Cbl-associated protein regulates assembly and function of ... · replaced with the 22 bp...

RESEARCH ARTICLE 627

Development 140, 627-638 (2013) doi:10.1242/dev.085100© 2013. Published by The Company of Biologists Ltd

INTRODUCTIONInteractions between cells and the extracellular matrix (ECM) arecrucial for many biological processes. These include cell migration,directed process outgrowth, basement membrane-mediated supportof tissues and maintenance of cell shape (Bökel and Brown, 2002;Hogg et al., 2011; Nakamoto et al., 2004; Watt and Fujiwara,2011). Communication between cells and ECM proteins oftenoccurs through the action of /-integrin heterodimers, a receptorcomplex that forms adhesive contacts, including focal adhesions,hemiadherens junctions, costameres and myotendinous junctions(Bökel and Brown, 2002). In response to extracellular forces, focaladhesions undergo structural changes and initiate signaling eventsthat allow adaptation to tensile stress (Geiger et al., 2009). Vinculinis thought to be the primary force sensor in the integrin complex,mediating homeostatic adaptation to external forces (Carisey andBallestrem, 2011; Grashoff et al., 2010; Hytönen and Vogel, 2008).

Vinculin-binding partners include proteins belonging to the CAP(Cbl-associated protein) protein family (Kioka et al., 1999).However, the physiological significance of this association isunknown. Mammalian CAP proteins are components of focaladhesions in cell culture (Kioka et al., 1999; Zhang et al., 2006). Inmyocytes, CAP localizes to integrin-containing complexes calledcostameres that anchor sarcomeres to muscle cell membranes

(Zhang et al., 2007). There are three mammalian CAP proteinfamily members: CAP, Vinexin and ArgBP2 (Kioka et al., 2002).CAP associates in vitro with many proteins, including thecytoskeletal regulators Paxillin, Afadin and Filamin, vesicletrafficking regulators such as Dynamin and Cbl, and the lipid raftprotein Flotillin (Chiang et al., 2001; Mandai et al., 1999; Zhang etal., 2006; Zhang et al., 2007). In vitro studies demonstrate that CAPregulates the reassembly of focal adhesions following nocodazoledissolution (Zhang et al., 2006). However, despite extensive studieson CAP (Kioka, 2002; Zhang et al., 2006), little is known about itsfunctions in vivo. Cap (Sorbs1) mutant mice are defective in fatmetabolism, and targeted deletion of the vinexin gene results inwound-healing defects (Kioka et al., 2010; Lesniewski et al., 2007).Drosophila CAP binds to axin and is implicated in glucosemetabolism (Yamazaki and Nusse, 2002; Yamazaki and Yanagawa,2003). Analysis of CAP function in mammals is complicated bypotential functional redundancy of the three related CAP proteins.Therefore, we have examined the function of Drosophila CAP, thesingle CAP family member in Drosophila, in vivo.

The Drosophila muscle attachment site (MAS) is an excellentsystem for studying integrin signaling. Somatic muscles in eachsegment of the fly embryo and larva are connected to the body wallthrough integrin-mediated hemiadherens junctions (Brown, 2000).Somatic muscles in flies lacking integrins lose their connection tothe body wall (Brown et al., 2000; Brown et al., 2002; Clark et al.,2003; Zervas et al., 2001). Surprisingly, flies lacking Vinculin, amajor component of cytosolic integrin signaling complexes, areviable and show no muscle defects (Alatortsev et al., 1997). Thus,unlike its mammalian counterpart, Drosophila Vinculin isapparently dispensable for the initial assembly of integrin-mediatedadhesion complexes at somatic MASs.

The fly MAS is structurally analogous to the fly chordotonalorgan. These organs transduce sensations from various stimuli,including vibration, sound, gravity, airflow and body wallmovements (Caldwell and Eberl, 2002; Kamikouchi et al., 2009;

1Solomon H. Snyder Department of Neuroscience, The Johns Hopkins UniversitySchool of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. 2HowardHughes Medical Institute, The Johns Hopkins University School of Medicine, 725North Wolfe Street, Baltimore, MD 21205, USA. 3Department of Neurology, TheJohns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore,MD 21205, USA. 4Department of Biology, The University of Iowa, Iowa City, IA52242, USA. 5Janelia Farm Research Campus, Howard Hughes Medical Institute,Ashburn, VA 20147, USA. 6Department of Cell Biology, The Johns HopkinsUniversity School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

*These authors contributed equally to this work‡Author for correspondence ([email protected])

Accepted 21 November 2012

SUMMARYCbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, itsphysiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-richstructures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, whichform an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrantjunctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse ofscolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the rolesof different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of theCAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopyDrosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at themembrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin proteincomplex in stretch-sensitive organ assembly and function.

KEY WORDS: CAP, Integrin, Vinculin, Muscle attachment site, Chordotonal organ, Drosophila

Cbl-associated protein regulates assembly and function oftwo tension-sensing structures in DrosophilaRajnish Bharadwaj1,2,3, Madhuparna Roy4,*, Tomoko Ohyama5,*, Elena Sivan-Loukianova4,*, Michael Delannoy6, Thomas E. Lloyd1,3, Marta Zlatic5, Daniel F. Eberl4 and Alex L. Kolodkin1,2,‡

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Kernan, 2007; Yack, 2004; Yorozu et al., 2009). The chordotonalorgan is composed of individual subunits called scolopidia, eachcontaining six cell types: neuron, scolopale, cap, ligament, capattachment and ligament attachment cells (Todi et al., 2004).Chordotonal neurons are monodendritic, and their dendrites arelocated in the scolopale space, a lymph-filled extracellular spacecompletely enveloped by the scolopale cell (Todi et al., 2004).Within the scolopale cell, a cage composed of actin bars, calledscolopale rods, facilitates scolopale cell envelopment of thescolopale space (Carlson et al., 1997; Todi et al., 2004). Thus, likethe MAS, the actin cytoskeleton plays a specialized role in definingchordotonal organ morphology. Similarities between MASs andchordotonal organs include the requirement during development inboth tendon and cap cells for the transcription factor Stripe (Inbalet al., 2004). Furthermore, both of these cell types maintainstructural integrity under force and so are likely to share commonmolecular components dedicated to this function.

