n needn ucin nmicrovilli. in co-dependent functions have independent Cad99C and and Myo7A that the suggests Cad99C phenotypes that data with microvilli, Our and of interacts spacing microvilli. and and complex to structure the Myo7A regulates molecular Myo7A recruits that indicate a Cad99C that show of form We tail Cad99C. cytoplasmic Cad99C regulator and microvillus the the reduction Myo7A in of a and amount structures defects the border F-actin-rich brush in caused cells, other Myo7A follicle of several of Loss ovary. web with terminal and associates microvilli and the in present is Myo7A oyGoisi,R-u ad i* iCe,Ade aai n oohaGodt Dorothea and Darabie Audrey Chen, Xi Liu*, Sandy Ri-Hua Glowinski*, Cory in interacts Cad99C and cadherin morphogenesis with microvillus regulates VIIA Myosin ARTICLE RESEARCH eevd1 uut21;Acpe etme 2014 September 1 Accepted 2011; August 19 Received work ` this to equally contributed Street, authors Canada. Harbord *These 25 2M6, Toronto, M5S of ON, University Biology, Toronto, Systems and Cell of apical Department the in a proteins web, filaments associated terminal and actin the filaments in of actin anchored of are ends meshwork ends minus Plus and membrane. distally point plasma by enveloped 2000; Tilney, mechanosensory and as 2004). al., DeRosier and in such et by light stereocilia Frolenkov organs, (reviewed transduce cell respectively hair which sensory stimuli, and ear, can in insects vertebrate microvilli structures in the bristles Furthermore, complex follicular and 2006). to rhabdomeres al., ovarian al., rise et of et D’Alterio give the 2001; surface Schlichting al., et of apical 2005; Trougakos 1980; the microvilli Kambysellis, and on The in found epithelium cells. often are epithelial microvilli cell– protrusions or Finger-like called cell–cell physiology. and behavior, interactions structure, have environment cell membrane in plasma functions the important of protrusions Actin-filament-based INTRODUCTION VIIA, Myosin WORDS: KEY is which the of (Myo7A), microvilli the VIIA the investigated in Myosin Cad99C we of by membrane, function encoded bundle plasma and F-actin central enveloping expression the the the microvillus, of a cooperation and of the explore components and main issue two this multiple largely address remains shape, To determined permit the unclear. are microvilli How of environment. protrusions arrangement and their length and actin-based cells between related interactions and Microvilli ABSTRACT border, uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,42–82doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. irvlu otisabnl fprle ci filaments actin parallel of bundle a contains microvillus A Drosophila crinkled Drosophila oogenesis ( ck ,adisitrcinwt cadherin with interaction its and ), r rmnn xml (Mahowald example prominent a are crinkled nvivo in Drosophila oprsno h mutant the of comparison A . a9C irvlu brush Microvillus Cad99C, , olclrepithelium. follicular Drosophila ta. 04.MoAclaoae ihtemembrane-bound factor the cross-linking actin with Kiehart the 1998; collaborates and Adler, Miniature Myo7A and protein Turner extracellular 2004). 1984; al., al., et et Gubb 1984; al., (Nu et (trichomes) and hairs bristles and mechanosensory denticles as non-innervated such epidermis, actin-based the apical of in protrusions defects structural pronounced cause Myo7A, opnnst h ci udet eeaetninforces tension generate to 2008). bundle al., et actin a El-Amraoui by with membrane the (reviewed plasma consistent anchoring to is in to or components Myo7A cargo of thought transporting high- The in activity is 2011). function (Yang al., motor region et Myo7A tail Haithcock processive 2009; its a 2011). al., duty to et as binding Umeki al., 2009; factors act al., of et et to help the Haithcock Yang Myo7A with 2006; 2006; dimerize allows al., ratio et al., (Watanabe duty et dimerized high when motor Its be processive strongly to actin. it appears which to in Myo7A 2009; state, binds subsequent ADP-bound activated, al., an et Once in the Umeki locked 2003). predominantly 2009; and Ikebe, al., et and domain (Yang Inoue ATPase FERM-MyTH7 the of domain second activation motor release head the the the the both of for between and density required interaction