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First published online 28 November 2007
doi: 10.1242/dev.011437
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1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA.
2 MRC-LMCB, University College London, London WC1E 6BT, UK.
Author for correspondence (e-mail:
susanp{at}fhcrc.org)
Accepted 28 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Unconventional myosin, Myosin XV, Dorsal closure, Filopodia, Microtubules, Actin, Cytoskeleton, DE-cadherin (Shotgun), EB1, 
-tubulin, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Raich et al., 1999;
Vasioukhin and Fuchs, 2001).
During dorsal closure (DC), a morphogenetic event that occurs late in
Drosophila embryogenesis, two lateral sheets of epithelial cells move
towards one another over a dorsally exposed region of extraembryonic tissue
and fuse together at the midline (Jacinto
et al., 2002). During this process, the dorsal-most face of
leading edge (LE) epithelial cells exhibit dynamic cellular protrusions,
lamellipodia and filopodia, required initially for sensing their environment
and finding their appropriate counterpart on the opposing epithelial sheet.
Subsequently, the opposing protrusions adhere to one another, facilitating the
formation of transient cell-cell contacts as the epithelial sheets zipper
together, followed by permanent cell-adhesion structures. Filopodia contain a
core of organized bundled actin filaments, oriented with their barbed (plus)
ends towards the tip (Faix and Rottner,
2006). Filopodia continuously assemble and disassemble, with
growth occurring via de novo actin nucleation and polymerization locally at
their tips. Although the dynamic rearrangements of cells and filopodia during
DC are mainly attributed to actin dynamics, a recent report has described a
role for microtubules in epithelial zippering, the final step of DC
(Faix and Rottner, 2006;
Jankovics and Brunner,
2006).
The coordination of environmental sensing, cell-cell recognition and
adhesion mediated by LE cell protrusions must require orchestrated movements
of structural, adhesive and regulatory molecules within filopodia.
Unconventional myosins have recently been implicated in the movement of such
cellular molecules/machineries within filopodia
(Mermall et al., 1998;
Berg and Cheney, 2002).
Unconventional myosins are actin-based motor proteins that can be subdivided
into at least 18 distinct classes (I-XVIII) based on their motor and tail
domain structural and functional characteristics
(Oliver et al., 1999;
Berg et al., 2001;
Tzolovsky et al., 2002;
Foth et al., 2006). One subset,
the `MyTH-FERM' unconventional myosins, includes classes VII, X, XII and XV
that share structurally conserved features in their tails: MyTH4 (myosin tail
homology 4) domains that bind to microtubules
(Weber et al., 2004) and FERM
(band 4.1, ezrin, radixin, moesin) domains that are involved in cargo binding
(Sousa et al., 2005; Sheetz,
1999). The tail regions are thought to determine where the myosins
are located and what cargos they transport
(Oliver et al., 1999;
Sheetz, 1999).
Mutations in MyTH-FERM unconventional myosins result in disorganized
stereocilia leading to deafness and vestibular dysfunction in humans and mice:
myosin VIIa is responsible for human Usher Syndrome type IB and the mouse
shaker 1 mutation, whereas myosin XV is linked to human non-syndromic
deafness, DFNB3, and the mouse shaker 2 mutation
(Gibson et al., 1995;
Weil et al., 1995;
Liang et al., 1999;
Libby and Steel, 2000). In
Dictyostelium, Myosin VIIa (MVII) localizes to filopodial tips and is
required for the formation of filopodia and cell attachment
(Titus, 1999;
Tuxworth et al., 2001). In
mammalian cells, Myosin X (Myo10) moves bidirectionally within filopodia and
accumulates at filopodial tips (Berg and
Cheney, 2002; Bohil et al.,
2006). Ectopic expression of Myo10 is sufficient to direct
assembly of filopodia in cells lacking them. The Myo10 FERM domain has been
shown to bind β-integrin and transport it to filopodial tips where it is
needed for proper filopodial extension and substratum adherence
(Zhang et al., 2004).
