicates that Dcp2 is enriched in P-granules ( Figure 2A ) . 133 : Dcp2 is also present at sites where P-granules are observed in C elegans germ cells during oogenesis ( see Figure 5A ) . 135 : Although Dcp2 is still observed in some P-granules in the pgl-1 ( ct131 , bn101 , and bn102 ) mutants at the restrictive temperature , this localization is reduced when compared with wild-type embryos ( Figure 2B ) . 136 : In contrast GLH-1 continued to be associated with P-granules in the pgl-1 ( ct131 as well as bn101 , and bn102 ) mutants , consistent with previous observations in gonads ( Figure 2B ; Kawasaki et al , 2004 ) . 138 : This is not the case in glh-1 ( ok439 as well as gk100 ) mutants , where Dcp2 immunolabeling in the P cell appears relatively normal ( unpublished data ) . 140 : In summary , Dcp2 is found in P-granules , showing that a component of the mRNA degradation machinery localizes to these sites in a manner that is compromised in pgl-1 mutants . 141 : C elegans DcpS Is Found throughout the Cytoplasm In both mammalian cells and yeast , there are two major and general pathways by which mRNA degradation occurs . 143 : Staining with multiple markers indicates that Dcp2 colocalizes with P-granules . 145 : Dcp2 is seen to colocalize with these markers in P-granules . 146 : ( B ) Dcp2 localization to P-granules is diminished in pgl-1 ( bn101 ) mutants . 149 : Although wild-type ( N2 ) embryo P-granules ( marked by GLH-1 ) contain Dcp2 , the levels of Dcp2 are diminished in P-granules of pgl-1 mutants ( bottom panels ) . 155 : By immunostaining C elegans embryos we found that there was a broad distribution of DcpS throughout the cytoplasm of all lineages ( Figure 3B ) . 157 : DcpS was not generally observed in cytoplasmic bodies , although we infrequently observed a region of more intense DcpS 5883 S Lall et al Figure 3 . 158 : C elegans DcpS is found throughout the cytoplasm during embryogenesis . 160 : ( B ) Immunolocalization of DcpS in embryos shows signal throughout the cytoplasm just after fertilization ( top row ) , with some intense staining observed in the majority of embryos around the male pronucleus . 161 : In older embryos , DcpS is observed throughout the cytoplasm , although sites containing higher levels of signal are observed , albeit infrequently ( arrows ) . 165 : Though it has previously been shown that mammalian DcpS is found in the nucleus , our observation of significant cytoplasmic DcpS in C elegans is consistent with previous biochemical evidence from the nematode A suum . 166 : In particular , the predominantly cytoplasmic location of DcpS is consistent with measurements of DcpS decapping activity in subcellular fractionation experiments using nematode A suum embryo extracts ( Cohen et al , 2004 ) . 167 : Our immunostaining allows us to conclude that the bulk of DcpS signal is observed cytoplasmically , a situation consistent with S cerevisiae ( Malys et al , 2004 ) and that enzymes involved in mRNA cap metabolism are not all enriched in cytoplasmic bodies . 168 : Therefore although some DcpS may be present in C elegans nuclei , we do not observe the predominantly nuclear localization suggested in other systems ( Salehi et al , 2002 ; Cougot et al , 2004a ; Liu et al , 2004 ) . 192 : Although we found that Dcp2 is localized to P-granules , the progeny of dcp2 RNAi-treated mothers did not show sterility ( unpublished data ) , showing that depletion of Dcp2 alone from the P-granules does not affect germ line development . 244 : The human CBP-20 antibody recognizes a nuclear enriched protein in C elegans ( Figure 6A ) . 245 : In C elegans embryos staining of CBP-20 is strikingly strong at the nuclear envelope ( arrow , Figure 6A ) , though some diffuse as well as punctate cytoplasmic staining is also observed . 259 : ( A ) Anti-CBP-20 immunostaining reveals localization at the nuclear envelope ( staining at nuclear periphery indicated by arrow ) . 261 : ( C ) Immunostaining with a cross-reactive antibody raised to A suum ( nematode ) eIFE-3 shows general cytoplasmic staining in C elegans embryos , rather than overt staining of cytoplasmic foci . 262 : The arrow indicates P-granule staining by the anti-eIF4E antisera . 266 : The peripheral nuclear staining is consistent with CBP-20 accompanying newly synthesized mRNAs to at least the nuclear envelope . 269 : We did not observe obvious somatic cytoplasmic foci with this antibody ( Figure 6C ) . 270 : Although we can not eliminate the possibility that this cross-reactive antisera is recognizing a generally localized eIF4E isoform that is masking localization of one isoform in cytoplasmic foci , these data do show that the C elegans eIF4E proteins are not entirely localized to cytoplasmic foci . 271 : In addition , we observe signal in P-granules ( Figure 6C , arrowhead ) . 272 : This localization is expected of at least the eIF4E isoform IFE-1 ( Amiri et al , 2001 ) . 273 : Hence although C elegans eIF4E signal is observed in P-granules , very general cytoplasmic localization is also observed in all embryos examined . 274 : Thus neither eIF4E nor CBP-20 are observed to be localized to foci to the degree that Dcp2 is . 275 : DISCUSSION We have shown that Dcp2 localizes to distinct cytoplasmic foci during early C elegans embryogenesis . 276 : In addition Dcp2 is also found in P-granules , colocalizing with the P-granule markers GLH-1 and the antigens recognized by the K76 and OIC1D4 antisera . 277 : In contrast the decapping enzyme of the 3 to 5 decay pathway , DcpS is more generally localized throughout the cytoplasm . 282 : Dcp2 foci are obvious around the nuclei of oocytes , but become difficult to detect in late oogenesis . 283 : Although it is possible that these bodies are sites of Dcp2 storage , strong labeling in the cytoplasm shortly after the onset of embryonic development , as well as somatic foci becoming obvious around the onset of zygotic transcription and at a time when there is turnover of maternal transcripts , suggests that Dcp2 foci may be functional sites . 285 : Because cytoplasmic Dcp2 foci become very evident at the four-cell stage when there is some embryonic transcription we favor the hypothesis that Dcp2 foci are active sites of decay rather than storage sites ( Edgar et al , 1994 ) . 287 : Although we do not know where P-bodies would sediment during such purification , the 130S fraction is where polysomes are found , suggesting that Dcp2 activity is enriched with multisubunit machinery . 288 : This suggests that in nematodes , Dcp2-like activity is strongest in a fraction that may possibly contain structures such as degradation foci . 302 : Although DcpS has previously been detected in the nucleus of yeast and mammalian cells ( Salehi et al , 2002 ; Cougot et al , 2004a ; Liu et al , 2004 ) and Dcp2 can decap trimethylated capped RNAs across species ( Cohen et al , 2005 ) and might be predicted to be involved in decapping snRNAs , the bulk of the immunostaining we observe is cytoplasmic . 