Here, we show that the Drosophila CAP protein is selectivelylocalized to both muscle attachment sites and chordotonal organs.In Drosophila CAP mutants we observe morphological defects thatare indicative of actin disorganization in both larval MASs and thescolopale cells of Johnston’s organ in the adult. The morphologicaldefects in scolopale cells result in vibration sensation defects inlarvae and hearing deficits in adults. We also find that, like itsmammalian homologues, Drosophila CAP interacts with Vinculinboth in vitro and in vivo. These results reveal novel CAP functionsrequired for actin-mediated organization of cellular morphology,lending insight into how CAP mediates muscle and sensory organdevelopment and function.

MATERIALS AND METHODSDrosophila geneticsDrosophila CAP deletion mutants were generated by imprecise excisionof the P-element CAPCA06924 inserted in the intron proximal to the SH3domain-coding exons. We generated multiple excisions, two deleting thefirst two SH3 domain-coding exons. These deletions are dCAP49e anddCAP42b, and delete 2.9 kb (genomic region 2R:6,190,378-6,193,350)and 2.7 kb (genomic region 2R:6,190,378-6,193,141), respectively,downstream of the CAPCA06924 P-element. A precise excision wegenerated called CAP49a was used as wild-type control.CAPCA06924,mys1, rhea1, flotillin-2KG00210 and tensin mutant by1 werefrom the Bloomington Stock Center; zasp9 and zasp56 mutants were agift from Frieder Schock (McGill University, Montreal, Canada).Cheerio and paxillin RNAi lines were obtained from the ViennaDrosophila Research Center.

Drosophila CAP miRNA constructs target the sequencesCAGCTATGTTGAGATTGTCAGT and TTCAATACGCTGAC -GCAAAATG in the CAP-E transcripts encoding the C-terminal CAP SH3domains. These constructs were generated as described previously (Chenet al., 2007). The stem-loop backbone and surrounding sequences wereunaltered, and the Drosophila Mir6.1 gene-targeting miRNA sequence wasreplaced with the 22 bp complementary to the CAP transcript. Two mRNAconstructs targeting distinct CAP sequences were inserted in tandem intothe pUAST vector and used to generate transgenic flies.

Drosophila CAP antibodyThe Drosophila CAP short isoform (CAP-E), which encodes the three SH3domains, was cloned into the pGEX4T-1 vector to generate a GST fusionprotein. Purified GST-CAP-E was injected into rabbits for polyclonalantibody production. GST-CAP-E-bound Affigel beads were used toaffinity-purify CAP-specific antibodies.

Immunostaining and microscopyDrosophila embryos were processed for immunostaining as describedpreviously (Patel, 1994). For most experiments, fixation used 4%

paraformaldehyde. For examination of larval muscle attachment sites, filletpreparations of wandering third instar larvae were prepared in PBS, asdescribed previously (Brent et al., 2009). For Johnston’s organ staining,adult heads with the proboscis removed were fixed in 4% PFA for90 minutes at 4°C. After three washes with PBS, heads were incubated in12% sucrose overnight and subsequently frozen in OCT. Cross-sections (20m) were cut using a cryostat. The following antibodies were used: anti-PS integrin at 1:100 (DSHB), 22C10 at 1:200 (DSHB), 21A6 at 1:200(DSHB), rabbit anti-85e-tubulin at 1:20 [a gift from T. C. Kaufman(Indiana University, Bloomington, IN, USA) and A. Salzberg (RappaportInstitute, Haifa, Israel)], rabbit anti-CAP at 1:1000 (our antibody), rabbitanti-Zasp at 1:1000 (a gift from F. Schock), rabbit anti-pinch at 1:500 (agift from M. C. Beckerle, University of Utah, Salt Lake City, UT, USA),anti-thrombospondin at 1:400 (a gift from T. Volk, Weizmann Institute ofScience, Rehovot, Israel), mouse anti-tiggrin at 1:200 (a gift from L.Fessler, UCLA, Los Angeles, CA, USA). Images were acquired using aZeiss LSM 510 confocal microscope.

Electron microscopyDrosophila third instar larval fillets from dCAP49e mutants and controlswere processed as described with minor variations (Ramachandran andBudnik, 2010). Larval fillets were fixed with 1% glutaraldehyde and 4%paraformaldehyde in cacodylate buffer at 4°C overnight, stained first with2% osmium tetroxide at 4°C for 1 hour and then with 2% uranyl acetatefor 30 minutes. Samples were dehydrated by incubating with increasingconcentrations of ethanol and equilibrated in varying concentrations ofpropylene oxide and Epon resin. Larval fillets were pinned to embeddingmolds using insect pins and embedded in Epon resin. Horizontal ultrathinsections of the larval fillet were cut using an ultramicrotome. Sections weresubsequently stained with uranyl acetate and lead citrate; TEM wasperformed using a Hitachi H-7000 transmission electron microscope.Ultrastructural analysis of Johnston’s organs was performed as describedpreviously (Todi et al., 2005).

ImmunopurificationFor each immunopurification experiment, 1.5 g of Drosophila embryos and100 g of Drosophila CAP antibody were used. Immunopurification wasperformed as described previously (Bharadwaj et al., 2004). The proteinbands specific to wild-type lysates were excised, pooled, subjected totrypsin digestion and used for MALDI-TOF mass spectrometry analysis(Johns Hopkins Medical School Mass Spectrometry facility).

Vibration sensation assayThe vibration sensation assay was performed as described previously(Swierczek et al., 2011; Wu et al., 2011). Approximately 96-hour-old thirdinstar larvae of the specified genotypes were selected using GFP-balancerchromosomes. Larvae were placed on an agar plate lying directly over aspeaker that provides pulses of vibration stimuli. Larvae were subjected toeight 1000 Hz, 1 V 1-second pulses followed by a 30-second pulse. Usingthe Multi-Worm Tracker (MWT) and choreography software (Swierczeket al., 2011), the behavior of the entire larval population (n=~100) on thedish was simultaneously tracked and analyzed. The hunch response wasassessed by measuring the average mean midline body length of the entirelarval population before and after vibration stimuli.

Auditory nerve electrophysiologyElectrophysiological recording of sound-evoked potentials (SEPs) wasperformed as described previously (Eberl et al., 2000; Eberl and Kernan,2011).