Drosophila has three MyTH-FERM myosin homologs: myosin VIIa, VIIb
and XV (Tzolovsky et al.,
2002). Functional data has only been reported for
crinkled (myosin VIIa), mutants of which are semi-lethal with escaper
adults exhibiting defects in actin-rich structures such as bristles and hairs
(Kiehart et al., 2004), and
deafness due to disruption of scolopidia auditory organ integrity required for
transducing auditory signals (Todi et al.,
2005).
In this study, we show that the Drosophila myosin XV homolog, which we named Sisyphus (Syph), is required for proper DC where it traffics sensory, cytoskeletal, and adhesion cargos within LE cells and their filopodial protrusions.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We attempted to generate imprecise excisions using three different viable P-element lines inserted in or near the Syph locus (EY00595, EP1321 and NP-4100). We used a combination Southern blot and PCR analyses of potential excision lines to determine the molecular nature of the excision. Although we were able to generate many precise excisions, no imprecise excisions affecting Syph were obtained after screening >500 excision lines.
Sisyphus-GFP and Sisyphus-RFP were created by fusing either GFP or RFP
[`Cherry' (Shaner et al.,
2004)] C-terminal to the Syph ORF. The resulting Sisyphus-GFP or
Sisyphus-RFP fusion constructs were cloned into pUASp as
KpnI-XbaI fragments and used to make germline transformants
as described previously (Spradling,
1986). RFP-actin was made by fusing mRFP N-terminal to the ATG of
the actin5C ORF and cloned into pUASp as a KpnI-BamHI
fragment (P. Martin and S.M.P., unpublished). All transformant lines used in
this study were mapped to a single chromosome and shown to have non-lethal
insertions.
Nomenclature
The `MyTH-FERM' subset of unconventional myosins, includes classes VII, X,
XII and XV. Drosophila has three recognized MyTH-FERM unconventional
myosins: a myosin VIIa homolog, Crinkled (CG7595); a myosin VIIb homolog,
Myo28B1 (CG6976); and a myosin XV homolog, Myo10A (CG2174)
(Tzolovsky et al., 2002).
Drosophila does not have a class X homolog. We refer to the fly
myosin XV homolog as Sisyphus, rather than its FlyBase name of Myo10A, to
avoid confusion between the `10A' in this name (which refers to its
cytological location on fly chromosomes) and class X unconventional myosins
(of which it is not a member).
Antibody production and characterization
Polyclonal mouse antiserum against Syph was generated by immunizing BALB/c
BYJ Rb(8.12) 5BNR/J mice (Jackson Labs) with a mixture of GST fusions to Syph
amino acids 1608-1650, 2083-2115 or 2565-2602. These regions of Syph contain
no homology to any other Drosophila gene. Antibody specificity was
tested via western blot using in vitro translated full-length and tail
fragments of Syph (see Fig. S1A in the supplementary material). This antibody
also recognizes a doublet in extracts from DC staged wild-type
Drosophila embryos (see Fig.
3B). Using this antibody (1:100 dilution), we find that Syph is
maternally contributed and ubiquitously distributed in embryos (see Fig. S1C
in the supplementary material and data not shown), and localizes to condensed
chromosomes on the mitotic spindle (see Fig. S1D in the supplementary
material).
Cell culture
Drosophila cell lines used in this study, S2R+, BG2 (ML-DmBG2) and
EB1-GFP (S2-Mt-EB1-GFP) were obtained from the Drosophila Genomics
Resource Center (DGRC; see
https://dgrc.cgb.indiana.edu/cells/store/catalog.html
for cell line description and references). S2R+ and EB1-GFP cells were grown
at 25°C in Schneider's medium (Invitrogen) supplemented with 10%
heat-inactivated FBS, 25 mM glutamine, penicillin and streptomycin. BG2 cells
were grown at 25°C in Shields and Sang M3 medium (Sigma) with 25%
bactopeptone (Difco), 10% yeast extract (BD), and supplemented as above.