304 : The fact that a large percentage of C elegans mRNAs are trimethylated in addition to snRNAs and some snoRNAs might explain why the vast majority of detectable decapping protein is observed in the cytoplasm in C elegans and does not exclude the possibility that functional levels of nuclear Dcp2 and DcpS are present , but difficult to detect over cytoplasmic signal Localization of Dcp2 to P-Granules Our data indicates that Dcp2 protein is also found in C elegans P-granules . 308 : The notion that there is active mRNA turnover occurring in P-granules might be strengthened by the observation that the C elegans ortholog of Dhh1 , an enhancer of S cerevisiae Dcp1/Dcp2 decapping , has been localized to P-granules ( Navarro et al , 2001 ) . 312 : Alternatively , it may be that Dcp2 localized to Pgranules is sequestered and inactive , promoting the stability of some mRNAs in the P-cell . 324 : Thus although CGH-1 is localized to the same cytoplasmic bodies as Dcp2 , it may have a distinct role . 326 : If , for example , CGH-1 was involved in translational control , it may appear to be localized to P-bodies and act as a promoter of decapping in yeast , even if it does not participate directly in mRNA decay . 330 : This would explain why the phenotypes of cgh-1 and dcp2 are so distinct and also why CGH-1 has a distinct temporal pattern of cytoplasmic localization in the C elegans embryo , appearing to leave Dcp2enriched foci during early development . PAPERDELIMITER PMID:16839187 5 : eak-6 and eak-5/sdf-9 encode protein phosphatase homologs ; eak-4 encodes a novel protein with an Nmyristoylation signal All three genes are expressed primarily in the two endocrine XXX cells , and their predicted gene products localize to the plasma membrane . 158 : In both strains containing GFP and RFP reporter constructs , GFP and RFP colocalized ( Figure 4A ) , indicating that eak-4 , sdf-9 , and eak-6 are all expressed in XXXL/R EAK-4 , SDF-9 , and EAK-6 : : GFP Fusion Proteins Localize to the Plasma Membrane In order to visualize the subcellular localization of EAK proteins , we made full-length translational GFP fusion constructs and expressed them in wild-type animals . 161 : Coexpression of translational GFP fusions with an sdf9p : : RFP promoter fusion indicated that all three GFP fusions localize to the plasma membrane of the XXX cells ( Figure 4B ) . 162 : Membrane localization was also apparent in intestinal cells ( unpublished data ) . 163 : We determined the role of the N-myristoylation consensus motif in EAK-4 plasma membrane localization by constructing an EAK-4 : : GFP mutant in which the invariant glycine residue at position 2 is mutated to alanine ( G2A ) . 164 : In contrast to wild-type EAK-4 : : GFP , which was localized to the plasma membrane ( Figure 4B and 4C ) , the EAK-4 G2A mutant GFP fusion protein exhibited diffuse cytoplasmic localization ( Figure 4C ) , indicating that an intact N-myristoylation motif is required for EAK-4 plasma membrane localization . 165 : To gain insight into the influence of DAF-2/InsR signaling on EAK plasma membrane localization , we examined SDF9 : : GFP subcellular localization in daf-2 ( e1370 ) mutant animals grown at 25 8C . 167 : SDF-9 : : GFP exhibited plasma membrane localization in both wild-type and daf2 ( e1370 ) animals ( Figure S6 ) , indicating that its localization does not require normal levels of DAF-2/InsR signaling . 169 : EAK-4 , SDF-9 , and EAK-6 Localize to the Plasma Membrane of the XXX Cells ( A ) eak-4 , sdf-9 , and eak-6 promoters drive expression in the same cells . 173 : ( B ) EAK-4 : : GFP , SDF-9 : : GFP , and EAK-6 : : GFP fusion proteins localize to the plasma membrane of XXX . 176 : ( C ) Mutation of the invariant glycine in the N-myristoylation motif of EAK-4 abrogates plasma membrane localization . 229 : Mechanisms of SDF-9 and EAK-6 Membrane Localization In contrast to EAK-4 , which likely localizes to the plasma membrane through N-myristoylation ( Figure 4C ) , the mechanisms underlying the membrane localization of SDF-9 and EAK-6 are not clear . 231 : SDF-9 and EAK-6 could associate with the plasma membrane by binding to membrane-associated proteins such PLoS Genetics | www . plosgenetics . org Figure 6 . 243 : The observation that SDF-9 : : GFP exhibits plasma membrane localization in the absence of intact DAF-2/InsR signaling ( Figure S6 ) is consistent with the lack of SDF-9 binding to tyrosine phosphoproteins in 293T cells and suggests that SDF-9 membrane association is independent of tyrosine phosphorylation . 284 : A Model for EAK Protein Function The data presented in this work are consistent with a model whereby EAK-4 , SDF-9 , and EAK-6 function in a single complex or pathway at the XXX plasma membrane in parallel with AKT-1 to inhibit dauer formation . PAPERDELIMITER PMID:16790495 1 : Molecular Biology of the Cell Vol 17 , 38323847 , September 2006 00027703 Caenorhabditis elegans UNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments D Kristina B Mercer , * Rachel K Miller , * Tina L Tinley , * Seema Sheth , * Hiroshi Qadota , * and Guy M Benian* *Department of Pathology and Graduate Division of Biological and Biomedical Sciences , Emory University , Atlanta , GA 30322 Submitted February 17 , 2006 ; Revised May 11 , 2006 ; Accepted June 9 , 2006 Monitoring Editor : Thomas Pollard To gain further insight into the molecular architecture , assembly , and maintenance of the sarcomere , we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans . 5 : Antibodies generated to UNC-96 localize the protein to the M-line , a region of the sarcomere in which thick filaments are cross-linked . 7 : Additionally , UNC-96 copurifies with native thick filaments . 58 : Specifically , we have found that UNC-96 localizes to the M-line where it is likely to play an important role in A-band integrity . 59 : This result is supported by data revealing that UNC-96 interacts with UNC-98 ( another M-line component ) and paramyosin ( a thick filament component ) and is present in a preparation of purified thick filaments . 60 : We present a model in which UNC-96 is required in adult muscle and acts as both an M-line structural component and as a facilitator of thick filament assembly and/or turnover , through its association with unincorporated paramyosin and UNC-98 . 305 : As shown in Figure 7A , in body wall muscle , UNC-96 is located in M-lines , identified by use of antibodies to the M-line marker UNC-89 . 307 : In such single sarcomere muscles , UNC-96 is found in the middle of the A-bands , a location similar to that found in body wall muscles . 315 : In the single sarcomere muscles , UNC-96 : : GFP is located in the middle of A-bands . 316 : In body wall muscle , UNC-96 : : GFP is specifically located at M-lines , and in addition , at dense bodies . 318 : UNC-96 Is Expressed in Embryonic Body Wall and Pharyngeal Muscle Embryos were costained with anti-UNC-96 and anti-MHC A As shown in Figure 8 , UNC-96 is first detectable at the 1 . 5-fold stage , in which UNC-96 , like MHC A , is diffusely localized in the cytoplasm of body wall muscle cells . 323 : UNC-96 resides in M-lines , but it does not seem to completely colocalize with UNC-89 . 329 : Embryos were costained with antiUNC-96 and anti-MHC A As shown in the top panel , UNC-96 is first detectable at the 1 . 