of 34
FGF Pyramus has a transmembrane domain and cell-
autonomous function in polarity
Vincent Stepanik
1,†
,
Jingjing Sun
1,†
,
Angelike Stathopoulos
1,*
1
California Institute of Technology, Division of Biology and Biological Engineering, 1200 East
California Blvd., Pasadena, CA 91125, United States of America
SUMMARY
Most FGFs function as receptor ligands through their conserved FGF domain, but sequences
outside this domain vary and are not well studied. This core domain of 120 amino acids (aa) is
flanked in all FGFs by highly divergent amino-terminal and carboxy-terminal sequences of
variable length.
Drosophila
has fewer FGF genes, with only three identified to date: Pyramus
(Pyr), Thisbe (Ths), and Branchless (Bnl), and all three are relatively large FGF proteins (~80
kDa). We hypothesized that the longer FGF proteins present in
Drosophila
and other organisms
may relate to an ancestral form in which multiple functions or regulatory properties are present
within a single polypeptide. Here we focused analysis on Pyr, finding that it harbors a
transmembrane domain (TMD) and extended C-terminal intracellular domain containing a degron.
The intracellular portion limits Pyr levels, whereas the TMD promotes spatial precision in the
paracrine activation of Heartless FGF receptor. Additionally, degron deletion mutants that
upregulate Pyr exhibit cell polarity defects that lead to invagination defects at gastrulation,
demonstrating a previously uncharacterized cell-autonomous role. In summary, our data show that
Pyr is the first demonstrated transmembrane FGF, that it has both extracellular and intracellular
functions, and that spatial distribution and levels of this particular FGF protein are tightly
regulated. Our results suggest that other FGFs may be membrane-tethered or multifunctional like
Pyr.
Graphical Abstract
*
corresponding author and lead contact: angelike@caltech.edu.
AUTHOR CONTRIBUTIONS
A.S., V.S., and J.S. conceived the project and planned the experimental approach. A.S. directed the project. V.S. performed all protein
and cell culture studies, bioinformatic analysis of proteins, and generated Pyr
intra
antibodies and CRISPR/Cas9 mutants. J.S.
performed all the stainings and quantitative analysis of imaging data, and viability studies. Data were analyzed by V.S., J.S., and A.S.
The manuscript was written by V.S., J.S., and A.S.
these authors contributed equally
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DECLARATION OF INTERESTS
The authors declare no competing interests.
HHS Public Access
Author manuscript
Curr Biol
. Author manuscript; available in PMC 2021 August 17.
Published in final edited form as:
Curr Biol
. 2020 August 17; 30(16): 3141–3153.e5. doi:10.1016/j.cub.2020.06.006.
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Keywords
Drosophila melanogaster
; Pyramus (Pyr); Thisbe (Ths); Branchless (Bnl); FGF signaling;
transmembrane domain (TMD); degron
INTRODUCTION
The length of sequences outside the core FGF domain varies greatly among FGF genes, of
which 22 have been identified in vertebrates [rev. in
1
]. The N- and C-terminal flanking
sequences are very short for genes FGF1, FGF2, FGF4, FGF6, FGF7, and FGF10. In
contrast, extended sequences are present in FGF3, FGF5, FGF8, FGF9, and FGF16–19, and
have properties such as supporting autoinhibition through homodimerization (FGF9) and
even supporting nuclear localization (FGF1 and FGF2) [
2
,
3
]. These observations suggest
that FGF proteins, especially those with extended sequences, may have additional functions
beyond those supported by the core FGF domain, which include receptor binding/activation.
In particular, relatively long regions of undefined functions are located at the C-termini of
Drosophila
FGF proteins Pyr and Ths [Figure 1A;
4
]. We showed that each contains a signal
peptide at the N-terminus and that the N-terminal portion of each protein is secreted into cell
culture supernatants [Figure S1A,B),
5
]. These results suggested that Ths can be secreted in
entirety (but sometimes without its C-terminus due to proteolytic cleavage), whereas the fate
of the Pyr C-terminus, which is not detectable in supernatants, was less clear.
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The extracellular portions of Pyr and Ths ligands both activate the Heartless (Htl) FGF
receptor [
5
7
].
pyr
and
ths
have both overlapping and distinct roles in supporting Htl-
dependent processes, including control of mesoderm spreading at gastrulation [
8
,
9
]. While
ths
and
pyr
genes usually share spatiotemporally similar expression patterns,
pyr
is generally
expressed in a more restricted domain relative to
ths
[
4
]. Our previous study suggested an
interaction between the membrane-tethered heparan sulfate proteoglycan (HSPG) Syndecan
with Pyr, whereas the secreted, extracellular HSPG Trol was found to genetically interact
with Ths [
10
,
11
]. Collectively, these results suggested that Pyr may signal to Htl through its
FGF domain in a more localized manner, whereas Ths may have a longer range of action.