RNA interference (RNAi)
Syph double-stranded RNA (dsRNA) was generated and either injected into
embryos or placed on cells as previously described
(Magie et al., 2002;
Magie and Parkhurst, 2005).
The regions of Syph used to generate dsRNA#1 (2480-2597aa) or dsRNA#2
(2391-2559aa) do not contain homology to other Drosophila genes as
assayed by BLAST. Non-relevant RNAi constructs (GFP, -catenin,
p120catenin, or Rho1) do not produce the same phenotypes as Syph dsRNA when
injected into embryos or put on cells
(Magie et al., 2002;
Magie and Parkhurst, 2005)
(data not shown).
Immunofluorescence
Immunofluorescence of embryos and cells was performed as described
previously (Magie and Parkhurst,
2005; Rosales-Nieves et al.,
2006). The following antibodies were used in this study:
-DE-cadherin (1:10, a gift from H. Oda; also known as Shotgun -
FlyBase), -Groucho (1:400, a gift from C. Delidakis), and
-tubulin (1:500-1000, Harlan Sera-lab). Alexa Fluor 568- and Alexa
Fluor 633-labeled phalloidin (Invitrogen) was used at 1-3 Units/assay and
SlowFade Gold with DAPI (Invitrogen/Molecular Probes) was used to stain
nuclei.
Confocal microscopy
Confocal microscopy was performed with a Zeiss LSM-510M with excitation at
488 nm, 543 nm, 633 nm or 780 nm (two-photon), and emission collection with
BP-500-550, BP-565-615, BP-650-710 filters or BP-435-485 filters,
respectively. An -Plan-Fluor 100x/1.45 oil immersion, a
Plan-Neofluor 40x/1.3 oil immersion, or a Plan-Apochromat 20x/0.75
dry objective was used for imaging. Post-acquisition images were processed
with a 3x3 median filter in ImageJ, and assembled with Adobe Photoshop
and Canvas software.
For live imaging, embryos were dechorionated and placed in halocarbon 700 oil on Greiner Lumox culture dishes made with hydrophilic gas permeable membranes (Sigma). For each movie, the figure panels corresponding to the movie, magnification, time interval between frames, and frame rate, are as follows. Movie 1: Fig. 2F-F''', 160x, 3-4 minutes/frame, 3 frames/second; Movie 2: Fig. 2G-J, 240x, 4 seconds/frame, 7 frames/second; Movie 3, Fig. 4K-M, 160x, 2 seconds/frame, 25 frames/second; Movie 4, Fig. 6A-C, 240x, 2 seconds/frame, 25 frames/second.
Plasmids and constructs
This study used the following constructs: Syph-FL (1-2602aa from CG2174;
accession no. AAF47983), Syph-tail (1295-2602aa), Syph-t1 (1295-2082aa),
Syph-t2 (2083-2273aa), Syph-t3 (2274-2602aa), Syph-L1 (2259-2373aa), Syph-L2
(2374-2476aa), Syph-L3 (2477-2573aa), Syph-E (2574-2602aa), Syph-L1a
(2259-2331aa), Syph-L1b (2332-2373aa), Syph-L1a1 (2259-2284aa), Syph-L1a2
(2285-2305aa), Syph-L1a3 (2306-2330aa), Syph-L3a (2477-2530aa), Syph-L3b
(2531-2573aa), cadICD (1346-1507aa from CG3722; accession no.
AAF46659), cad-A (1346-1390aa), cad-B (1391-1438aa), cad-C (1439-1466aa),
cad-D (1467-1507aa), Katanin-60 (1-572aa from CG10229; accession no.