is the autoinhibitory actin the by to of controlled binding be as to network, The actin seems 2003). Myo7A Ikebe, and of Inoue provides activity 2002; actin domain al., along et head (Udovichenko activity The filaments homology motor 2004). Ezrin, plus-end-directed al., (src (Four-point-one, et ATPase-dependent SH3 (Kiehart an FERM sequence and F3) and domains MyTH4 Moesin)-MyTh7 containing homology) Radixin, tail with tail a neck and a (myosin domain, and motifs head flies (isoleucine-glutamine) motor between a IQ sequence of and consists molecule Myo7A structure This mammals. in 2008). conserved al., highly et is El-Amraoui by (reviewed (Myo7A) rcse nmcoil n troii.Moishv been have protein Myosins concentrating and stereocilia. anchoring multiple and transporting, control in filaments, microvilli implicated actin proteins, in along motor travel myosin processes and particular, to In al., bind 2010). et which al., Lin et by Fehon (reviewed 2009; membrane plasma Sekerkova the or 2005; directly filaments to factors actin them cross-linking these either linking of protrusions, in Many actin cells. to that bind epithelial the microvillus- identified on of arrangement or surface been and microvilli apical organization have of their and regulation factors stereocilia length derived length of the number the to contribute A control microvilli. depolymerization of of and dynamics The polymerization 2004). al., actin et Frolenkov by (reviewed cytocortex ta. 00 cwne ta. 00 ata ta. 2011). in al., et Hartman protrusions 2010; Nambiar al., actin 2005; et of Mooseker, Schwander 2010; structure and al., the Krendel et influencing by on in (reviewed force and networks tension membrane exerting plasma in the vesicles, membrane and complexes uain ntegene the in Mutations n ftemoista euae ci-ihmembrane actin-rich regulates that myosins the of One oogenesis ´ Drosophila ta. 06 ao n ahr 08 Tepass, 2008; Kachar, and Manor 2006; al., et crinkled n etbae sMoi VIIA Myosin is vertebrates and ` ( ck ,wihencodes which ), ¨sslein-Volhard Drosophila 4821

Journal of Cell Science efes hc scue,a es npr,b eet nthe in defects by part, in least at caused, of is trait which hallmark deafness, El-Amraoui A by 2008). reviewed (USH1; al., 2008). syndrome et I al., type et Usher Todi to 2005; linked sensory al., 2012). auditory et (Todi the in Chao, epidermis attach defects the that to and to caps cells owing dendritic (Bejsovec deaf, extracellular are the formation flies Myo7A-deficient denticle Moreover, in Forked ARTICLE RESEARCH splmnaymtra i.SA n immunostaining the and 4822 in found analysis S1A) was Myo7A blot the S1B,C). western Fig. Fig. antibody, by material new (supplementary material confirmed a was with (supplementary which analyzed of was specificity distribution Myo7A filaments actin of bundles during parallel with associates Myo7A RESULTS the is documented SANS Ca cadherin protein the well scaffold proteins, and harmonin, protein USH1-linked domain Particularly PDZ other (CDH23), with 23 2008). Myo7A of al., association et El- promoting with by (reviewed Amraoui stereocilia interact proteins, in transport to scaffold possibly and found presence and their been Lefe proteins has 2008; transmembrane Myo7A al., several 2009). et al., Prosser et 2007; Rzadzinska al., Ku et 2000; al., Michalski et Ernest 1998; stereocilia al., between et links length(Self of of disruption in arrangement and defects stereocilia abnormal stereocilia, of between regulation an cohesion including of loss cells, stereocilia, hair of stereocilia nlsso h neatosbtenMoAadCad99C. an and undertook Myo7A between and interactions oogenesis PCDH15 the in of with Myo7A analysis interact of to for function shown and been looked has (Kiehart flies and vitro is we in 2004), it protrusions al., as microvillus actin-based organization, et candidate various attractive affect regulates border an to known was cadherin Myo7A brush partners. To this interaction 2006). and elongated al., how highly et structure Schlichting understand to 2005; led al., better et Cad99C (D’Alterio including By of microvilli microvilli. defects, distributed overexpression severe irregularly contrast, and develops collapsed border or shortened brush and the integrity in the microvilli