5-fold embryonic stage , in which UNC-96 , like myosin chain A , is localized to the cytoplasm of body wall muscle cells . 405 : The top panel shows the localization of UNC-96 to M-lines in wild-type muscle . 424 : UNC-96 copurifies with nematode thick filaments . 428 : Notice that UNC-96 copurifies with thick filaments in fraction F14 . 434 : Given myosin and paramyosin are the primary components of thick filaments , this indicates that UNC-96 copurifies with thick filaments . 454 : UNC-96 Copurifies with Nematode Thick Filaments Significant mislocalization of paramyosin in unc-96 mutants , and the interaction between UNC-96 and paramyosin , prompted us to investigate the possible association of UNC-96 with thick filaments . 457 : As shown in Figure 12B , UNC-96 occurs in the 15K pellet containing thick filaments and low levels of thin filaments , ribosomes , and nuclear fragments . 458 : This indicates that UNC-96 may be associated with thick filaments . 459 : Further purification of these thick filaments by sucrose gradient sedimentation of the 5K supernatant material shows that UNC-96 follows known markers of the thick filament , namely , myosin heavy chains and paramyosin ( Figure 12C ) . 471 : Antibodies generated to UNC-96 show that the protein is located at the M-lines in adult body wall muscle and in the middle of A-bands in the single sarcomere pharyngeal and anal depressor muscles . 473 : However , in body wall muscle , in addition to being localized to M-lines , the GFP fusion protein is also located at dense bodies . 477 : Another possibility is that indeed UNC-96 is normally present in dense bodies but at such a low concentration that it is undetectable by immunofluorescence . 480 : Despite some similarities in localization to UNC-98 ( Mercer et al , 2003 ) and UNC-97 ( Hobert et al , 1999 ) , we found no antibody or GFP fusion protein evidence for UNC-96 being located in muscle cell nuclei . 481 : Given that we have localized UNC-96 to the M-line region at the light microscope level and that EM images of vertebrate sarcomeres show that the M-line is a region in which thick filaments are cross-linked by M-bridges ( Knappeis and Carlsen , 1968 ; Luther and Squire , 1978 ) , it is not surprising that UNC-96 copurifies with thick filaments . 488 : We propose that UNC-96 normally has two locations within muscle cells : some of it is a fixed component of the M-line ( which may be associated with paramyosin ) , and some of it is associated with paramyosin that has either dissociated from , or has not yet been incorporated into , thick filaments . PAPERDELIMITER PMID:12808046 1 : Molecular Biology of the Cell Vol 14 , 24922507 , June 2003 Caenorhabditis elegans UNC-98 , a C2H2 Zn Finger Protein , Is a Novel Partner of UNC-97/PINCH in Muscle Adhesion Complexes D Kristina B Mercer , * Denise B Flaherty , * Rachel K Miller , * Hiroshi Qadota , Tina L Tinley , * Donald G Moerman , and Guy M Benian* *Department of Pathology , Emory University , Atlanta , Georgia 30322 ; Graduate Division of Biological and Biomedical Sciences , Emory University , Atlanta , Georgia 30322 ; and Department of Zoology , University of British Columbia , Vancouver , British Columbia V6T1Z4 , Canada Submitted October 22 , 2002 ; Revised January 29 , 2003 ; Accepted February 26 , 2003 Monitoring Editor : Mary C Beckerle To further understand the assembly and maintenance of the muscle contractile apparatus , we have identified a new protein , UNC-98 , in the muscle of Caenorhabditis elegans . 6 : By use of UNC-98 antibodies and green fluorescent protein fusions ( to full-length UNC-98 and UNC-98 fragments ) , we have shown that UNC-98 resides at M-lines , muscle cell nuclei , and possibly at dense bodies . 7 : Furthermore , we demonstrated that 1 ) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization , and 2 ) the C-terminal ( fourth ) Zn finger is required for localization to M-lines and dense bodies . 9 : We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression . 64 : Consistent with the mutant phenotype , UNC-98 is localized to M-lines and probably to dense bodies . 65 : Surprisingly , UNC-98 is also present in muscle cell nuclei . 233 : UNC-98 : : GFP Is localized to Muscle M-Lines , Dense Bodies , and the Nucleus To determine where the unc-98 gene is expressed and where the UNC-98 protein is localized , we made transgenic lines carrying unc-98 with a GFP translational fusion ( unc-98 : : GFP ; Figure 6A ) . 243 : ( B ) Fluorescence micrograph of localization of the unc-98 : : GFP within a body wall muscle cell , suggesting that UNC-98 is located in dense bodies ( seen as dashes ) and M-lines ( seen as lines composed of fine dots ) . 245 : ( C and D ) Fluorescence micrographs showing localization of unc-98 : : GFP to muscle cell nuclei . 254 : Expression was first observed in the developing embryo at the 1 . 5 to 2-fold stage , in which the UNC-98-GFP protein seemed localized to filamentous tracts , which generally follow myosin chain A antibody staining , and therefore are interpreted to be located in myofibrils . 255 : Additionally , expression is seen in undefined puncta located along the filamentous tracts . 257 : As shown in Figure 6B , within adult body wall muscle , the UNC-98-GFP fusion localizes to dense bodies and M-lines . 258 : Within these muscle cells , we also see prominent localization of the UNC-98-GFP in the nucleus . 259 : As Vol 14 , June 2003 2499 KB Mercer et al shown in Figure 6 , C and D , the UNC-98-GFP colocalizes with the DNA-binding dye DAPI . 260 : The signal is excluded from what seems to be the nucleolus and as shown under higher resolution in Figure 6E , the UNC-98-GFP signal is discontinuous . 262 : During development , the nuclear localization of UNC-98 : : GFP begins at the late L2 to early L3 larval stages ( our unpublished data ) . 278 : Anti-UNC-98 Antibodies Localize to Muscle MLines and Nuclei These same anti-UNC-98 antibodies ( EU131 ) were used to detect UNC-98 in the muscle of wild-type and unc-98 mutant worms by using immunofluorescence microscopy . 279 : When used against wild-type embryos , these antibodies stain filamentous structures , presumably myofibrils , as early as the 1 . 5 to 2-fold stage . 280 : As shown in Figure 8 , AC , in wild-type adult animals fixed with methanol and paraformaldehyde , these antibodies stain the M-line region , colocalizing with UNC-89 , a previously Figure 7 . 295 : In wild-type muscle fixed with methanol and paraformaldehyde on whole worms , or alternatively , ethanol or methanol on worm frozen sections , anti-UNC-98 staining was only found at the M-line ; no staining was observed at the dense bodies or in nuclei . 296 : However , when frozen sections were fixed with n-heptane , we saw staining of M-lines and , in addition , the muscle cell nuclei ( Figure 8J ) . 297 : Thus , it would seem that there is indeed UNC-98 in the nucleus , but the epitopes detected by our antibodies are inactivated by the usual fixatives . 303 : Merged images are presented in C , F , and I In wild-type ( AC ) , UNC-98 colocalizes with UNC-89 , a known component of the M-line region ( Benian et al , 1996 ) . 