AAF52059), aPKC (1-606aa from CG10261; accession no. AAF58177), Milton
(1-1122aa from CG13777; accession no.AAN10622), and EB1 (1-291aa from CG3265;
accession no.AAM70826). Syph-L1 substitution point mutations
(LGVE*, SEAEQ*, QEF*, SLYC*,
IVQG* and DAFT*) were made within the Syph-L1 fragment
(2259-2373aa) using primers that substitute alanines for the amino acids
indicated. Syph-L3 substitution point mutations (HWS*,
STR* and DMK*) were made within the Syph-L3 fragment
(2477-2573aa) using primers that substitute alanines for the amino acids
indicated. These constructs were cloned into pCite (Novagen), pGEX (GE) or
pRSET (Stratagene) vectors using standard PCR cloning techniques.
Protein expression, GST-pulldown assays and immunoprecipitations
Protein expression, GST pulldown assays and immunoprecipitations were
performed as previously described (Magie
and Parkhurst, 2005;
Rosales-Nieves et al.,
2006).
Yeast two-hybrid interactions and screening
Yeast two-hybrid interactions and screening were performed with a LexA
fusion to the Syph C-terminal tail (1295-2602aa) using a Drosophila
early embryo library as previously described
(Poortinga et al., 1998).
Putative Syph interactors were confirmed in a yeast mating assay as previously
described (Poortinga et al.,
1998), with a subset of the interactors also confirmed using GST
pulldown assays (see Fig. 3,
Table 1, and Fig. S2A in the
supplementary material).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Berg and Cheney,
2002). We examined Syph localization in Drosophila S2R+
and BG2 cells (see Materials and methods) and observed Syph localization in
puncta along filopodia, as well as throughout the cell body
(Fig. 1E-G;
Fig. 4H,H'). Similar to
other MyTH-FERM myosins, Syph localizes to the ends of filopodia, but unlike
them, it does not accumulate preferentially at their tips
(Fig. 1E',G').
Dynamic localization of Syph during dorsal closure
To further investigate the role of Syph in LE cells of the embryo in vivo,
we generated Drosophila lines carrying GFP or RFP fusions to Syph
under the control of the conditional UAS promoter. Driving expression of the
fluorescent fusion proteins in epithelial stripes with an Engrailed (En)-Gal4
driver (Brand and Perrimon,
1993), we observed accumulation of Syph at the leading edge during
DC (Fig. 2A-E'). As seen
with antibody staining, this accumulation intensifies just prior to and during
epithelial edge matching (Fig.
2A',F-F'''), but subsides after the edges have met and
fused (Fig. 2E'). At
higher magnifications we observed punctate accumulations of Syph at the
dorsal-most edge of LE cells and in filopodia extending from their dorsal-most
edge (Fig. 2F-L''). As
filopodia from opposing epithelial edges meet and adhere, the punctate
accumulation appears to migrate into the newly formed junction
(Fig. 2F-F''' and see
Movie 1 in the supplementary material).
To examine the dynamics of Syph localization within filopodia, we captured time-lapse confocal images of LE filopodia (Fig. 2G-J' and see Movie 2 in the supplementary material). The filopodia themselves were very dynamic, growing and moving between frames. Within filopodia, we observed punctate dots and bands of Syph that move along the length of the filopodia. Although Syph can be observed at the tips of filopodia, this accumulation is not permanent; rather Syph appears to be in constant motion along the filopodia.
Identification of Syph cargo proteins
Since Syph protein localization suggests that it traffics proteins within
LE cells and their filopodia, we performed a yeast two-hybrid screen with the
C-terminal cargo-binding (tail) region of Syph to identify its cargos (see
Materials and methods). We identified 25 potential cargos, including
DE-cadherin and several microtubule (MT)-regulating proteins
(Table 1). DE-cadherin was of
particular interest because mutations in human cadherin-23 and
protocadherin-15 are also linked to Usher syndrome
(Reiners et al., 2006). The
MT-associated cargos, including -tubulin, atypical protein kinase C
(aPKC), the MT severing protein Katanin-60, the MT transport protein Milton
(Milt), and the MT plus-end (+TIP) binding protein EB1, were also of interest
because MTs have been recently been shown to play an essential role in DC
(Jankovics and Brunner, 2006)
and in filopodial dynamics (Rodriguez et
al., 2003; Schober et al.,
2007).