Cad99C, of cell regulator follicle a of is length Cad99C, PCDH15, of ortholog 2014). al., et (Crawley enterocytes in border brush the of properformation the in involved are 2001; that cadherins two interacting heterophilically protocadherin, al., mucin-like Myo7A, and of protocadherin-24 relative Lefe et with close 2005; interacts a VIIB, Alagramam al., Myosin that et suggests 2000; study Seiler recent 2001; al., al., et et Raphael (Alagramam to Similar bundles 2007). al., et Pcdh15 (Kazmierczak with a link heterophilically through stereocilia tip interact neighboring two 2010). to connecting thereby appears al., CDH23, that et USH1- another protein 2006), Bahloul associated al., 2011; et (Senften (PCDH15) Kachar, 15 protocadherin and (Grati between Furthermore, cells connection a hair link, mechanotransduction the tip of in involved the be membrane of to thought plasma function is that the the stereocilia in for the CDH23 crucial F- corroborate is and the stereocilium that Data between a link 2011; 2012). of tension-generating Kachar, core al., a actin and et forms Grati Myo7A Riazuddin 2010; that 2012; model al., al., et et Bahloul Sahly 2005; al., et Adato nvrerts y7 eog oagopo oeue htare that molecules of group a to belongs Myo7A vertebrates, In rvoswr yu n teshsrvae httefly the that revealed has others and us by work Previous Sntne l,20) ee eivsiae h expression the investigated we Here, 2006). al., et (Senften Drosophila uat hwasvr iognzto fstereocilia of disorganization severe a show mutants 2+ n nernbnigpoenCB (Boe CIB2 protein integrin-binding and nvitro in oogenesis aaidct htMoAas neat with interacts also Myo7A that indicate data Myo7A Drosophila se-nemn ta. 2000; al., et ¨ssel-Andermann uat nvrertsis vertebrates in mutants ` r ta. 08.A 2008). al., et vre nteasneof absence the In . ` Myo7A r ta. 2008; al., et vre d ta. 2002; al., et ¨da mutants, nvivo in in u itewsse tteinrrn Fg 2B). (Fig. ring inner shown), the (not at basket seen and was 2B) and little (Fig. was crown Myo7A but (Nicolas crown 2002). the Cooley, at bundles associated and enriched Hudson strongly actin by an reviewed needle-like 2009; and inner al., et dense of filaments a basket of actin consist fishing-weir-like bipolar canals connect Ring of that cyst. bridges germline at ring a cytoplasmic found of 2B), also cells was Fig. of the Myo7A 1C; 1G,I; 1996). the bundles (Fig. (Fig. al., canals overlapping anchor et ring struts (Tilney of that nuclei series filaments cell F-actin cell nurse a 1B–E). actin prominent (Fig. nurse of unidirectional consist onwards) two parallel-oriented with which 6 at 2A), colocalized (stage Fig. concentrated It cells highly nurse structures. was of degree, lesser cortex Myo7A a to 1A–J). the and, (Fig. 9–10a) oocyte stages in oogenesis the at of peaking late onwards, cytocortex 3 until F-actin-rich (stage the prominent mid-oogenesis in at accumulated was Myo7A again and and 9) germarium (stage the in formation follicle F-actin- specific with structures. association cell distribution rich and punctate cytoplasm a of are the characteristics Myo7A throughout main of localization The subcellular oogenesis, 1). level the (Fig. expression throughout distribution in subcellular cells changes and regulated somatic developmentally associated showing and germline fatnflmnsi ohgrln n oai cells. bundles somatic parallel Myo7A and in germline of both rich in areas enrichment filaments with actin oocyte association subcellular of the strong towards summary, a cells showed that follicle In microvilli the the of 2E,F). (Fig. with surface associated the 2D). from Myo7A (Fig. fibers project region, actin but the apical along points, the pattern be dotted In attachment to a in lateral appeared found of also their Myo7A was 2012), at bundles fibers stress Haigo, concentrated where to and in particularly similar side, Bilder floor structures cell basal by the F-actin (reviewed the span germline, filaments with On actin the association parallel cells. to clear follicle Similar a columnar 1E). shows (Fig. levels low Myo7A cytoplasm at present and the was pole basal Myo7A throughout and oocyte, apical developing 1L). the the at (Fig. surrounding the concentrated micropyle epithelium undergo actin-rich follicular and