RESULTSDrosophila CAP genomic organization and CAPmutant generationDrosophila CAP is the only member of the CAP gene family inDrosophila. Like its mammalian counterparts, Drosophila CAPpossesses three C-terminal SH3 domains and an N-terminal SoHo(sorbin homology) domain (Fig. 1A). There are three mammalianCAP proteins: CAP, vinexin and ArgBP2. Drosophila CAP shows

RESEARCH ARTICLE Development 140 (3)

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the highest amino acid sequence similarity to mammalian CAP(Fig. 1B) (Yamazaki and Nusse, 2002). Genomic DNA sequence,northern blot and western blot analyses reveal that there aremultiple CAP isoforms in Drosophila, similar to mammalian CAPs(Fig. 1C) (Yamazaki and Nusse, 2002). The only coding regionshared by all predicted Drosophila CAP protein isoforms is at theC terminus and encodes the SH3 domains. Through impreciseexcision of a P transposable element (CAPCA06924), we generatedtwo deletions, dCAP42b and dCAP49e, that remove the first two ofthe three conserved SH3 domains, and also a precise excision,dCAP49a, that serves as a control (Fig. 1A,C). Drosophila CAPmutants are viable and fertile. Western blot analysis of Drosophilaembryonic and adult whole-body lysates using a CAP polyclonalantibody we raised against the Drosophila CAP C-terminal SH3domains reveals multiple protein isoforms in wild-type flies thatprobably correspond to different Drosophila CAP isoforms; thesebands are absent in dCAP42b and dCAP49e whole-body lysates(Fig. 1D).

The imprecise CAP excision mutants dCAP49e and dCAP42b

likely result in complete loss of CAP protein, but there remains thepossibility that the N-terminal region of CAP might be generatedin these mutants. Therefore, we also used a miRNA-mediatedknock-down strategy to assess CAP loss-of-function (Chen et al.,2007). We generated transgenic fly lines harboring a constructcontaining two miRNAs, in tandem, that targeted two distinct C-terminal CAP sequences in the open reading frame, a regionconserved in all predicted CAP miRNA products (Fig. 1C).Western blot analysis of adult whole fly body lysates from flies

expressing the CAP tandem miRNAs under control of an actin-Gal4 driver did not detect any CAP protein (Fig. 1D). FunctionalmiRNAs targeting any site in a mRNA result in degradation, orblockade of translation, of the entire mRNA (Du and Zamore,2007); flies ubiquitously expressing these miRNAs probably lackall N-terminal CAP protein.

CAP localizes to muscle attachment sites andchordotonal organ scolopale cellsWe next analyzed CAP protein expression throughout developmentusing our CAP polyclonal antibody, which most probablyrecognizes all predicted Drosophila CAP isoforms. In stage 16embryos, CAP is expressed predominantly in chordotonal sensoryorgans, somatic muscles, the dorsal vessel (the Drosophila heart)and the gut (Fig. 2A,C,E). CAP immunostaining is specific forendogenous CAP protein as dCAP49e mutants, which lack thegenomic region encoding the CAP SH3 domains, exhibit no CAPprotein (Fig. 2B,D,F). In somatic muscles, CAP colocalizes withthe PS integrin subunit at muscle attachment sites (MASs)(Fig. 2E). In third instar larvae, CAP protein expression persists atMASs and also at Z-lines of somatic muscles, although Z-lineexpression is lower in comparison with MAS expression (seebelow: Fig. 5A; supplementary material Fig. S1C).

Prominent CAP localization is also found in embryonicchordotonal sensory organs, identified by the five scolopale cellsof the pentascolopidial organ in each embryonic hemisegment(Fig. 2C). We immunostained wild-type and dCAP49e embryos withCAP antibodies and the 22C10 monoclonal antibody, which stains

629RESEARCH ARTICLECAP-mediated tension sensing

Fig. 1. Drosophila CAP protein domainorganization, CAP genomic organization andgeneration of CAP loss-of-function mutants. (A) Domain organization of CAP protein domains, withCAP mutants shown below protein domain schematic.(B) Sequence alignment of CAP and its mammalianhomologs. (C) Schematic of predicted CAP cDNAtranscripts and CAP deletion mutants. The P-elementCAPCA06924 (blue triangles) was used to generate twoCAP deletion mutants: CAP42b and dCAP49e; the preciseexcision dCAP49a was used as a wild-type control. Anenlarged image of isoform CAP-D is shown: blue linesindicate the extent of each deletion. (D) Whole-bodylysates from adult flies of indicated genotypes wereimmunoblotted with anti-CAP (upper panel) and anti-actin (lower panel).

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chordotonal neurons (Fujita et al., 1982). These experiments showthat CAP is expressed specifically in scolopale cells, which envelopthe dendrites of chordotonal neurons (Fig. 2C). In dCAP49e

embryos, we observed no anti-CAP chordotonal immunostaining(Fig. 2D). In third instar larvae, CAP is also localized inchordotonal organs, predominantly within scolopale cells but alsoweakly in cap cells (Fig. 3A,B). The MAS, Z-disk and scolopalecell-specific localization of CAP is also recapitulated by a CAPGFP-trap transgenic line (Buszczak et al., 2007), which harbors aGFP transgene in the endogenous CAP genomic locus thatexpresses a CAP-GFP fusion protein (supplementary material Fig.S1B,C). Thus, in embryonic and larval developmental stages, CAPlocalizes to two actin-rich structures: MASs and chordotonal organscolopale cells (Todi et al., 2004).