To confirm that the proteins we identified are bona fide Syph cargos, we performed GST pulldown assays with GST fusions to a subset of these proteins (Fig. 3A; see Fig. S2A,B in the supplementary material). Consistent with their predicted roles as Syph cargos, Katanin-60, aPKC, Milt, EB1 and the intracellular domain of DE-cadherin (cadICD) all bind the C-terminal tail region of Syph (Fig. 3A). To verify that these interactions occur in vivo, we co-immunoprecipitated Syph-containing complexes from Drosophila embryo lysates. Given the disease relevance of E-cadherin and the availability of reagents, we focused on the interaction between Syph and cadICD. Western blot analysis of these Syph-immunoprecipitated complexes reveals the presence of DE-cadherin (Fig. 3B), confirming the in vivo relevance of this interaction.
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), which
shows that the regions corresponding to L1 and L3 are structurally adjacent
(Fig. 3J-J''), forming a
surface fit for binding cargo. In parallel, we mapped the binding of Katanin-60, aPKC and Milt to the same two distinct regions of the Syph FERM domain (Fig. 3F-G; data not shown). We attempted to generate mutants that were defective in binding of specific cargos using a series of targeted three to four amino acid substitution mutations within the context of Syph protein fragments encompassing either the L1 or L3 regions (Fig. 3C; data not shown). Point mutations that abolish cadICD binding (IVQG*, DAFT*, STR* and DMK*) also abolish the binding of Katanin-60 and Milt, but not that of aPKC (Fig. 3F,G). Thus, our results suggest that although Syph could transport some of its cargos simultaneously, transport of others is probably mutually exclusive.
Conversely, we mapped the region of cadICD that binds Syph. We divided the cadICD into four pieces (cadA-D) (Fig. 3H; see Fig. S2C in the supplementary material), and find that Syph binds the most C-terminal 40aa piece, cad-D (aa1467-1507) (Fig. 3H,I). This interaction domain has not previously been assigned a function, but is conserved in human cadherins (see Fig. S2D in the supplementary material).
Syph is required for proper dorsal closure
To determine whether Syph protein accumulation in LE cells and filopodia
has a functional role, we examined DC in syph-deficient embryos.
Since specific syph mutations have not yet been reported and we were
unable to generate one using imprecise P-element excision (see Materials and
methods), we used dsRNA interference (RNAi) to specifically reduce Syph
protein levels and disrupt Syph function (see Materials and methods; see Fig.
S3 in the supplementary material). We injected two different Syph dsRNAs, an
unrelated dsRNA (i.e. GFP), or buffer alone into embryos expressing a
UAS-GFP-actin fusion construct (Verkhusha
et al., 1999) under the control of the En-Gal4 driver to express
the GFP-actin fusion protein in alternating epidermal stripes, and followed
their development during DC. In buffer-injected embryos, DC proceeded normally
and was complete in 2.5-3 hours. Cells within a given segment matched with
their counterpart on the opposing side during epithelial zippering, as
indicated by the correct alignment of En pattern stripes in 96%
(n=248) of embryos (Fig.
4A-A''''). Injection of an unrelated dsRNA (GFP) also
resulted in correct alignment of En pattern stripes in 98% (n=64) of
embryos (see Fig. S3B-C in the supplementary material). In syph
dsRNA-injected embryos, although DC was complete within 3 hours, we observed
mismatching of segments in 
38% of embryos (39%, n=229 for dsRNA
#1; 37%, n=99 for dsRNA #2). As the two Syph dsRNAs produce identical
phenotypes, we show only dsRNA #1 from this point. In these mismatching cases,
one stripe (four cells), or even a single LE cell, would broaden and contact
several cells on the opposing epithelium
(Fig. 4B-C'''',F,G).
Thus, Syph is required for proper environmental sensing and cell-cell
recognition during DC.
