Current Biology, Volume
30
Supplemental Information
FGF Pyramus Has a Transmembrane Domain
and Cell-Autonomous Function in Polarity
Vincent Stepanik, Jingjing Sun, and Angelike Stathopoulos
Figure S1. Longer exposures and extended molecular weight ranges of western blots.
Related to
Figures 1 and 3.
(A) Schematic showing the domain structure of Ths and Pyr, including the signal peptide (SP), FGF
domain, and transmembrane domain (TMD) of
Pyr. Truncations expressed in S2 cells for Fig
ure
1D
-
G
and D
-
F below are shown, with a 6xHis,mCh tag inserted before the FGF domain of each, and a 3xFLAG
tag at the C
-
terminus, including the full
-
length Ths and Pyr shown in B.
(B) Western blots of pulldowns
from supernatants of S2 cell transfections of dual
-
tagged, full
-
length
Ths and Pyr. Black arrowhead is cleaved, N
-
terminal fragment
-
only Ths, and red arrowhead is full
-
length Ths.
(C
-
E) Western blots of pulldowns from supernatants and total cell extracts
of S2 transfected with Pyr
truncations. Short and long exposures for each western are shown. Arrowheads indicate isoforms
common to longer truncations and are presumably mature products. Pyr truncations ending at T440 and
beyond, tagged at their N
-
termini
with mCh and at their C
-
termini with 3xFLAG, show FLAG signals
that are only detectable intracellularly. In contrast, truncations ending at A370 and T399 show FLAG
signals in both the supernatant and intracellularly (Fig
ure
S1C,D). Also, for truncations exte
nding
beyond T399, the strong correspondence of mCh and FLAG signals is lost, suggesting that at least one
proteolytic cleavage site exists in the interval of aa 370
-
466. (D) Red arrowheads indicate MW of bands
with corresponding RFP and FLAG signals of lo
nger truncations that are only present intracellularly.
Black arrowhead indicates N
-
terminal product cleaved from C
-
terminal sequence, common to
truncations C
-
terminal to aa 370. Dotted line indicates products with range of MW likely due to
differential po
st
-
translational modifications, and black arrows on longer exposure are shorter Pyr
intra
forms resulting from the truncations. For the cell pellet samples of Pyr 1
-
600 and 1
-
680, red arrowheads
indicate FLAG
-
reactive, non
-
RFP reactive bands indicative of f
ree, intracellular Pyr C
-
terminal
fragments. For the 1
-
466 cell pellet sample in Fig 1D, although a weaker FLAG signal is seen relative
to the other truncations tested, there is a readily detectable FLAG signal with this longer exposure. We
have noticed so
me variability of FLAG signal detection in western blots depending on amino acid
context, which is indicated for 1
-
466 with the equal strength of RFP signal compared to the other
truncations. For the anti
-
FLAG blot of supernatants in C, the dotted line ind
icates lower
-
abundance,
higher molecular weight isoforms of the truncations that are FLAG
-
reactive. The absence of
corresponding RFP
-
reactive signal is likely due to weaker antibody RFP performance.
(E) Longer exposure and extended molecular weight ranges
of the western blot in Figure 3B.
FIGURE S2. The C
-
terminus of Pyr is undetectable when tagged after amino acid 730. Related to
Figure 2.
(A) Schematic of Pyr truncations near the Pyr degron, with a C
-
terminal GFP tag. SP=signal peptide,
TMD= trans
membrane domain, red box = degron. Triangles and dashed lines denote approximate
location of proteolytic cleavage sites.
(B) Western blot probed with GFP of cell pellet extracts of S2 cells transfected with the indicated
truncations, full length (1
-
766) o
r non
-
transfected (
-
). All lanes were present on the same blot. Panel has
been cropped between 1
-
766 and (
-
) samples to exclude non
-
relevant lanes. Red arrowhead indicates
intact “full length” mCh
-
Pyr
-
GFP truncations, and the black arrowhead indicates Pyr
i
ntra
-
GFP separated
from the rest of the Pyr molecule.
Figure S3. Multi
-
sequence alignment of Pyr orthologs in Drosophila and Dipteran flies in the
Tephritidae and Muscidae family members. Related to Figures 1 and 2.
MUltiple Sequence Comparison by
Log
-
Expectation (MUSCLE) alignment of Pyr orthologs across
representative species of the Drosophilidae, Tephritidae, and Muscidae families of Dipterans. Regions
of high conservation of interest are noted including the FGF domain, transmembrane domain and
post
-
TMD stretch of basic residues, degron as well as positions of truncation mutants created using
CRISPR/Cas9 (stop sign symbols). Amino acid color code consists of blue=hydrophobic, red=basic,
purple=acidic,
polar=green,
yellow=proline,
pink=cysteine,
c
yan=histidine
and
tyrosine,
orange=glycine.
Figure S4. Pyr TMD when connected with the N
-
terminus supports Mys localization which
possibly limits the protrusive activity of mesoderm cells. Related to Figure 5.
(AB,D
,E) To assay a role of TMD for localized Mys expression at the dorsal ectoderm
-
mesoderm
interface, either wild
-
type or truncated
pyr
399
and
pyr
430
alleles were also crossed into
Df(2R)pyr36
mutant background to eliminate the possible effect of a second
-
sit
e mutation in homozygous
backgrounds (See Methods). Mesoderm cells are marked by anti
-
Twi immunofluorescence (red), and
Mys levels assayed by anti
-
Mys immunofluorescence signal (white).
(C) Relative difference in Mys expression within the dorsal ectoderm
(see boxed region in A) for
transheterozygous combinations between CRISPR/Cas9 mutants and a
pyr
null (i.e.
Df(2R)pyr36
) is
plotted. The Mys level at the dorsal ectoderm
-
mesoderm interface in
pyr
399
/Df(2R)pyr36
(D) is
significantly lower compared to those
in
+/Df(2R)pyr36
(B) or
pyr
430
/Df(2R)pyr36
(E). n≥8, p<0.
01.
Genotypes and antibody combination are as indicated. Scale bar, 20
μ
m.
(F,G)
Example of protrusion measurement in Zen software (F). More mesoderm protrusions in
pyr
N+C
mutants compared to wild
-
type embryos suggests that disconnecting of TMD from the N
-
terminal FGF
domain and/or higher levels of Pyr
intra
promotes the protrusive activity of mesoderm cells (n
≥
8, p<0.005)
(G).
Figure S5. Pyr
intra
functions cell
-
autonomously to regulate apicobasal polarity.
Related to Figure
7.
(A
-
F’) Expression of Crb (blue) and Baz in stage 6 embryos with magnified views of boxed areas to the
right. Baz localization in the dorsal ectoderm extends basally upon ubi
quitous overexpression of
pyr
(A’), while it is not affected in the same region in
pyr
N+C
mutant embryo (B’). It is also not affected in
the neuroectoderm in loss of function
pyr
mutants (C’,D’,E’,F’). Htl antibody (green) is used to mark
the mesoderm. Gen
otypes and antibody combinations are as indicated. Scale bar, 20
μ
m.