Here we investigated whether the extended Pyr sequence outside the FGF core domain
contributes to its signaling potential.
RESULTS
Pyr, but not Ths, has a transmembrane domain following its FGF domain
Phobius computational analysis [see Methods;
12
] predicts a transmembrane domain (TMD)
from aa 400–425 in Pyr, but not for Ths (Figure 1B, top vs. bottom). These residues are
highly-conserved among
Drosophila
species and Tephritidae and Muscidae fly families,
while adjacent sequences lack conservation (Figure 1C). A block of nearly-invariant basic
residues immediately follows from R427 to R435, consistent with the “positive inside” rule
for efficient membrane insertion of transmembrane helices [
13
,
14
]. Live imaging of the mCh
fusions in cell culture shows evidence of a Pyr TMD. While both Ths and Pyr localize to
vesicles in transfected cultured
Drosophila
S2 cells, Pyr also shows membrane localization
(Figure 1D–E’). Pyr 1–440 localizes to the membrane, while Pyr 1–399 does not, further
indicating aa A400-A425 act as a TMD (Figure 1F–G’).
Adding this region and adjacent sequences necessary to retain the Phobius TMD prediction
to a tagged portion of the extracellular domain of Pyr (i.e. mCh-Pyr aa 1–292) promotes
tethering to the membrane similar to CD2 (positive control), while 3xFLAG (negative
control) does not (Figure 1 J–L). Additionally, deletion of A400-A425 results in the
appearance of an additional, higher molecular weight band in supernatants (Figure 1H),
demonstrating that this sequence normally prevents secretion of the polypeptide following
T425. These data demonstrate that Pyr has a TMD, a feature not previously identified in
FGFs.
The C-terminus of Pyr has a potent degradation sequence
When a GFP tag (~27 kDa) is inserted at the Pyr C-terminus, it is not detectable by western
blot or microscopy (Figure 2A,C,D and Figure S2A,B), suggesting that the Pyr C-terminus
is destabilizing. To define a putative degradation sequence, we surveyed a series of
truncations near the Pyr C-terminus, tagged at their C-termini with GFP (Figure 2A). The
longest C-terminal truncation retaining GFP signal is 1–715 (Figure 2D, bottom), indicating
a destabilizing sequence near aa 716–730. Total Pyr levels (i.e. extracellular/supernatant and
intracellular/cell pellet) further increase when aa 681–693 or aa 694–715 are further deleted
(Figure 2C,D respectively), indicating a secondary degradation sequence. aa 710–730 of Pyr
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are highly conserved while sequences just N-terminal to this region are less so (Fig 2G,
Figure S3).
To test if these sequences have intrinsic degradative capacity, we expressed GFP fused to
fragments of the Pyr C-terminus in S2 cells (Figure 2B,E). Strong GFP expression is
observed with all but the Pyr
632–766
-GFP fusion, which shows no signal (Figure 2E). This
demonstrates that residues 716–766 support efficient degradation as a “degron” (Figure
2A,C,D) that is portable to non-Pyr proteins and effective even outside of the secretory
pathway. Despite the potent degradative capacity of these residues, the N-terminus of Pyr
remains detectable when expressed in the context of full-length protein (e.g. Figure 2C,
Figure S1B,C,D top, 1–766). It is therefore likely that this degron is cleaved from the rest of
Pyr. In support of this, fusion of either aa 688–766 or 688–715 to the C-terminus of GFP
downregulates but does not eliminate GFP fluorescence, whereas fusion to aa 716–766 does
(Figure 2F).
Interestingly, when GFP is inserted in-frame within Pyr
intra
between the TMD and C-
terminal degron, signal is detected (i.e. stable within the context of full-length Pyr), but not
when placed at the very C-terminus (Figure 2I’ vs. K’). It is associated with mCh signal in
vesicles, and at cellular protrusions and independently in the cytoplasm and in vesicles,
indicating that cleavage from the N-terminus occurs intracellularly (Figure 2K”).
Intracellular Pyr is separable from the N-terminus and is detectable in vivo
To characterize Pyr
intra
, we generated an antibody to aa 452–715 (Figure 3A). Anti-Pyr
intra
recognizes 140–170 kDa isoforms in S2 cells transfected with dual-tagged Pyr that
correspond to the same bands recognized when detecting the N-terminus, indicative of full-
length protein (i.e. Pyr
N-term
+Pyr
intra
) (Figure 3B and S1E, compare with Figure S1D cell
pellet, anti-RFP). Lower molecular weight Pyr
intra
bands separated from the N-terminal
fragments are also detected for the full length and truncation constructs, and in the case of
full length, separate from the C-terminal degron (i.e. ~50 kDa for 1–680 and ~52 kDa for 1–
766; Figure 3B).