Syph is required for proper localization of cadherin to the leading edge
Since Syph and cadherin show direct molecular association, we examined
their subcellular localization in embryos and cells. Live imaging of DC-staged
embryos expressing cadherin-GFP and Syph-RFP showed that both proteins are
present in LE cell protrusions, albeit at lower levels than actin-GFP (data
not shown). However, as the embryo LE cell filopodia were too dynamic for
capture of live dual-fluor confocal images, we co-stained Drosophila
BG2 and S2R+ cells, both of which exhibit numerous filopodia. Consistent with
their direct molecular interaction in vitro, Syph and cadherin co-localize in
puncta along the filopodia (Fig.
4H-J'; data not shown).
To test whether the syph-deficient embryos exhibit mismatching
because of cadherin mislocalization, we injected Syph dsRNA or buffer alone
into embryos expressing a GFP-DE-cadherin
(Oda and Tsukita, 1999) fusion
protein. In buffer-injected embryos, 98% (n=50) developed normally
(no-mismatching). We observed localized accumulation of GFP-cadherin at the
dorsal-most surface and in filopodia of the LE cells
(Fig. 4D-D''',K; see Movie
3 in the supplementary material). In syph dsRNA-injected embryos, we
observed a mismatching phenotype similar to that observed with the
GFP-actin-expressing embryos, as well as abnormal constriction of the LE cells
in opposing segments as a result of cells reaching laterally and matching with
other adjacent cells (68% exhibit mutant phenotypes; n=117;
Fig. 4E-E'''').
Surprisingly, these embryos progressed more slowly and failed to close during
the time they were observed (about 9 hours), which was 4-5 hours longer than
wild-type or the buffer-injected control embryos.
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9 hours) (Jankovics and
Brunner, 2006). Although this ectopic spastin expression did not
affect filopodial environment sensing or cell-cell recognition during DC, it
did disrupt the final step of zippering when adhesions are formed.
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- and β-tubulin; lack of a UAS-β-tubulin fly line permitted
us to only inject into embryos overexpressing GFP--tubulin. In control
buffer-injected embryos, the opposing segments matched up correctly and closed
within 3 hours in 99% (n=83) of embryos
(Fig. 5C-C''''). In
embryos injected with Syph dsRNA (69%, n=121) we observed an
intermediate phenotype. DC progression was slowed (
5 hours) and advancing
segment stripes puckered, similar to that observed with embryos expressing
GFP-cadherin (Fig.
4D-D'''') or GFP alone
(Fig. 5B-B''''').
However, after about 5 hours, fusion of the stripes occurred and a midline
seam was observed. We conclude that overexpression of GFP--tubulin does
not rescue the abnormal phenotypes to the extent that overexpression of
GFP-actin does. The apparent rescue of the closure phenotype, though, leaves
open the intriguing possibility that overexpression of both - and
β-tubulin, and thus MTs, may result in stronger rescue.
Syph is required for filopodia dynamics and cargo localization
The above results suggest that Syph has important roles in: (1) the
transport of essential cargo proteins required for proper segmental matching
and zippering to the leading edge, and (2) the modulation of cytoskeletal
architecture and dynamics. To further explore the first half of this
hypothesis, we took advantage of a newly created line of EB1-EYFP-expressing
flies (Jankovics and Brunner,
2006) to test the effect of Syph removal on the trafficking of
EB1, a putative Syph cargo. In buffer-injected embryos, EB1-EYFP could be seen
to move dynamically within the cells expressing it and within the numerous
filopodial extensions at the dorsal-most edge of the LE cells
(Fig. 6A).
Syph-deficient embryos displayed slower movement of EB1-EYFP within
the cells expressing it (9.0±2.4 µm/minute, compared with
11.4±2.9 µm/minute for wild-type, as determined by kymographic
analysis; P<0.0003). In addition, syph-deficient embryos
have fewer filopodia extensions with a concomitant increase in lamellipodia
(Fig. 6B; see Movie 4 in the
supplementary material). In the most severely disrupted
syph-deficient embryos, trafficking of EB1-EYFP within LE cells is
disrupted such that the predominantly apical-basal movement seen in control
embryos becomes randomly oriented (7.1±2.0 µm/minute;
Fig. 6C; see Movie 4 in the
supplementary material). Since EB1 is a microtubule tip binding (+TIP)
protein, this may also reflect a disruption of the cytoskeleton and suggests a
role for Syph in regulating cytoskeletal architecture.