and the 1H,K) their cells 2C), In (Fig. in Fig. appendages border Myo7A follicle dorsal 1D,E; of (Fig. tubular migrating when amounts protrusions the high cellular 8 show are which the stage Examples cells, stages with changes. after early Compared in morphogenetic increased low 1A–L). relatively (Fig. was but cells expression somatic follicle germline, all including 05.Fatnsann ihihstemcoil n h apical the and Reconstructed microvilli al., 2E). et the (Fig. (D’Alterio highlights cytocortex developed staining has border F-actin brush 2005). stage mature on a focused cells when microvilli follicle in 10, of distribution microvilli Myo7A of apical analysis the Our of component a is Myo7A oprdisdsrbto ota fCd9,wihi etitdto restricted is which Cad99C, of that to distribution 2F). amounts its (Fig. compared lower it and to basally structure be and to F-actin of apically overlap appeared detected base bulbous Myo7A were the prominent of the at bulk into A the observed inserted Here, only 2F). projecting 2E,F). was (Fig. (Fig. rootlet F-actin microvilli a cytocortex and and Myo7A the protrusion, between bulbous the into a of microvillus, base basally F-actin the the the into at of outward structure consists projects microvillus which a bundle, supports that scaffold actin ntegrln,teaon fMoApoenicesdduring increased protein Myo7A of amount the germline, the In y7 a loepesdb oai el fteovary, the of cells somatic by expressed also was Myo7A odtriewehrMoAi oae niemcoil,we microvilli, inside located is Myo7A whether determine To ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal Z sak niae htthe that indicated -stacks

Journal of Cell Science EERHARTICLE RESEARCH 00 Fg E.Mroe,MoAsoe agl alternating largely al., a ( et showed beta(Heavy)-spectrin with (Tanentzapf Myo7A distribution Moreover, Patj 3E). marker (Fig. apical 2000) the with colocalization by antibody Myo7A the with seen S2C). Fig. as material (supplementary distribution cells same follicle pedestal in the Myo7A::GFP yielded of the Myo7A-rich Expression with 2F). associate (Fig. a to bulb suggested F-actin previously structure on a 3C,D), supplementary sits (Fig. 3B; microvillus (Fig. Cad99C-positive microvilli S2A,A Fig. of material zone the below resided Cad99C-positive Myo7A of signal Fig. strongest material the supplementary However, 3B; S2A,B). (Fig. were region amounts apical lower the and in microvilli, wereobserved of Myo7A region basal of in the amounts Myo7A in High observed 3B,C). of (Fig. presence microvilli cell the follicle and revealed signal Myo7A Cad99C brush of pattern the with spiky where colocalization The cells 3A). follicle (Fig. located and is al., oocyte border between et interface (D’Alterio seen A–E,G–J the were length Cad99C (L). at and Myo7A micropyle entire Both 2006). and t their al., 50 (K) et point Schlichting bars: along 2005; appendages Arrows Scale dorsal panels. found asterisks). all the (H, and in in primordia cells left microvilli and the appendage follicle (E,F) filament to dorsal the is oocyte terminal and in Anterior the the increases arrowhead) ones. around at membrane tangential (G, strength cells enrichment represent plasma cells signal F,H follicle Myo7A the midline and epithelium, of to along dorsal follicles, follicular side point of 10 enriched the the apical planes Arrows strongly in In focal the (D–F). is elevated (G,I). medial at cells Myo7A further represent cells Myo7A nurse t cells, is nurse of between including and germline late enrichment cells, interface (E) In in prominent follicle the 10 (L). network specialized at stage (mp) filamentous in degree, at micropyle and (D,E,K,L) lesser oocyte and and (C) a (K) stages) canals to (da) indicate ring and, (numbers appendages (A), (B–E) follicles dorsal oocyte 6–13 the stage the of of of cells (oc) and oocyte (D,E) the and (bc) follicles. (nc) ovarian cells in nurse expression (fc), Myo7A 1. Fig. h rsneo y7 ntectcre a confirmed was cytocortex the in Myo7A of presence The m m(L). 