Pyr
intra
was not readily detectable by western blot in extracts from a variety of
developmental timepoints (e.g. embryo, larva, adult, ovary), indicating that it is expressed at
low levels, likely due to the presence of the C-terminal degron. We therefore enriched for
Pyr
intra
through immunoprecipitation (IP) from 3–7 h embryo extracts, a window in which
Pyr function has been characterized [
8
,
15
]. Pyr
intra
was detected as a smear from 49–57 kDa,
shorter than if present as full-length Pyr or still connected to the TMD, neither of which are
expected to be extracted by our IP-compatible conditions (Figure 3C, Pyr 1–430, see below).
The resulting smear of the Pyr
intra
-specific signal may relate to post-translational
modification(s) or the lability of Pyr
intra
at this stage. When Pyr
intra
was immunoprecipitated
from extracts of S2 cells constitutively expressing full-length Pyr, similar signal was
detected with a predominant band of ~57kDa that was absent from control S2 cells (Figure
3D).
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Pyr truncation mutants exhibit phenotypes including increased survivability consistent
with gain-of-function
To test the functional significance of the TMD and degron of Pyr
in vivo
, we introduced stop
codons into the endogenous
pyr
locus to remove the functions of these domains using the
CRISPR/Cas9 system [see Methods;
16
].
pyr
399
and
pyr
430
both delete Pyr
intra
, while
pyr
399
also deletes the TMD (Figure 3E).
pyr
715
removes the potent degron, while
pyr
680
removes
additional sequence that further reduces total Pyr and Pyr
intra
levels in cultured S2 cells
when present (Figure 3E; see also Figure 2D).
pyr
null mutants that delete the entire gene coding sequence are lethal [
17
]. All
pyr
mutants
that delete the TMD and/or intracellular domain created by CRISPR/Cas9 are viable and
fertile, suggesting that loss of Pyr N-terminal function is the underlying cause of the lethality
of null mutants. However, these
pyr
truncation mutants are severely compromised in health
and exhibit decreased fertility with some stocks producing less than 25% of the expected
progeny (i.e.
pyr
715
), requiring the stocks be kept as heterozygotes (i.e. over balancer; see
Methods). When
pyr
mutations are assayed in trans to a
pyr
null allele [i.e. Df(2R)pyr36;
17
],
pyr
430
/
Df
and
pyr
680
/
Df
display a better survival rate compared to +/
Df
(Figure 3H).
Using the Pyr
intra
antibody, no staining is observed in
pyr-
null embryos (i.e.
Df(2R)pyr36)
or
the C-terminal truncation (i.e.
pyr
399
and
pyr
430
) mutants (Figure 3K,M) demonstrating
antibody specificity. This antibody detects in wild-type embryos a pattern indistinguishable
from the highly dynamic
pyr in situ
pattern [Figure 3I–J’, compared with 3F–G’;
4
].
Furthermore, anti-Pyr
intra
staining appears to be stronger in the mutant that lacks the C-
terminal degron (i.e.
pyr
680
, Figure 3L vs. 3J), suggesting that Pyr
680
protein, and likely also
Pyr
430
, may be expressed at higher levels due to lack of the C-terminal degron.
Collectively, these findings demonstrate these truncations (i.e.
pyr
430
and
pyr
680
) retain
some function(s), and support the view that the TMD is a critical component of Pyr function.
The Pyr TMD and degron contribute to localization of Htl receptor and protrusion
formation during mesoderm migration
Activation of Htl by Pyr and Ths triggers cell shape changes in the mesoderm, initiating a
slow epithelial to mesenchymal transition (EMT) that continues as mesoderm cells spread
upon the
pyr-
expressing ectoderm [
9
,
18
,
19
]. Htl is membrane-associated and becomes
concentrated at the tissue interface (arrowheads, Figure 4A,B’,C’) [
18
,
20
]. We hypothesized
that localized expression of Pyr, possibly supported by its TMD, may be required for the
concentration of Htl receptor.
We first investigated how Pyr expression changes over time. No antibody is available for
direct localization of the Pyr extracellular region that contains the core FGF domain;
however, anti-Pyr
intra
is expected to recognize the full-length protein prior to the proteolytic
cleavage that separates the extracellular portion from the TMD and C-terminal Pyr
intra
.
Therefore, Pyr
intra
antibody was used as the best available proxy for full-length protein.
Depending on developmental stages and cell types, Pyr localizes to both the cytoplasm and
the membrane (Figure 4A–D’). Specifically, until stage 9, Pyr is detected only within the
cytoplasm in the neuroectoderm (Figure 4A–C’). Later during neurogenesis when
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