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). Similar
to embryos, we find that S2R+ cells treated with Syph dsRNA exhibit fewer
cellular extensions than their untreated counterparts. In addition, the
regular latticework of MTs observed in untreated cells
(Fig. 6H,H') is disrupted
in Syph dsRNA-treated cells, with many aggregates of tubulin accumulating
within the cell body (Fig.
6I,I'). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Transport of proteins to and within these protrusions and the leading edge is
essential for the environmental sensing, cell-cell recognition and adhesion
events that take place during DC. Here we present the first characterization
of the Drosophila myosin XV homolog, Sisyphus, and show that it is
required for DC during at least two key steps: for proper epithelial
alignment, and for zippering and fusion of the two epithelial sheets. Live
imaging of Syph shows that this motor protein accumulates at the leading edge
during DC, whereas our mapping and RNAi studies demonstrate interactions with
its cargo proteins (Fig. 3),
consistent with a role as a transport protein. Our results also suggest a
possible role for Syph in the coordination of the actin and MT networks
required for the dynamic protrusions during DC.
The MyoX MyTH-FERM myosin has been proposed to play a structural role by
facilitating actin polymerization at filopodia tips by pushing the plasma
membrane away from the growing actin filament barbed ends to create a space
for actin monomer addition (cf. Sousa and
Cheney, 2005). Although Syph is present at filopodium ends and
could perform a similar role for actin or MT assembly, unlike MyoX, it is not
preferentially found at tips. Instead, Syph moves bi-directionally within LE
cells and their protrusions (Fig.
2F-J'; see Movies 1 and 2 in the supplementary material).
Reduction of Syph by RNAi disrupts filopodia formation, and this probably
contributes to the segment mismatching and zippering/fusion phenotypes
observed in syph-deficient embryos. Dictyostelium cells
mutant for the myosin-VII MyTH-FERM protein have also been shown to exhibit
loss of filopodia and adhesion defects
(Titus, 1999;
Tuxworth et al., 2001). How
filopodial formation is disturbed in syph-deficient embryos remains
to be answered, though improper distribution of cargo proteins required for
proper filopodial dynamics and integrity of the leading edge is a strong
possibility. Another interesting and nonexclusive model is that disruption of
the actin-microtubule network caused by Syph knockdown in turn disrupts
filopodial formation and/or integrity of the leading edge. Additional studies
will be required to differentiate between these two possibilities.
Our mapping data shows that Syph appears to play a key role in transporting
structural, adhesive and regulatory molecules within filopodia via its
C-terminal FERM domain (Fig.
3). This is consistent with studies in mammals that show that, in
addition to the motor domain, the FERM domain of MyoXV is critical for
development of stereocilia required for normal hearing and balance
(Anderson et al., 2000). One
Syph cargo we identified that binds to the FERM domain of Syph is DE-cadherin
(Table 1). Cadherin is required
at filopodium ends where it forms transient cell-cell contacts, followed by
more permanent cell adhesion ones. Here we show that syph-deficient
embryos are defective in epithelial fusion during DC, presumably due to the
failure of cadherin to correctly accumulate at the dorsal-most edge of LE
cells to mediate fusion and adhesion. Interestingly, this `failure to close'
phenotype is reminiscent of that observed in Rac GTPase mutants proposed to
interfere with the contact-inhibition machinery
(Woolner et al., 2005). Our
results may have clinical relevance, since mutations in the X, VIIA and XV
classes of MyTH-FERM unconventional myosins have been shown to cause deafness
and aberrant morphology of stereocilia in inner-ear hair cells. Interestingly,
in addition to MyoVIIa alleles, mapping of recessive mutations for the most
common form of hereditary deaf-blindness in humans, Usher syndrome, has
identified alleles of cadherin-23 and protocadherin-15
(Libby and Steel, 2000;
Reiners et al., 2006). The
MyoVIIa MyTH-FERM myosin has also been shown to interact with cadherin
complexes through the vezatin adaptor protein
(Kussel-Andermann et al.,
2000). Together, these observations suggest that proper
distribution and localization of cadherin is essential for normal stereocilia
formation or maintenance and may be a conserved task among MyTH-FERM
unconventional myosins.
