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Molecular Analysis of Core Kinetochore Composition and Assembly in Drosophila melanogaster

Przewloka, Marcin R. and Zhang, Wei and Costa, Patricia and Archambault, Vincent and D'Avino, Pier Paolo and Lilley, Kathryn S. and Laue, Ernest D. and McAinsh, Andrew D. and Glover, David M. (2007) Molecular Analysis of Core Kinetochore Composition and Assembly in Drosophila melanogaster. PLoS ONE, 2 (5). Art. No. e478. ISSN 1932-6203. PMCID PMC1868777. doi:10.1371/journal.pone.0000478. https://resolver.caltech.edu/CaltechAUTHORS:20200807-170351789

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Abstract

Background. Kinetochores are large multiprotein complexes indispensable for proper chromosome segregation. Although Drosophila is a classical model organism for studies of chromosome segregation, little is known about the organization of its kinetochores. Methodology/Principal Findings. We employed bioinformatics, proteomics and cell biology methods to identify and analyze the interaction network of Drosophila kinetochore proteins. We have shown that three Drosophila proteins highly diverged from human and yeast Ndc80, Nuf2 and Mis12 are indeed their orthologues. Affinity purification of these proteins from cultured Drosophila cells identified a further five interacting proteins with weak similarity to subunits of the SPC105/KNL-1, MIND/MIS12 and NDC80 kinetochore complexes together with known kinetochore associated proteins such as dynein/dynactin, spindle assembly checkpoint components and heterochromatin proteins. All eight kinetochore complex proteins were present at the kinetochore during mitosis and MIND/MIS12 complex proteins were also centromeric during interphase. Their down-regulation led to dramatic defects in chromosome congression/segregation frequently accompanied by mitotic spindle elongation. The systematic depletion of each individual protein allowed us to establish dependency relationships for their recruitment onto the kinetochore. This revealed the sequential recruitment of individual members of first, the MIND/MIS12 and then, NDC80 complex. Conclusions/Significance. The Drosophila MIND/MIS12 and NDC80 complexes and the Spc105 protein, like their counterparts from other eukaryotic species, are essential for chromosome congression and segregation, but are highly diverged in sequence. Hierarchical dependence relationships of individual proteins regulate the assembly of Drosophila kinetochore complexes in a manner similar, but not identical, to other organisms.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1371/journal.pone.0000478DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868777/PubMed CentralArticle
ORCID:
AuthorORCID
Przewloka, Marcin R.0000-0002-0329-9162
Archambault, Vincent0000-0002-2857-7667
D'Avino, Pier Paolo0000-0002-4773-6950
Lilley, Kathryn S.0000-0003-0594-6543
Laue, Ernest D.0000-0002-7476-4148
McAinsh, Andrew D.0000-0001-6808-0711
Glover, David M.0000-0003-0956-0103
Additional Information:© 2007 Przewloka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. March 27, 2007; Accepted: May 1, 2007; Published: May 30, 2007. The work presented here was supported by BBSRC grants BB/C508926/1 and BB/E011586/1. P.C. was supported by funding from the EC FW5 Network “Molecular analysis of Drosophila cell division” (HPRN-CT-2006-00260). V.A. holds a long-term fellowship from the Human Frontier Science Program. Programme grant support from Cancer Research UK to D.M.G. also helped support this work. A.D.M was supported by Marie Curie Cancer Care. We would like to thank members of our laboratories for helpful discussions and Tania Minns and Elizabeth Button for technical assistance. We thank Dr Patrick Meraldi for sharing sequence data prior to publication. We are also grateful for anti-CENP-C antibodies supplied by Christian Lehner. Note in Proof. While this manuscript was being prepared, two other publications appeared describing data that partially overlap with and are in agreement with our findings: Goshima et al. (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316:417–421; and Schittenhelm et al. Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. (2007) Chromosoma DOI 10.1007/s00412-007-0103-y [in press]. Author Contributions. Conceived and designed the experiments: MP WZ AM DG. Performed the experiments: MP WZ PC. Analyzed the data: EL MP WZ PC AM DG. Contributed reagents/materials/analysis tools: MP VA WZ KL PC PD AM. Wrote the paper: MP AM DG. The authors have declared that no competing interests exist.
Funders:
Funding AgencyGrant Number
Biotechnology and Biological Sciences Research Council (BBSRC)BB/C508926/1
Biotechnology and Biological Sciences Research Council (BBSRC)BB/E011586/1
Marie Curie FellowshipHPRN-CT-2006-00260
Human Frontier Science ProgramUNSPECIFIED
Cancer Research UKUNSPECIFIED
Marie Curie Cancer CareUNSPECIFIED
Issue or Number:5
PubMed Central ID:PMC1868777
DOI:10.1371/journal.pone.0000478
Record Number:CaltechAUTHORS:20200807-170351789
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200807-170351789
Official Citation:Przewloka MR, Zhang W, Costa P, Archambault V, D'Avino PP, Lilley KS, et al. (2007) Molecular Analysis of Core Kinetochore Composition and Assembly in Drosophila melanogaster. PLoS ONE 2(5): e478. https://doi.org/10.1371/journal.pone.0000478
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104850
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:11 Aug 2020 21:35
Last Modified:16 Nov 2021 18:36

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