308 : Note localization to the muscle cell nucleus ( marked with an arrow ) and M-lines in which structure was poorly maintained in this fixative . 311 : Note localization to what seem to be M-lines , dense bodies , and the nucleus ( marked with an arrow ) . 317 : As shown in Figure 8K , in these unc-98-rescued animals , anti-UNC-98 antibodies localize to all three structures labeled with UNC-98-GFP : M-lines , nuclei , and dense bodies . 318 : Mapping of Regions Required for Nuclear Localization versus M-Line and Dense Body Localization Curious as to how important it was to have all Zn fingers for proper localization of UNC-98 , we generated four UNC-98 : : GFP constructs each missing one , two , three , or four Zn fingers ( Figure 9A ) . 320 : Removal of as little as the C-terminal Zn finger ( construct A ) resulted in lack of proper localization to the M-line region and dense bodies . 323 : We conclude that the fourth Zn finger is necessary for localization of UNC-98 to these focal adhesion-like structures . 328 : As shown in Figure 9B ( right-most box ) , this protein does not accumulate in the nucleus , but remarkably , localizes to M-lines and dense bodies . 329 : We conclude that the N-terminal 106 residues are both necessary and sufficient for nuclear localization , yet are not required for proper localization to M-lines and dense bodies . 331 : Mapping regions of UNC-98 required for nuclear versus M-line and dense body localization . 339 : In construct B , note the nuclear localization but the lack of proper localization to M-lines and dense bodies . 340 : In construct E , note the lack of nuclear signal , but normal localization to M-lines and dense bodies . 343 : tionally , the ability to create a GFP construct that removes the nuclear localization strongly suggests that nuclear localization is not merely due to the presence of a GFP fusion or overexpression . 345 : Remarkably , construct E , which fails to localize to the nucleus but localizes to M-lines and dense bodies , fully rescues the polarized light defects of unc-98 ( su130 ) . 346 : Construct A , which is missing the last Zn finger , localizes to nuclei but not properly to M-lines and dense bodies in a wild-type background . 380 : We found UNC-98 protein localized to M-lines and possibly to dense bodies . 402 : In wild-type animals expressing full-length UNC-98 fused to GFP , GFP signal was detected in body wall muscle Mlines , dense bodies , and nuclei . 404 : Antibodies raised to UNC-98 protein localized only to the M-line when animals were fixed with the usual fixatives ( methanol/paraformaldehyde , ethanol , or methanol ) . 405 : However , when n-heptane was used as fixative , anti-UNC-98 labeling was seen at M-lines and in muscle cell nuclei . 406 : This suggests that there is indeed endogenous UNC-98 in nuclei , but the usual fixatives destroy enough epitopes so that the UNC-98 antigen can not be detected . 407 : Further support for a nuclear , in addition to M-line location for UNC-98 , is that when anti-UNC-98 antibodies were used to stain animals that were genotypically unc-98 but rescued for the Unc-98 muscle structure defect , labeling was seen at M-lines , nuclei , and dense bodies . 409 : Although it is possible that high-level expression might result in ectopic localization of UNC-98 , the distinct pattern of dense body labeling seems more than coincidental Thus , our interpretation is that in wild-type animals , UNC-98 resides at M-lines , and in muscle cell nuclei and dense bodies . 410 : However , there may be low concentrations of UNC-98 in nuclei and at dense bodies . 411 : This explanation seems plausible for the nuclear location , because UNC-98 , with NLS and NES sequences , might be quickly shuttling in and out of the nucleus , never achieving a high nuclear concentration at any one time . 412 : We determined some of the regions of UNC-98 that are required for nuclear versus focal adhesion localization . 420 : In contrast , a construct containing all four Zn fingers , but lacking the N-terminal 106 amino acids ( construct E ) assembled into M-lines and dense bodies , but no longer localized to nuclei . 426 : To our knowledge , UNC-98 is the first C2H2 ( TFIIIA or Kruppel-like ) Zn finger domain containing protein that has been shown to be localized to discrete regions outside the nucleus . 427 : UNC-98 and UNC-97 ( to which UNC-98 interacts ) are also the only known components of C elegans muscle to have a dual intracellular residence ( to the nucleus and focal adhesion structures ) . 440 : Several lines of evidence indicate that UNC-98 interacts directly with UNC-97 in nematode muscle : 1 ) the colocalization of each GFP-fusion protein to focal adhesions and nuclei ; 2 ) the identification of subdomains of UNC-98 and UNC-97 that are necessary and sufficient for interaction in yeast two-hybrid assays ; and 3 ) recombinant UNC-98 and UNC-97 form a protein complex , in vitro . 450 : The fascinating dual location of UNC-98 , focal adhesionlike structures , and nuclei leads us to the following speculation . 455 : We can speculate that the strength of attachment of the muscle cell , via its dense bodies and M-lines , is reported by UNC-98 moving from the adhesion sites to the nucleus . PAPERDELIMITER PMID:16111945 27 : The BEC-1 : : GFP fusion protein was located in the cytoplasm of many cell types and often accumulated in vesicle-like structures ( Figure 1B ) . 28 : Some expression was also observed in the nucleus . 46 : Fluorescent microscopy shows accumulation of BEC-1 : : GFP in the cytoplasm of seam hypodermal ( S ) and neuronal ( N ) cells of an early transgenic L3 larva . PAPERDELIMITER PMID:17476212 69 : Using a full-length pha-4 complementaryDNA translationfusiontoGFPunderthe pha-4 promoter , we observed nuclear localization of PHA-4 during development and adulthood within the same cells ( Fig 3b and see Methods ) and also found pha-4 expression in the adult worm expanded to a few neuronal cells in the head and tail , which were not found in the developing animal ( Fig 3b ) . 70 : This expression pattern did not change in response to dietary restriction ( data not shown ) , and PHA-4 seemed constitutively nuclear under all conditions tested ( Fig 3c ) . 96 : B , a , AD84 worms reveal PHA4GFP nuclear localization in intestinal cells ( b and c , red arrows ) , head neurons ( d and e , white arrows ) and tail neurons ( f and g , yellow arrows ) . 98 : Nuclear localization of PHA-4GFP in intestinal nuclei ( red arrows ) remained constant under both conditions ( see Methods ) . PAPERDELIMITER PMID:9422779 13 : PKC3 is enriched in particulate fractions of disrupted embryos and larvae . 14 : Immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/ plasma membrane at the apical surface of intestinal cells and in embryonic cells . 54 : A high proportion of PKC3 is anchored/targeted in the vicinity of cortical actin cytoskeleton at all stages of development . 224 : The antibodies also complexed a protein of the same size in cytosol derived from hamster AV-12 cells that were stably transfected with a PKC3 transgene ( Fig 3B , lane 1 ) . 300 : L1 larvae have the highest level of particulate PKC3 but also contain a similar amount of the kinase in cytosol . 