In addition, we mapped the binding site for Syph on DE-cadherin to a
C-terminal 40aa fragment of the cadICD
(Fig. 3I). The
Drosophila genome contains 17 cadherin proteins in addition to
DE-cadherin (Hill et al.,
2001); we found that three of these show conservation within this
C-terminal 40aa motif. Interestingly, this region of DE-cadherin has not been
previously assigned a function, but shows conservation with over 15 human
cadherins (see Fig. S2D in the supplementary material), and thus may define a
novel unconventional myosin-binding domain by which cadherins are tethered and
transported.
A remarkable recent finding is the presence of and requirement for MTs in
filopodia, as well as in the final stages of zippering during fly DC
(Jankovics and Brunner, 2006;
Schober et al., 2007).
Although unconventional myosins have been generally considered as actin-based
motor proteins, members of the MyTH-FERM class were recently shown to bind to
and travel along MTs via their MyTH4 domains
(Weber et al., 2004). Thus, in
addition to trafficking on actin, Syph could traffic on MTs through its two
MyTH4 domains. Dynamic MTs have been proposed to work by regulating local
concentrations of the cadherin needed to establish and maintain cell-cell
contacts (Stehbens et al.,
2006), or by delivering actin-organizing proteins to filopodia
tips (Basu and Chang, 2007).
Thus, Syph could use MTs both as a transport substrate, and be involved in
their dynamic assembly/disassembly.
Another appealing possibility is that Syph may play a role in coordinating
the two cytoskeletons. Several of the putative cargos we identified for Syph
are MT-associated proteins: -tubulin, Katanin-60, Milt and EB1 are all
MT components or binding partners (Table
1). Transport of the MT subunit, -tubulin, and the
MT-severing protein, Katanin-60, suggest a role for Syph in the assembly and
disassembly of MTs, whereas the plus end MT-binding protein EB1 suggests a
role in stabilization and regulation. A recent study has shown that ectopic
expression of another Drosophila MT-severing protein, spastin,
resulted in delayed epithelial hole closure [taking about 9 hours instead of 3
hours to reaching completion (Jankovics
and Brunner, 2006)]. The similar phenotype that we observe in
syph-deficient embryos suggests that Syph modulates MT cytoskeleton
regulation by transporting cargo proteins essential for its regulation.
Our identification of cadherin and several MT-linked proteins as Syph
cargos and the mutually exclusive - and perhaps competitive - binding for
these cargos on the Syph FERM domain (Fig.
3), leads us to propose that one role of Syph is to coordinate the
actin and MT networks during filopodial dynamics through differential cargo
transport. Consistent with this possibility, we find that both -tubulin
and actin overexpression rescues the Syph `failure to close' phenotype, and in
the case of actin, the delayed closure phenotype as well. These results
suggest that the actin network can partially compensate for disruption of the
MT one, and that Syph, in addition to serving as a delivery system, may play a
role in the regulation of actin and MT cytoskeleton cross-talk during
processes such as DC. The finding that the binding sites for putative cargo
proteins are mutually exclusive implies that Syph itself must be regulated by
proteins that help it `choose' particular proteins to transport. Future
studies aimed at uncovering and deciphering the rules governing the choice of
cargo and transport substrate for unconventional myosin motors are likely to
provide exciting new insight into the coordinate regulation of and cross-talk
between the actin and MT cytoskeletons during highly orchestrated
morphogenetic events such as DC.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/53/DC1
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ACKNOWLEDGMENTS |
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