CAP is required for larval chordotonal organfunctionWe next performed a recently described behavioral assay thatprovides a sensitive readout of chordotonal organ-mediatedlocomotor response to vibration (Swierczek et al., 2011; Wu et al.,2011). Fly chordotonal organs are mechanosensory organs thatmediate proprioception in larvae and transduction of acousticsignals in adults (Caldwell et al., 2003; Eberl, 1999). Wild-typethird instar larvae exhibit a characteristic biphasic vibrationresponse. Larvae first display a fast startle response to vibration byhunching their bodies, followed by a second, relatively slower,head-turning response. These responses are largely dependent onthe chordotonal sensory neurons and can be precisely quantified(Wu et al., 2011). The ‘hunch’ response to vibration is quantifiedby measuring the average larval body length reduction. The meannormalized larval body length is measured over the larval midlinebefore and after a stimulus: for control dCAP49a (precise excision)larvae, this was 1.01±0.11 (before) and 0.94±0.1 (after); fordCAP42b heterozygotes, it was 0.99±0.07 (before) and 0.91±0.07(after) (n=100 larvae for each genotype) (Fig. 3C). Thus, wild-typelarvae show a significant (P<0.01) reduction in mean normalizedbody length in response to vibration. By contrast, dCAP49e anddCAP42b mutant larvae do not exhibit a significant hunchingresponse to vibration (Fig. 3C). The mean normalized midline bodylength before and after stimuli for dCAP49e was 0.99±0.08 (before)and 0.98±0.1 (after); and for dCAP42b mutants it was 0.99±0.08(before) and 1±0.09 (after) (n=100 larvae per genotype). This samephenotype was also observed when dCAP42b or dCAP49e alleleswere placed in trans to a large chromosomal deficiency spanningthe entire CAP locus (Fig. 3C). Scolopale cell-specific expressionof CAP rescues this vibration-sensation defect observed in CAPmutants, and scolopale cell depletion of CAP by driving CAPmiRNA using the NompA-Gal4 driver (Chung et al., 2001)phenocopies CAP mutants (Fig. 3C).

CAP is also enriched at the cap cell attachment site to theectoderm (but not in ligament cells) (supplementary material Fig.S2A-B�). Integrin is also enriched at these sites. We examined PSintegrin localization in cap cells of dCAP49e mutants, and indCAP49e mutants integrin localization in cap cells and the overallmorphology of chordotonal organs, revealed by anti-85e-tubulinlabeling, is apparently normal (supplementary material Fig. S2C-D�). These results suggest chordotonal defects observed in dCAP49e

mutants arise primarily from the lack of scolopale cell CAP.

Structural and functional Johnston’s organdefects in adult CAP mutantsWe next examined a distinct chordotonal organ class: the adultJohnston’s organ. The Johnston’s organ resides in the secondantennal segment and is composed of ~250 scolopidia (Todi et al.,2004). Johnston’s organ scolopidia and larval chordotonal organsshow remarkable morphological similarities. Several proteins,including Atonal and Beethoven, serve specialized functions inboth of these organs (Caldwell et al., 2003; Todi et al., 2004). CAPis strongly expressed in wild-type Johnston’s organs but not indCAP49e mutants (Fig. 4A). Immunostaining with phalloidin,which illuminates Johnston’s organ scolopale rods (Todi et al.,2004), and with the 22C10 mAb, which labels sensory neurons,reveals CAP in Johnston’s organ scolopale cells, similar to ourobservations in embryonic chordotonal organs (Fig. 4A).

Electrophysiological analyses to detect auditory responses wereperformed by recording from the auditory nerve following a matingsong stimulus (Eberl et al., 2000; Eberl and Kernan, 2011). The


Fig. 2. Embryonic expression of CAP protein. (A-D) Wild-type (A,C)and dCAP49e (B,D) embryos stained with anti-CAP and 22C10 antibody(which labels all sensory neurons). (A,B) Wild-type embryos show CAPstaining in chordotonal organs (arrowheads), dorsal vessels (thinarrows) and gut (thick arrows); this staining is absent in CAP mutants.(C,D) Higher magnification image of CAP localization in chordotonalorgan scolopale cells (arrows) in wild type, but not dCAP49e,embryos.(E,F) Wild-type and dCAP49e embryos immunostained with anti-CAPand anti-PS integrin; CAP colocalizes with PS integrin at MASs(arrows). Scale bars: 60 µm in A,B; 20 µm in C-F.

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electrophysiological response to mating songs was significantlydiminished in dCAP49e mutants. The average response amplitudefor dCAP49a (precise excision control) was 843±52 V, comparedwith 603±46 V in CAP mutants (n=27 animals per genotype)(Fig. 4C). We next depleted CAP from all scolopale cells in adultflies by expressing CAP miRNA under the control of the NompA-Gal4 driver (Chung et al., 2001). This resulted in a significantreduction in response amplitude: 703±63 V, compared with991±65 V when the same CAP miRNA is driven in muscles(n=21 animals for each genotype).

We next examined larval chordotonal and adult Johnston’s organmorphologies using several markers, including the 21A6 mAb(described above), NompA-GFP (which labels the extracellularmatrix in the scolopale space, called the dendritic cap) (Chung etal., 2001), and anti-neurexin (which labels cell junctions betweenscolopale cells and also those between scolopale and CAP cells)(Baumgartner et al., 1996). None of these markers revealedobvious abnormalities in CAP mutant embryos or larvae(supplementary material Fig. S3A; data not shown). However,ultrastructural transmission electron microscopy (TEM) analysis ofJohnston’s organ revealed that CAP mutant scolopale cells aredysmorphic and appear collapsed and ruptured (Fig. 4B). This is in

sharp contrast to wild-type Johnston’s organs, in which scolopalecells appear circular in cross-section, are of equal size and showfairly regular spacing (Fig. 4B). This morphological defect isconsistent with CAP maintaining cytoskeletal structural integrity.In addition, sensory neurons in CAP mutant Johnston’s organsoften show accumulation of vacuolar structures (supplementarymaterial Fig. S3B). As CAP localizes predominantly to scolopalecells, these defects are probably secondary to scolopale cellmorphological defects and may result from partial disruption of thelymph environment surrounding sensory neuron dendrites in theJohnston’s organ. Taken together, these data show that CAPmutants exhibit functional and morphological Johnston’s organdeficits; the highly localized expression of CAP in scolopale cells,both in Johnston’s organ and the larval chordotonal organ,implicates CAP in the regulation and/or maintenance of scolopalecell membrane morphology.