301 : PKC3 synthesized in AV-12 cells accumulates predominantly in cytosol and can be purified as a soluble protein ( see Results above ) . 302 : Association of PKC3 with the organelles and cytoskeleton of C elegans at six consecutive stages of development suggests that the enzyme may be targeted to specific intracellular locations via interactions with anchoring proteins and/or lipids . 303 : Accumulation of PKC3 in cytosol is developmentally regu 1138 Structure , Expression , and Properties of C elegans PKC3 FIG . 314 : The atypical kinase is principally a cytosolic enzyme ( of moderate abundance ) only in adult egg-laying nematodes ( Fig 4 ) . 345 : Selective and targeted accumulation of PKC3 at the apical surfaces of intestinal cells ( in an L1 larva ) is evident in D PKC3-derived immunofluorescence outlines the lumen of the intestine . 349 : In addition , affinity purified anti-PKC3 IgGs selectively decorated the apical surfaces of all intestinal cells , thereby outlining the lumen of the gut ( Fig 6D ) . 350 : The highly polarized concentration of the kinase near the apical plasma membrane and the absence of antigen in cytoplasm and all other membranes/organelles of the large intestinal cells strongly suggest that PKC3 is targeted and anchored in situ ( Fig 6D ) . 351 : When immunochemical analysis was extended to the level of electron microscopy , PKC3 molecules were readily detected at the apical surface ( villi ) of C elegans intestinal and pharyngeal cells , 4 in proximity with the cortical actin cytoskeleton that lies under the plasma membrane . 357 : During early embryogenesis ( 1 200 cells , 03 h post-fertilization ) PKC3 accumulates in numerous cells , and a substantial portion of the kinase is typically clustered and enriched in the vicinity of the cell periphery and cell-cell junctions ( Fig 7 , A , B , and D ) . 358 : Double immunostaining with IgGs directed against PKC3 and actin ( Fig 7 , D and E ) suggests that PKC3 is ( in part ) co-localized with the cortical actin cytoskeleton . 377 : D and E show the partially overlapping distribution of PKC3 ( fluorescein signal ) and F-actin ( rhodamine signal ) , respectively , in a doubly stained embryo . 463 : A high proportion of PKC3 is tightly bound to organelles/cytoskeleton in six of the seven developmental stages of C elegans . 464 : In contrast , accumulation of PKC3 in cytoplasm is restricted to L1 larvae and adult nematodes ( Fig 4 ) . 467 : This is evident in the enrichment of PKC3 near the periphery of embryonic cells and the appearance of the kinase near the apical surfaces of cells comprising the intestine . 468 : The subcellular location of PKC3 , co-immunostaining with actin in embryos , immunoelectron microscopy , 4 and the resistance of PKC3 to solubilization with non-ionic detergents5 suggest that the kinase is associated with the cortical actin cytoskeleton . 469 : In intestine , only those portions of the cortical cytoskeleton/cell membrane complex that abut the lumen of the gut contain PKC3 , and immunostaining outlines the entire length of C elegans digestive system . PAPERDELIMITER PMID:17218529 1 : 00028993 The C elegans RSA Complex Localizes Protein Phosphatase 2A to Centrosomes and Regulates Mitotic Spindle Assembly Anne-Lore Schlaitz , 1 Martin Srayko , 1 , 6 , 4 Alexander Dammermann , 2 , 6 Sophie Quintin , 1 , 6 , 5 Natalie Wielsch , 1 Ian MacLeod , 3 Quentin de Robillard , 1 Andrea Zinke , 1 John R Yates , III , 3 Thomas Mu ller-Reichert , 1 Andrei Shevchenko , 1 Karen Oegema , 2 and Anthony A Hyman1 , * 1 2 Max-Planck-Institute of Molecular Cell Biology and Genetics ( MPI-CBG ) , Pfotenhauerstrasse 108 , 01307 Dresden , Germany Department of Cellular and Molecular Medicine , Ludwig Institute for Cancer Research , School of Medicine , University of California , San Diego , La Jolla , CA 92093 , USA 3 4 5 Department of Cell Biology , The Scripps Research Institute , La Jolla , CA 92037 , USA Present address : Department of Biological Sciences , University of Alberta , Edmonton , Alberta T6G 2E9 , Canada . 6 : We describe a complex that targets the Protein Phosphatase 2A holoenzyme ( PP2A ) to centrosomes in C elegans embryos . 9 : The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors : the microtubule destabilizer KLP-7 and the C elegans regulator of spindle assembly TPXL-1 . 51 : GFP : : RSA-1 coprecipitated with the PP2A catalytic and structural subunits LET-92 and PAA-1 , indicating that RSA-1 functions as part of a PP2A heterotrimeric complex In addition to the PP2A core heterodimer , we consistently coimmunoprecipitated the core centrosomal protein SPD-5 and an uncharacterized protein , Y48A6B . 11 ( Table 1 ) . PAPERDELIMITER PMID:17761536 4 : Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors . 6 : Further , PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles , respectively , and the lowest levels of these proteins correlate with high LET-99 accumulation . 21 : In response to these initial asymmetries , PAR-1 , a serine/threonine kinase , is recruited to the posterior cortex and restricts the effectors MEX-5 and -6 ( MEX5/6 ) to the opposite anterior cytoplasm . 24 : The PAR-4 serine/threonine kinase is uniformly localized to the cortex Although PAR-4 is not required for polarity establishment , it is required to maintain the normal position of the PAR-3/ PAR-2 boundary in the late one-cell embryo , and has been suggested to act downstream of PAR-1 and MEX-5/6 in the generation of cytoplasmic asymmetries . 179 : ( B ) Averaged cortical intensities of PAR-3 and -1 in prophase wild-type ( n 10 ) and par-4 ( n 9 ) embryos . 302 : These observations suggest a simple mechanism for formation of the LET-99 band : The presence of high levels of cortical PAR-3 inhibits LET-99 accumulation at the anterior cortex ( independently of PAR-1 ) , and high levels of PAR-1 inhibit LET-99 accumulation at the posteriormost cortex ( Figure 10B ) . 303 : It would appear that a threshold of PAR-1 levels are required for complete inhibition at the cortex , because the LET-99 and PAR-1 domains overlap . PAPERDELIMITER PMID:11784094 238 : PAR-5 Protein Is Present in the Cytoplasm of Gonads , Oocytes , and Early Embryos To determine the distribution of PAR-5 protein in C elegans , we made antibodies against a peptide representing the carboxy-terminal sequence of the predicted par-5 gene product . 255 : appears to be both cytoplasmically and cortically distributed ( Fig 6A ) . 256 : The cytoplasm of oocytes and early embryos also stains brightly . 265 : Thus , it is likely that the cytoplasmic stain in gonads and early embryos revealed by the PAR-5 antibody reflects the in vivo distribution of PAR-5 . 304 : ( A ) Adult hermaphrodite gonad showing PAR-5 at the cortices and in the cytoplasm between the germ nuclei . 305 : ( B ) One-cell embryo showing PAR-5 staining throughout the cytoplasm . PAPERDELIMITER 00002179 302 : In the four quadrants of body wall muscle used for locomotion , each of which is composed of two rows of obliquely oriented uninucleate cells , pat-3 , is localized to the dense bodies , the M-lines , and the borders between cells ( Fig 6 , A and B ) . 313 : More anteriorly along the intestinal-rectal tract , /3pat-3 was detected on the ventral surface of the sphincter muscle and the basal surface of the intestinal muscles ( Fig 6 C ) . 319 : All six coelomocytes , which have gland-like morphologies and may act as scavenger cells , have pat-3 localized on the surface of the cell in a punctate pattern ( Fig 6 , C and D ) . 322 : Lastly , the apical surface of the intestine was frequently strongly stained in wild-type animals . 375 : Arrows indicate staining localizedto the longitudinalcell boundaries . 376 : ( B ) Enlarged view of body wall muscle cells showing/pat-3 staining localized to the dense bodies ( arrows ) and M lines ( arrowheads ) . 377 : ( C ) Lateral view , showing/pat-3 staining of the ventral attachment sites ( apposed to the anus ) and the middle strut of the anal depressor muscle ( ADM ) , the ventral attachment sites of the sphincter muscle ( Sph ) , the basal surface of the intestinal muscles ( IM ) , and the cell surface of the left postembryonically derived coelomocyte ( CC ) . 390 : ( G ) Threefold embryo , showing/3pat-3 staining in a neuronal cell body and process located laterally in the anterior portion of the embryo , with the process extending anteriorly . 482 : In body wall muscle , 8pat-3 was concentrated in the dense bodies , the M lines , and the obliquely oriented borders between muscle cells . 485 : Our demonstration that MH25 recognizes 8pat-3 validates the proposal of Francis and Waterston ( 1985 ) that MH25 binds to a membrane or cell surface component of the dense body . PAPERDELIMITER 00029303 269 : In wild-type embryos , ZYG-1 can be detected as a single dot at the poles of the first mitotic spindle until late anaphase when the two centrioles of a pair separate giving rise to two dots ( Figure 4A ) . 271 : During the second cell cycle of wild-type embryos , ZYG-1 can be detected at the center of each centrosome/spindle pole as either one dot representing a centriole pair prior to anaphase or later two dots representing the separated centrioles ( Figure 4B ) . 272 : In zyg-1 ( it25 ) mutants , we found that ZYG-1 could still localize to centrioles ( n 41 embryos ) . 276 : Insets show ZYG-1 staining at spindle poles . 288 : cycle stages , we found that there were no discernable differences between wild-type and zyg-1 ( it25 ) mutants in the centriole levels of ZYG-1 . 289 : In anaphase , however , most wild-type embryos appeared to possess more centriole-associated ZYG-1 than did the zyg-1 ( it25 ) mutants ( Figure 4A ) . 290 : This difference appeared transient as the wild type and the mutant invariably possessed similar centriole levels at telophase . 301 : single dot of ZYG-1 at the center of each centrosome , but we never observed more than one microtubuleorganizing center per blastomere ( Figure 4B ) . 303 : For all seven strains , we observed that the first bipolar spindle often contained poles with two ZYG-1 dots ( Figure 4A ) , indicating that the first round of centrosome duplication had been executed properly . 306 : As in the wild type , ZYG-1 staining indicated that all spindles in the double mutants contained centrioles at the poles ( Figure 4B and our unpublished observations ) . 311 : We found that at all stages examined , all of the zyg-1 ( it25 ) ; szy double mutants possessed centrosome-associated levels of ZYG-1 that were similar to that of the parental zyg-1 ( it25 ) line . 422 : Insets show threefold magnified views of ZYG-1 staining at centrosomes . 430 : In most instances , we could clearly detect ZYG-1 staining at the center of these centrosomes , indicating that centrioles were present and that suppression by sun-1 ( RNAi ) involved restoration of centriole replication . 432 : If suppression is due to an increase in ZYG-1 levels at the centrosome , we might expect that centrioles in zyg-1 ( it25 ) ; sun-1 ( RNAi ) embryos would stain more intensely than controls . 433 : However , we found that zyg-1 ( it25 ) embryos subject to sun-1 ( RNAi ) did not 110 C A Kemp et al appear to possess any more centrosome-associated ZYG-1 than controls . 434 : In fact , some embryos subject to sun-1 ( RNAi ) appeared to have less ZYG-1 present at centrioles than control embryos ( Figure 6A ) . 435 : Thus , we do not find evidence that loss of SUN-1 activity results in elevated levels of ZYG-1 at the centrosome . PAPERDELIMITER PMID:10903169 125 : LAG-1 appeared to be expressed in the nuclei of all embryonic cells during the stages that the intestine forms , and was thus not informative ( data not shown ) . PAPERDELIMITER PMID:9003776 80 : First , we examined myc-tagged LAG-1 ( 48-673 ) , which is primarily nuclear on its own ( Figure 3A ) , and GLP-1 ( ANK ) , which is primarily cytoplasmic on its own ( Figure 3D ; Roehl and Kimble , 1993 ) . 82 : LAG-1 remained in the nucleus ( Figure 3E ) while GLP-1 ( ANK ) became nuclear ( Figure 3F ) . 87 : Second , we examined myc-tagged LAG-1 ( 199-673 ) . which is cytoplasmic on its own ( Figure 4A ) , and GLP-1 ( RAM/ANK-792-1171 ) , which is nuclear on its own ( Figure 4D ) . 88 : When myc-LAG-1 ( 199-673 ) and GLP1 ( RAM/ANK ) were co-expressed , myc-LAG-1 ( 199-673 ) became nuclear ( Figure 4E ) and GLP-1 ( RAM/ANK ) remained in the nucleus ( Figure 4F ) . 125 : ( A ) and ( B ) Transgenic animal caring myc-LAG-1 ( 199-673 ) : ( C ) ) and ( D ) transgenic animal cam ing GLP-1 ( RAM/ANK-792-1171 ) ; ( E ) and ( F ) Transgenic animal carrying both myc-LAG-1 ( 199-673 ) and GLP 1 ( RAM/ANK ) . myc-LAG-1 ( 199-673 ) is predominantly cytoplasmic when expressed on its own ( A ) . but is predominantly nuclear when co-expressed with Arrows point to the same intestinal nucleus in ( E ) and ( F ) . 132 : ( A ) and ( C ) c-myc-LAG-1 ( 48-673 ) is nuclear : ( B ) GLP-1 ( ANKq224 ) is cytoplasmic : ( D ) GLP-1 ( ANKe2144 ) is cytoplasmic . PAPERDELIMITER PMID:17586485 152 : Throughout development DPY-7 localises to the furrows that define the annuli in the wild type cuticle ( Fig 3A ) . 163 : ( A and B ) The regular annular furrow staining pattern in wild type N2 strain . PAPERDELIMITER PMID:16139225 73 : ( A ) In wild-type XX embryos , DPY-27 and SDC-3 localize specifically to X chromosomes . 166 : At the 30-cell stage , the dosage compensation protein SDC-3 is present in nuclei but has not yet localized to the X chromosomes . PAPERDELIMITER PMID:17719547 51 : To investigate mechanisms that instruct DA9 neuromuscular synaptogenesis , we used the mig-13 and itr-1 pB promoters to express the fluorescently conjugated synaptic vesicle markers rab-3 ( Mahoney et al , 2006 ) and snb-1 ( synaptobrevin ) ( Nonet , 1999 ) in addition to the active zone markers syd-2 ( Liprin-a ) ( Yeh et al , 2005 ) and ccb-1 ( putative b-subunit of voltage-gated calcium channels ) within DA9 . 109 : We examined whether the ectopic synaptic vesicle puncta observed in lin-44 and lin-17 mutants colocalize with the active zone markers SYD-2 ( Liprin-a ) , a gene essential for the assembly of presynaptic terminals ( Zhen and Jin , 1999 ; Kaufmann et al , 2002 ) , or CCB-1 , a putative b-subunit of voltage-gated calcium channels . 