CAP mutants exhibit morphological defects atsomatic body wall muscle attachment sitesAs CAP is robustly localized to embryonic and larval muscleattachment sites (MASs), we next examined the morphology ofMASs in CAP mutants. As shown by immunostaining for PSintegrin (an integral component of the MAS) and also for actin,stage 16 embryonic MASs in CAP mutants appear normal (Fig. 2F;data not shown). However, in third instar larvae, we find significantmorphological abnormalities in CAP mutants. Filleted preparationsof dCAP49e third instar larvae were stained with rhodamine-phalloidin to determine MAS actin localization. In wild-typelarvae, the somatic body wall MAS is bounded on either side by asmooth band of actin (Fig. 5B). Phalloidin staining also reveals thatadjacent muscles are apposed to one another with a small gap, ofuniform width over its entire length, separating adjacent body wallmuscles. By contrast, actin organization at the MAS in CAP mutantlarvae is severely disrupted, resulting in a jagged boundary at thegap where the MAS is located (Fig. 5B); this is in stark contrast tothe smooth boundaries at this gap observed in wild-type larvae.Furthermore, we observed a significantly enlarged gap betweenactin filaments from adjacent muscles at most MASs in thesemutants (Fig. 5B). We examined MAS morphology betweenlateral-ventral somatic muscles 6 and 7 in abdominal segments(A2-A5). For each genotype, 30 hemisegments from five to sevenlarvae were scored, revealing that this phenotype is fully penetrantin both dCAP49e and dCAP42b mutants.

At wild-type MASs, integrin subunits are localized to a smooth,compact band lining the narrow gap between muscles of adjacentsegments (Fig. 5B). However, in CAP mutants this band of integrinstaining is much broader and includes numerous invaginations intoadjacent muscles (Fig. 5B). The average length of the integrin-positive invaginations into the muscles adjacent to the MAS inwild-type larvae is 2±0.18 M; in dCAP49e mutants it isdramatically increased to 13±0.42 M (n=30 hemisegments pergenotype) (Fig. 5D). The penetrance and expressivity of thisphenotype were not enhanced when the dCAP49e allele was placedin trans to a deficiency spanning the entire CAP genomic region,indicating that dCAP49e is either a null, or a very strong loss-of-function, allele (Fig. 5D). In addition, MASs in CAP mutants showpartial detachments in ~25% of larval hemisegments, a phenotypesuggesting partial disruption of integrin function.

The MAS is composed of two different cell types:multinucleated muscle fibers and tendon cells that connect themuscle fiber to the body wall (Brown, 2000). To determine whichof these cell types require CAP function, we selectively depleted


Fig. 3. Vibration sensation defects in CAP mutants. (A,B) Wild-typeand dCAP49e larvae immunostained with anti-CAP (green) and 21A6antibody (red) (which labels Eys/Spam, an extracellular matrix proteininside the scolopale space). Anti-CAP labels scolopale cells and lateraltransverse muscle (LTM1) MASs. (C) Normalized midline length of larvaeof indicated genotypes before and after exposure to vibration stimuli(n=100 larvae per genotype); data are mean±s.e.m. **P<0.01. Scalebar: 30 µm.

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CAP by driving expression of the CAP tandem miRNAs transgenein muscle cells using the Mef2-Gal4 driver (Ranganayakulu et al.,1996), or in tendon cells using the stripe-Gal4 driver (Dorfman etal., 2002). Strikingly, CAP MAS localization was almostcompletely abolished when CAP miRNAs were expressed undercontrol of Mef2-Gal4, whereas CAP localization was unalteredwhen CAP miRNAs were driven by stripe-Gal4 (Fig. 5C).Therefore most, if not all, MAS CAP protein is derived frommuscles in third instar larvae. miRNA-mediated muscle-specificdepletion of CAP phenocopies all attributes of the dCAP49e

phenotype, both qualitatively and quantitatively, providing furtherevidence that CAP function in muscles is required for MAScytoskeletal organization (Fig. 5C,D). Selective removal of CAP intendon cells did not result in actin organization defects, but it didresult in a somewhat broader integrin distribution. However, thisphenotype is mild compared with muscle-specific depletion ofCAP, or with CAP mutants (Fig. 5B-D). We speculate that the mildintegrin localization defects we observed using stripe-Gal4 drivenCAP miRNA arise from loss of CAP function in tendon cells atearlier stages of development. Importantly, we do not observe leakyexpression of stripe-Gal4 in muscle cells (data not shown). We alsoexamined dCAP49e mutant MASs in lateral transverse muscles.Unlike longitudinal muscles, which are attached both to tendoncells and to the muscles in the next segment, lateral transversemuscles are attached to the body wall only through tendon cells.Lateral transverse MASs appear normal in dCAP49e mutants(supplementary material Fig. S4A), suggesting that CAP isessential for the assembly of muscle-muscle junctions but isdispensable for muscle-tendon cell junctions.

CAP mutants also exhibit two additional defects. First, near themuscle cell surface, aberrant muscle structure (supplementarymaterial Fig. S4B, arrow), with Z-lines oriented perpendicular to

the MAS (as opposed to the horizontally aligned Z-lines observedin control animals), is observed in ~30% of dCAP49e larvae (n=30)(supplementary material Fig. S4B). Second, close to the musclemembrane, the parallel arrangement of actomyosin filaments isdisrupted (arrowhead) in ~15% of CAP mutants (n=30)(supplementary material Fig. S4B, arrowhead). Lateral adhesionsbetween muscles are normal in CAP mutants. Taken together, thesedata show that CAP, unlike certain integrin signaling components,is dispensable for initial assembly of the MAS but is required forMAS development and maybe also maintenance later indevelopment.

Altered distribution of integrin signalingcomponents at the MAS in CAP mutantsMammalian CAP proteins are implicated in integrin signaling(Kioka et al., 2002; Zhang et al., 2006), so we next investigatedwhether CAP functions to assemble integrin signaling complexcomponents. We examined the distribution of MAS extracellularmatrix proteins tiggrin and thrombospondin (Bunch et al., 1998;Subramanian et al., 2007), integrin subunits and the integrinsignaling proteins pinch, Ilk and zasp (Geiger and Yamada, 2011;Jani and Schöck, 2007) at the CAP mutant MAS (Fig. 6A,B;supplementary material Fig. S5; and data not shown). We observedno difference in the levels or colocalization of these proteins atMASs in CAP mutants. However, each of these integrin-associatedproteins displays the aberrant, broad, MAS distributionaccompanied by numerous aberrant invaginations into adjacentmuscles that we observe for PS integrin expression in CAPmutants and CAP miRNA knock-down larvae (Fig. 6A,B;supplementary material Fig. S5; and data not shown).

To better characterize the ectopic CAP mutant MAS junctionalinvaginations revealed by anti-PS integrin immunostaining, we


Fig. 4. Audition defects in CAP mutants. (A) Antennalsections at the level of Johnston’s organ from wild type anddCAP49e mutants immunostained with phalloidin (red), anti-CAP(green) and 22C10 antibody (blue). CAP colocalizes withphalloidin, which stains actin rods in scolopale cells (arrows). (B) Electron microscopic images of ultrathin sections from wild-type and dCAP49e Johnston’s organs. CAP mutants showdysmorphic and collapsed scolopale cells (arrows). (C) Sound-evoked potentials recorded from the auditory nerves of adultflies. dCAP49e mutants (n=27 animals) and flies lacking CAPspecifically in scolopale cells (n=21 animals) show a reduction inamplitude of response to sound when compared with controls (t-test: ***P<0.005). Data are mean±s.e.m. Scale bars: 30 µm in A;1 µm in B.

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examined MAS ultrastructure in wild-type and CAP mutant thirdinstar larvae using TEM. We found that close to the muscle cellmembrane, myofilaments at MASs are disorganized in CAPmutants (Fig. 6C, thick arrow). Furthermore, we observe numerousjunctional invaginations in CAP mutant animals that are larger thanthose seen in wild-type larvae (Fig. 6C). This is commensuratewith our light-level observations of MASs in CAP mutants(Fig. 5B). The presence of these significantly enlargedmembranous infoldings, and also the cytoskeletal defects observedin our ultrastructural analysis, suggests that CAP regulatescytoskeletal organization and affects muscle membranemorphology at MASs.

Distinct functions of CAP isoforms at larval MASsCAP protein isoforms are observed from insects to mammals(Kioka et al., 2002). Therefore, we next assessed different CAPisoforms for rescue of CAP mutant MAS defects using cell-type-specific expression in vivo. We assessed rescue ability of a CAPisoform called CAP-C, which includes all CAP protein domains,and also the smaller CAP isoform, CAP-E, which includes only theC-terminal SH3 domains (Fig. 7A). In our immunoblottingexperiments, the presence of 100 kDa and 30 kDa bands, which areclose to the expected sizes of CAP isoforms C and E, respectively,suggests that these isoforms are found in vivo (Fig. 1D). Whenexpressed in muscles using the Mef2-Gal4 driver, myc-tagged

CAP-C fully rescues the MAS defects observed in CAP mutants(Fig. 7B,D). However, there are differences in the distribution ofthe Mef2-Gal4-driven CAP-C isoform and endogenous CAPprotein. The CAP-C isoform is localized evenly to both Z-lines andMASs, whereas endogenous CAP protein shows preferentialtargeting to MASs (compare Fig. 7B and Fig. 5A). The CAP-Eisoform possesses only the three CAP SH3 domains and noadditional CAP N-terminal domains, and it does not rescue theCAP MAS phenotype (Fig. 7A,B,D). Interestingly, this isoform islocalized predominantly to MASs and not to the Z-lines (Fig. 7B).Therefore, regions of N-terminal CAP are required for targetingCAP protein to Z-lines, whereas the CAP C-terminal SH3 domainsare sufficient for targeting to the MAS. Furthermore, though the N-terminal CAP protein motifs are dispensable for targeting to theMAS, these motifs are required for CAP function at MAS, giventhe inability of the CAP-E isoform to rescue CAP mutants. We alsoobserved that a CAP isoform lacking the conserved SoHo domainis able to rescue the CAP MAS phenotype (Fig. 7B,D). Therefore,the SoHo domain is not required for CAP function at the MAS.

Muscle-specific overexpression of full-length CAP-C, or theCAP-C isoform lacking the SoHo domain, did not result in MASdefects, either at embryonic or third instar larval stages (Fig. 7C,D;data not shown). However, overexpression of the short CAPisoform (CAP-E), which includes only the C-terminal SH3domains, did lead to defects in actin organization and integrin


Fig. 5. Muscle-attachment defects in CAP mutants.(A) Wild-type third instar larval fillet preparationsimmunostained with CAP antibody show CAP localizationat the muscle Z-lines (arrowheads) and muscle-attachmentsites (MASs) of muscles 6 and 7 (arrows). (B) Confocalimages of larval MASs at muscles 6 and 7 stained withphalloidin and anti-PS integrin. Arrowheads indicateMASs. Phalloidin staining reveals larger MAS gaps in CAPmutants. Numerous integrin-labeled junctionalinvaginations are presents in CAP mutants (arrows). (C) Larval fillets with cell-type-specific depletion of CAP,stained with indicated antibodies. (D) Quantification ofMAS defects in larvae of indicated genotypes. Data aremean±s.e.m. Thirty abdominal segments (A2-A5) wereanalyzed for each experiment. ***P<0.005. Scale bars: 60µm in A; 30 µm in B,C.

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distribution at MASs that were very similar to those observed inCAP mutants (Fig. 7C,D). Though these CAP-E gain-of-functiondefects are not enhanced by removal of one copy of CAP(supplementary material Fig. S6), co-expression of full-lengthCAP-C and also CAP-E suppresses these defects (supplementarymaterial Fig. S6). Therefore, the short CAP isoform acts in vivo ina dominant-negative fashion, probably through sequestration ofCAP-associated proteins, thereby inhibiting endogenous CAPfunction.

Integrin and Talin are required for CAPrecruitment to the embryonic MASTo examine CAP recruitment, or stabilization, at the larval MAS,we examined its localization in embryos lacking integrin-signalingpathway components. Wild-type embryos show robust CAPlocalization at MASs (Fig. 2E; Fig. 8A�). PS integrin mutants(mys1) exhibit the classic myospheroid phenotype (Brown et al.,2000). MAS CAP localization is missing in mys1 mutants (Fig. 8B-B�). This is also consistent with integrin recruitment to MASs priorto CAP recruitment, whereas CAP recruitment to scolopale cells(arrowheads) is independent of integrins (Fig. 8E-G�). In stage 14embryos, integrin is expressed at the MAS but CAP is not. At stage15, CAP expression is observed in regions of the MAS, whereas,at stage 16, CAP expression completely colocalizes with PSintegrin (Fig. 8E-G�).