111 : Furthermore , these ectopic presynapses also contain the putative calcium channel subunit CCB-1 ( Figure S4 ) . PAPERDELIMITER PMID:10811831 235 : Depletion of either -G spectrin ( Fig 7 A , middle column ) or both -G and -H spectrin subunits ( Fig 7 A , right column ) does not affect localization of the MH27 antigen to the apicolateral domains of epidermal ( arrowheads ) or gut cells ( arrows ) , which indicates that at least some adherens junction structures are assembled normally . 274 : Depletion of -G spectrin ( middle ) did not affect localization of MH27 to the adherens junctions at the apicolateral domain of the epidermal epithelia ( arrowheads ) or the intestinal cells ( arrows ) . PAPERDELIMITER PMID:17551574 6 : Strikingly , we find that during the first cell cycle , EEA-1 positive EEs become enriched at the anterior cortex In contrast , the Golgi compartment shows no asymmetry in distribution . 24 : In this report , we focus on the distribution of EEA-1 positive EEs during this first embryonic cell cycle , where polarity is established . 25 : RESULTS AND DISCUSSION Early endosomes are asymmetrically localised in the early Celegans embryo We visualised early endosomes ( EEs ) using the marker EEA1 , a Rab5 effector protein that has a role in early endosome docking/ fusion [ 8 , 9 ] . 26 : We used a characterized antibody against C elegans EEA-1 that was previously shown to mark early endosomes in other it issues [ 1013 ] . 28 : Individual meiotic EEA-1 PLoS ONE | www . plosone . org positive puncta appear variable in size and shape ; the majority are approximately spherical , but there is a significant population of puncta which appear irregularly shaped . 31 : As sperm asters become visible , we found that EEA-1 positive EEs begin to be concentrated in the anterior and depleted from the posterior of the embryo ( Figure 1C , D ) . 36 : Previous work showed that the endoplasmic reticulum shows a similar anterior enrichment to that seen above for EEA-1 positive EEs [ 15 ] . 46 : Shown are projections of EEs during the first two cell cycles visualised using anti-EEA-1 and a single focal plane of microtubules visualised using anti-tubulin . 65 : Unlike the EEA-1 positive EEs , the Golgi puncta show no asymmetry in distribution during the first cell cycle ( Figure 2A , D ; n = 13 ) . 67 : Early endosome asymmetry requires functional NMY-2 and PAR proteins The cortically associated anterior polarity proteins PAR-3 , PAR-6 , and PKC-3 become anteriorly enriched at the same time as the EEA-1 positive EEs [ 1820 ] . 72 : Asymmetry of early endosomes is coincident with NMY-2 asymmetry GFP : : NMY-2 embryos co-stained for EEs ( anti-EEA-1 ) and NMY-2 ( anti-GFP ) . 75 : To look at the relationship between EEA-1 positive EEs and NMY-2 asymmetry , we co-stained embryos for EEA-1 and NMY-2 . 80 : We found that asymmetry of EEA-1 positive EEs is lost in nmy-2 ( RNAi ) embryos ; there is an even distribution of EEs across the embryonic cortex throughout the first cell cycle ( Figure 4B , C ) . 85 : The requirement for NMY2 and the cortical colocalisation of NMY-2 and EEs suggest that EE asymmetry may be driven by anterior movement of the actomyosin cortex Early endosomes movement in the early embryo It was previously shown that the tandem FYVE domains of EEA1/T10G3 . 5 would target GFP to early endosomes in C elegans somatic it issues [ 24 ] . 86 : To generate a live EE marker for early embryos , we fused this region to GFP and placed it under the control of the germline pie-1 promoter to generate a GFP : : EEA1 ( FYVE*2 ) strain . 101 : EEs visualied live using GFP : EEA-1 ( FYVE*2 ) . 105 : Similar to the localization of EEA-1 positive EEs reported here , Poteryaev et al , 2005 showed that the endoplasmic reticulum also becomes anteriorly enriched during the first cell cycle [ 15 ] . PAPERDELIMITER PMID:11927551 5 : Using a GFP : : 2xFYVE fusion protein we found that the phosphatidylinositol 3-phosphate ( PtdIns 3-P ) product of LET-512/VPS34 is associated with a multitude of intracellular membranes and vesicles located at the periphery , including endocytic vesicles . PAPERDELIMITER PMID:17591921 50 : During interphase , before pronuclear migration , GFP : SP-12 was present in the pronuclear envelopes as well as in a network of ne tubules and punctate structures dispersed throughout the cytoplasm . 71 : Arrowheads point to a region of the nuclear envelope , illustrating the relative exclusion of RET-1 compared with the lumenal marker SP-12 from this ER domain . 92 : ( D ) Metaphase control ( left ) and rab5 ( RNAi ) ( right ) embryos were fixed and stained with antibodies to the endosome marker RAB-5 ( top ) and the Golgi marker SQV-8 ( bottom ) . 141 : Expression of the RFP fusion with RAB-5Q78L , at 25% of the level of endogenous RAB-5 ( Fig 4 A ) , reduced the normal anterior bias of endosomes in the polarized one-cell embryo but did not dramatically alter endosome morphology as assessed by examination of the localization of endogenous RAB-5 ( Fig 4 B ) , or a GFP fusion with the endosomal marker EEA-1 ( Fig S2 F ) . 186 : ( CF ) Spinning-disk confocal optics were used to image control ( n = 9 ) , rab5 ( RNAi ) ( n = 11 ) , yop-1 , ret-1 ( RNAi ) ( n = 10 ) , and npp-12 ( RNAi ) ( n = 10 ) embryos coexpressing RFP : histone and a GFP fusion with the resident INM protein LEM-2 . 242 : To analyze the removal of nuclear pores from the envelope , we lmed embryos expressing a GFP fusion with NUP155 , a stable component of the pore wall ( Franz et al , 2005 ) . 245 : Although a YFP fusion with the C elegans B-type lamin LMN-1 is nearly completely removed from the nuclear envelope by anaphase onset in control embryos , substantial amounts of LMN-1 remained at anaphase onset in RAB-5 or NPP-12 depleted embryos ( Fig 6 D ) . 248 : In the C elegans embryo , the INM protein LEM-2 is present in the peripheral ER throughout the cell cycle . 253 : In embryos depleted of SPD-5 alone , LEM-2 is completely dispersed into the surrounding ER and then reaccumulates around the chromatin as it begins to decondense . PAPERDELIMITER PMID:17276345 44 : We also found that APR-1 as well as DSH-2/Dsh , MIG-5/Dsh , and PRY-1/ Axin localized asymmetrically to the cortex prior to cell division , in a Wnt-dependent manner , suggesting that Wnt proteins regulate WRM-1 nuclear asymmetry through the asymmetric cortical localization of their downstream components . 153 : These GFP fusion constructs rescued their respective mutants ( see Supplemental Experimental Procedures ) , indicating that the fusion proteins were functional Using these constructs , we found that both APR-1 : : GFP and PRY-1 : : GFP localized to the anterior cortex in all seam cells ( Figures 4A4C and 4F ; data not shown ) , except for the T cell , in which APR-1 : : GFP , but not PRY-1 : : GFP , was asymmetric ( data not shown ) . 154 : Just after the completion of the divisions , APR-1 : : GFP and PRY-1 : : GFP remained on the cortex of the anterior daughter cells ( Figures 4D and 4G ) . 155 : They also localized symmetrically to the cytoplasm throughout the cell cycle . 