CAP localization in talin mutants (rhea1) (Brown et al., 2002)reveals a dramatic decrease in CAP MAS levels, though a lowlevel of CAP expression is still detectable at MASs in thesemutants. These mutants often show MAS splitting, observableby CAP localization in two bands, not one (Fig. 8C�, whitearrows). This may reflect partial detachment of muscles from thebody wall. We also examined CAP localization in embryoslacking zasp, which is implicated in integrin signaling (Jani andSchöck, 2007), and we find that CAP MAS localization is

unperturbed, both at stage 16 and 17 (Fig. 8D). Thus, bothintegrin and Talin are required for proper localization of CAP atthe embryonic MASs, suggesting CAP signals downstream ofintegrins at the MAS.

Depletion of Vinculin results in MAS defects anddisruption of CAP MAS localizationThere are several known CAP-interacting proteins, includingFlotillin, Tensin, Filamin and Paxillin (Kioka et al., 2002; Zhang etal., 2007). We used null mutants or RNAi-mediated knockdown todetermine whether any of these proteins phenocopy CAP mutants;however, none did (supplementary material Fig. S7A). We thensought to identify proteins required for CAP function,immunopurifying endogenous CAP from wild-type and CAPDrosophila embryo lysates using our CAP polyclonal antibody. Weisolated protein bands present in wild-type, but not the CAPmutant, immunoprecipitates (Fig. 9A). These bands were pooledand sequenced using MALDI TOF mass spectrometry. The mostenriched protein in CAP immunoprecipitates (aside from CAPitself), as indicated by the number of peptides corresponding to anyparticular protein, was Vinculin. We also identified Talin as a CAP-interacting protein. The other proteins identified in CAPimmunoprecipitates were 5-oxo-prolinase, Nervous wreck, 14-3-3and histone H2A.

Vinculin interacts with all mammalian CAP proteins in vitro(Kioka, 2002; Kioka et al., 2002; Zhang et al., 2006); however,the functional significance of this interaction is unknown. Weexpressed HA-tagged Vinculin with, or without, Myc-CAP-E(the CAP isoform consisting only SH3 domains) in S2R+ cellsand immunoprecipitated CAP with Myc antibodies. We foundthat immunoprecipitation of Myc-CAP-E using anti-Mycrobustly co-immunoprecipitated HA-Vinculin (Fig. 9B). We nextexamined a chromosomal inversion (In(1LR)pn2a) that disruptsthe vinculin gene (Alatortsev et al., 1997). We first assessed the


Fig. 6. MASs in CAP mutant larvae exhibit altereddistribution of integrin signaling components.(A) Wild-type and dCAP49e larvae stained with anti-Pinch and phalloidin. (B) Wild-type and dCAP49e larvaestained with anti-Zasp; both Pinch and Zasp show abroader distribution in CAP mutants compared with thecontrol animals (arrows in A,B). (C) Electron microscopicimages of muscle-attachment sites from wild-type anddCAP49e 3rd instar larvae reveal larger junctionalinvaginations in the dCAP49e animals (arrows) and alsomyofilament disorganization (thick arrow). Scale bars:30 µm in A,B; 2 µm in C.

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significance of Vinculin-CAP interactions in audition byelectrophysiological recordings from auditory nerves of wild-type and vinculin mutant flies, and did not observe anysignificant difference in sound evoked potentials between wildtype and vinculin mutants (supplementary material Fig. S7B).This suggests that CAP functions in chordotonal organs in aVinculin-independent manner. However, MASs in vinculinmutants show with full penetrance a marked reduction in CAPlevels, though CAP Z-band localization remains unaltered(Fig. 9C). We observed similar morphological defects inIn(1LR)pn2a homozygous mutants and in animals carrying thismutation over a deficiency that includes the entire vinculin gene,suggesting the phenotypic defects in In(1LR)pn2a homozygousmutants result from complete disruption of vinculin (data notshown). This suggests Vinculin plays a crucial role in therecruitment of CAP to the MAS, reminiscent of Vinculin-dependent recruitment of CAP to mammalian focal adhesions(Takahashi et al., 2005). Examination of actin staining invinculin mutants reveals that, as in CAP mutants, the actin bandslining the MAS are serrated and flank a gap wider than thatobserved in wild-type animals (Fig. 9C,D). However, this gap isnot as wide as in CAP mutants, and vinculin mutants show fewerPS integrin-labeled junctional invaginations relative to CAPmutants (Fig. 9C,D). These results show that CAP and itsbinding partner Vinculin function together as key regulators ofcytoskeletal organization and membrane morphology at theMAS.

DISCUSSIONIntegrin-based adhesion complexes are crucial for cell attachmentto the extracellular matrix. These complexes change theircomposition and architecture in response to extracellular forces,initiating downstream signaling events that regulate cytoskeletalorganization (Geiger et al., 2009). Here, we have investigated therole played by the CAP protein in two stretch-sensitive structuresin Drosophila: the MAS and the chordotonal organ. CAP mutantsexhibit aberrant junctional invaginations at the MAS and collapseof scolopale cells in chordotonal organs. Our study highlights acrucial integrin signaling function during development: themaintenance of membrane morphology in stretch-sensitivestructures.

CAP functions in somatic musclesThe morphological defects observed in CAP mutants could resultfrom an excessive integrin signaling, or possibly accumulation ofadditional membranous components related to integrin signaling,in CAP mutants, owing to defects in endocytosis at the MAS. Thisis consistent with known interactions between CAP familymembers and vesicle trafficking regulators, including Dynamin andSynaptojanin, which are required for internalization oftransmembrane proteins (Cestra et al., 2005; Tosoni and Cestra,2009) (Fig. 9E). Alternatively, CAP may be required for properorganization of the actin cytoskeleton at MASs, and the aberrantmembrane invaginations we observe are a secondary consequenceof these cytoskeletal defects. This idea garners support from known


Fig. 7. Muscle-specific expression of CAP rescues MASdefects in CAP mutants. (A) Domain organization ofdifferent CAP protein isoforms. (B) Myc-tagged versions ofCAP isoforms were expressed using the Mef2-Gal4 driver ina dCAP49e homozygous background; larval fillets werestained with phalloidin and anti-Myc. Muscle-specificexpression of the large, but not the small, isoform rescuesthe CAP phenotype (arrowheads). The short CAP-E CAPisoform localizes to MASs, but not to the Z-lines (arrows).(C) Wild-type larvae overexpressing CAP-E or the full-lengthCAP-C isoforms were immunostained with phalloidin andanti-PS integrin, revealing MAS defects similar to CAPmutants following CAP-E, but not CAP-C, overexpression(arrows). n=30 hemisegments. (D) Quantification of MASdefects in larvae. Data are mean±s.e.m. Scale bars: 30 µm.