156 : The asymmetric cortical distribution was observed just prior to the onset of division and during the division , but not in interphase cells ( data not shown ) , consistent with previous localization analyses of these proteins in interphase cells ( Hoier et al , 2000 ; Korswagen et al , 2002 ) . 158 : Therefore , the asymmetric cortical localization of these proteins was established only just prior to the onset of mitosis . 159 : Regarding nuclear localization , APR-1 was nearly excluded from the nucleus in interphase cells ( Figures 4B and 4C ) . 160 : At telophase ( data not shown ) or just after division , however , APR-1 : : GFP was clearly seen in both nuclei ( compare Figure 4B and Figure 4D ) . 161 : In addition , we found that a region near the N terminus of APR-1 ( amino acid residues 112333 ) containing the Arm repeatlike sequence strongly localized to the nucleus ( Figure 4E ) . 162 : Considering the defects in the nuclear export of WRM-1 in the apr-1 ( RNAi ) animals described above , these results suggested that APR-1 shuttles between the cytoplasm and nucleus to export WRM-1/b-catenin from the nucleus at telophase , like mammalian APC . 164 : Consistent with the reported direct interaction between APR-1 and PRY-1 ( Korswagen et al , 2002 ) , most APR-1 and PRY-1 signals overlapped at the cell cortex as well as in the cytoplasm ( Figures S3 ) . 165 : Similarly , many HA : : APR-1 or Flag : : PRY-1 signals overlapped with those of WRM1 : : GFP at the cortex and even in the cytoplasm , although some signals did not overlap . 225 : Nonetheless , WRM-1 appears to play a central role in this targeting mechanism , because , as we showed here , cortical WRM-1 affects the targeting of APR-1 and LIT-1 to the cortex Functions of APR-1 in Asymmetric Division Our results strongly suggest that APR-1 shuttles between the cytoplasm and nucleus and exports WRM-1 from both the anterior and posterior nuclei at telophase , when WRM-1 nuclear asymmetry is established . PAPERDELIMITER PMID:14622579 88 : Endophilin Is Localized to Synapses If endophilin is required for synaptic vesicle recycling , then it should be expressed in neurons and localized to synapses . 93 : GFP-tagged endophilin was enriched in puncta in all neuropil and colocalized with synaptobrevin , demonstrating that endophilin is localized to synapses ( Figure 3B ) . 95 : Furthermore , analysis of a genotype that mislocalizes synaptic vesicles suggests that endophilin may be associated with vesicles . 99 : The simplest interpretation of this result is that endophilin is associated with synaptic vesicles . 110 : ( B ) Endophilin is localized to synapses . 114 : Bracket : punctate staining is observed at the neuromuscular junctions of the SAB neurons . 117 : Bottom , merged image demonstrates that endophilin-GFP colocalizes with synaptobrevin at synapses . 119 : ( C ) Endophilin is localized to neuromuscular junctions . 121 : The ventral and dorsal cords show punctate localization of the GFPtagged endophilin protein ( small arrows ) , consistent with a localization to neuromuscular junctions . 281 : GFP-tagged endophilin was localized to puncta likely to be neuromuscular junctions in the dorsal nerve cord of wild-type animals ( genotype lin-15 ( n765 ) oxIs91 X ) . 296 : GFP-tagged endophilin was localized correctly to synapses in unc-26 ( s1710 ) mutant animals and was in fact brighter than in wild-type animals ( Figure 8A ) . PAPERDELIMITER PMID:9876188 6 : Staining of whole mounts with specific monoclonal antibodies reveals that the protein is expressed on the apical surface of hyp7 . 178 : LRP-1 is present on the apical surface of hyp7 By means of monoclonal antibodies that had been produced against the carboxyl terminus , expression of the lrp-1 gene product was investigated in mixed-stage populations of N2 worms using conditions ( Bettinger et al , 1996 ) in which 99% of whole mounts stained with both anti-LRP-1 antibodies and MH27 . 192 : Because MH27 recognizes an epitope associated with zonulae adherens that form at the apical junctions of polarized epithelia such as hyp6 , hyp7 , and the lateral seam cells ( Waterston , 1988 ; Podbilewicz and White , 1994 ) , a final conclusion is possible : location of 90% of the LRP-1 staining to the same focal plane as the MH27 staining demonstrates that LRP-1 is located in the apical region of hyp6 and hyp7 ( Fig 7 ) . 194 : Thus , the resemblance between LRP-1 and mammalian megalin extends to their localization at the apical surface of polarized epithelia . 197 : Expression of the lrp-1 gene product in apical regions of hyp7 . 201 : ( A ) Bands of apical , punctate staining of LRP-1 are present along the entire length of hyp7 ( only an anterior section is shown ) in an adult N2 hemaphrodite . 228 : The presence of LRP-1 on the apical surface of hyp6 and hyp7 is completely consistent with the failure of the mutants to shed and degrade old cuticle . 231 : Before a cycle of endocytosis is initiated , the large extracellular part of LRP-1 must therefore be located just beneath the overlying cuticle or in contact with it . 236 : The presence of LRP-1 in the apical region of hyp7 may appear at first glance to be inconsistent with such a function , as the sterols would have to pass through the cuticle before encountering the receptor . PAPERDELIMITER PMID:11927551 4 : It accumulates at a perinuclear region , and mutations in let-512/vps34 result in an expansion of the outer nuclear membrane as well as in a mislocalization and subsequent complete lack of expression of LRP-1 , a Celegans LDL receptor normally associated with the apical surface of hypodermal cells . 169 : LET-512/VPS34 is required for the expression and localization of LRP-1 at the apical surface of hyp7 The severe defects in molting and in the proper shedding of the old cuticle displayed by arrested homozygous let-512 ( h510 ) mutant worms resembled the defects caused by mutations in the Celegans gene lrp-1 ( Yochem et al , 1999 ) . 171 : lrp-1 encodes a gp330/megalinrelated member of the LDL receptor superfamily and is predominately located to the apical surface of the hypodermal syncytia hyp6 and hyp7 ( Yochem et al , 1999 ) . 174 : In wild-type animals the antibodies stained vesicles at the apical surface of the hyp7 syncytium ( Figure 4G ) ( Yochem et al , 1999 ) . 176 : Double staining with the GFP : : 2xT10G3 . 5 ( FYVE ) fusion protein revealed a co-localization of the LRP-1 receptor with PtdIns 3-P in vesicles ( Figure 4FH ) , showing that they represent endocytic structures . 177 : However , no co-localization of LRP-1 and LET-512/VPS34 , which mainly reside at a perinuclear position ( see above ) , could be observed . 178 : Co-localization of the LRP-1 receptor with PtdIns 3-P in endocytic vesicles suggests a possible role of PtdIns 3-P ( and hence LET-512/VPS34 ) in receptor-mediated endocytosis at the hyp7 plasma membrane of Celegans . 181 : During the developmental arrest of dpy-5 ( e61 ) ; let-512 ( h510 ) homozygous animals , LRP-1 gradually became less abundant and less regularly distributed at the apical surface of hyp7 than in control animals that were wild type for let-512 . 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