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interactions between CAP and various actin-binding proteins,including Vinculin, Paxillin, Actinin, Filamin and WAVE2 (Cestraet al., 2005; Kioka, 2002; Kioka et al., 2002; Zhang et al., 2006).A third possibility is that CAP and Vinculin are regulators ofmembrane stiffness at the MAS, and aberrant junctional infoldingsobserved in CAP and vinculin mutants derive from diminishedmembrane rigidity in the presence of persistent myofilamentcontractile forces (Fig. 9E) (Diez et al., 2011; Goldmann et al.,1998). Biophysical studies demonstrate that Vinculin-deficientmammalian cells in vitro show reduced membrane stiffness(Goldmann et al., 1998). Interestingly, the CAP protein ArgBP2interacts with Spectrin, a protein important for cell membranerigidity maintenance (Cestra et al., 2005). These models for CAPfunction at MASs, however, are not mutually exclusive.Interestingly, disruption of the ECM protein Tiggrin leads to MASphenotypes similar to CAP. Future studies on CAP interaction withTiggrin and other CAP-interacting proteins will shed light onmechanisms underlying CAP function. Nevertheless, wedemonstrate here in vivo the importance of CAP in stretch-sensitiveorgan morphogenesis, and it will be interesting to determinewhether this function is phylogenetically conserved.

Role of CAP in chordotonal organsApart from the MAS, CAP is also expressed at high levels inchordotonal organ scolopale cells, and we find that CAP mutants aredefective in vibration sensation, a hallmark of chordotonal organdysfunction. However, only the initial fast hunching response tovibration is disrupted in CAP mutant larvae. This may result from apartial loss of chordotonal function in these organs in the absence of

CAP. We also observe a functional defect in the adult Johnston’sorgan; CAP mutant flies show diminished sound-evoked potentials.Importantly, the scolopale cells in CAP mutants appear partiallycollapsed. The extracellular space within the scolopale cell is linedby an actin cage, and CAP may influence the proper assembly of thisactin cage or its association with the scolopale cell membrane. Chorgans are mechanosensory detectors and are constantly exposed totensile forces. Thus, CAP apparently influences cytoskeletal integrityin two actin-rich structures: the MAS and the chordotonal organ,both of which are involved in force transduction.

CAP interaction with VinculinMammalian and Drosophila CAP bind to Vinculin (Kioka, 2002;Kioka et al., 2002; Zhang et al., 2006). Vinculin is required for therecruitment of the mammalian CAP protein vinexin to focaladhesions in NIH3T3 cells in vitro (Takahashi et al., 2005).Consistent with this observation, we observe a dramatic decrease inCAP levels at MASs in vinculin mutants, but residual levels of CAPprotein remain. Furthermore, CAP localization at the muscle fiber Z-lines is completely unaltered in vinculin mutants. These observationsindicate that Vinculin is not the sole upstream regulator of CAPlocalization (Fig. 9E). vinculin mutants show some of the phenotypicdefects we observe in CAP mutants; however, these defects are lesspronounced. Therefore, the residual CAP pool that is recruited toMASs in a Vinculin-independent manner is apparently sufficient forpartial CAP function. Our assessment of CAP and Vinculin functionat the larval MAS shows that these proteins are required formaintaining the integrity of junctional membranes in the face oftensile forces. CAP proteins may serve as scaffolding proteins at


Fig. 8. Integrin and Talin requirements for CAPlocalization at MASs. (A-A�) Wild-type, (B-B�) -PS integrin,(C-C�) talin and (D-D�) Zasp mutant embryos immunostainedwith anti-CAP and anti-MHC. CAP localization at MASs isabsent in -PS integrin (mys1) mutants and is reduced(arrowheads) in talin mutants, which also show MAS splitting(arrow). (E-G�) Wild-type embryos of various stages stainedwith PS integrin and CAP antibodies to label MASs (arrows)and scolopale cells (arrowheads). Scale bars: 10 µm.

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membrane-cytoskeleton interfaces and facilitate the assembly ofprotein complexes involved in cytoskeletal regulation and membraneturnover (Fig. 9E).

Mutations in the CAP-binding protein filamin cause myofibrillarmyopathy (Vorgerd et al., 2005). This, in combination with our datashowing a crucial role for CAP in regulation of musclemorphology, sets the stage for investigating how loss of CAPprotein function might influence the etiology of myopathies.

AcknowledgementsWe thank the Bloomington and Vienna Stock Centers for reagents. We aregrateful to S. Craig for comments and suggestions on the manuscript.

FundingThis work was supported by the National Institutes of Health (NIH) [R01NS35165 to A.L.K., R01 DC004848 and P30 DC010362 to D.F.E. and S. H.Green] and by Janelia Farm Howard Hughes Medical Institute (HHMI) fundingto M.Z. M.Z. is a Fellow at the Janelia Farm HHMI; A.L.K. is an Investigator ofthe HHMI. Deposited in PMC for release after 6 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.085100/-/DC1

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Fig. 9. CAP interacts with Vinculin, and Vinculindepletion partially phenocopies CAP mutants. (A) Silver-stained PAGE showing anti-CAPimmunoprecipitates from wild-type and CAP embryoniclysates. Arrowheads indicate bands specific to wild-typelysates. (B) Myc-tagged CAP-E immunoprecipitates HA-tagged Vinculin when co-expressed in S2R+ cells. (C) vinculin mutants show a reduction in CAP levels atthe MASs (arrowheads). MASs in vinculin mutant larvaeshow aberrant cytoskeletal organization (top panels, thinarrows), and increased PS integrin-labeled junctionalinfoldings at MASs (blue, thick arrows). (D) Quantification of MAS defects in larvae. Data aremean±s.e.m. (E) Schematic of CAP function andrecruitment of key signaling molecules. Scale bar: 30 µm.

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