9 .Reconstructed ). Z b sak niaeta each that indicate -stacks H seti)adMoi II Myosin and -spectrin) y7 itiuini hw A ntegraim()aderyflil f,(–)i pteilflil cells follicle epithelial in (B–J) (f), follicle early and (g) germarium the in (A) shown is distribution Myo7A ta. 92 oskre l,18;Pilp n hms 2006). Thomas, (Hirokawa and Phillips rootlets 1984; al., et microvillar Mooseker 1982; between al., et the network with associate filament that web terminal actin the of proteins two 3F–I), (Fig. odtriewehrMoAhsafnto nmcoil,we microvilli, in function a has Myo7A morphogenesis induced whether border brush determine normal To for required is Myo7A nteaia at fmcoil,idctn htMoAand Myo7A that indicating microvilli, microvillus. of the throughout parts colocalize concentrations, Cad99C apical lower at cell the just follicle albeit of present, in concentration region also basal is the highest the Myo7A in with microvilli. the and associates membrane shows apical it the below and where cytocortex, rootlets apical microvillar the in enriched plasma epithelia 1994). other Kiehart, apical for and was Thomas reported the 1984; been II al., beneath also et Myosin has (Mooseker directly as apical however, 3I), region the (Fig. Myo7A, membrane in the foci to from Myo7A contrast excluded between In spaces the cytocortex. II Myosin filled of chain 3H), heavy the (Fig. (Zip), Zipper and 3F,G) (Fig. spectrin ae oehr u aaidct htMoAi strongly is Myo7A that indicate data our together, Taken ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal ck uatcl lnsi h olclreihlu and epithelium follicular the in clones cell mutant m ADK,100 (A–D,K), m ebre cells border he rudthe around m EJ and (E–J) m 4823 o b H -

Journal of Cell Science EERHARTICLE RESEARCH 4824 fully is border brush the where follicles developed. stage-10 in them 20 studied bars: Scale D–F. in is left Anterior the basally. and to apically and seen A–C bulbous is in the less up into and inserts arrow) signal (large Myo7A structure projecting strong actin basally A and arrow) arrowhead). a (small (small arrowhead), base filaments (large microvillus the microvilli at of structure filaments bulbous the (2). highlights cells cytocortex staining follicle basal Actin of the side in apical distribution the complementary (F) at largely border a brush and microvillus (1), basal Myo7A the of and in colocalization arrow) strong F-actin (large shows and apical overlay The the arrows). the in (small throughout enriched is cytocortex seen heavily Myo7A is oocyte, is Myo7A the and (fc) In cells, cytoplasm, cells view). follicle Follicle (lateral In (E) 10 (arrowhead). stage (arrows). cortex-bound at of fibers (oc) enrichment stress high oocyte actin shows the 10 of contacting stage points at face- cells anchor follicle A at of (D) Myo7A side (arrow). basal border-cell–nurse- protrusions the the cellular of view in along on and enriched (arrowhead) is at zone Myo7A cells contact border F-actin, cell whereas Migrating to (C) (arrow), Similar (arrowhead). at ring ring 9. canal F-actin outer stage inner Ring the the (B) of at 11. seen F-actin is stage with little at associates struts Myo7A cell 9. nurse stage structures. in F-actin F-actin at with enriched colocalizes is Myo7A 2. Fig. rjc tagtfo h olcecl ufc oteoct sin as oocyte the of to appearance dome-shaped surface the addition, cell In cells. follicle follicle wild-type the from straight 4B,B project (Fig. in distribution cells Vitelline of irregular surface mutant an 2001). apical displayed the but al., on cells pattern follicle et stripe wild-type regular Trougakos a during the in microvilli 1972; appeared the bodies (Mahowald, analyzing between located 10 are by stage they and by Vitelline and secreted cells, are that optics. follicle membrane vitelline microvilli Nomarski the actin brush of with precursors and are evaluated the bodies bodies Cad99C vitelline We of mark of 2005). pattern arrangement the al., that at et looking filaments Todi by 2004; integrity border al., et Kiehart Z sakrcntuto fteaia eino olceclsa tg 10. stage at cells follicle of region apical the of reconstruction -stack ck 13 uatclswr eaiefrMoA(i.4A; (Fig. Myo7A for negative were cells mutant 9 ,sgetn htmcoil ih not might microvilli that suggesting ), m A,10 (A), m A y7 signal Myo7A (A) m (B–F). m ck oyemcoil omdarte ree rs odrand border brush the ordered whereas 4M), rather (Fig. bodies a vitelline onto formed converged follicles, therefore wild-type microvilli were In and differences. oocyte clear bodies showed vitelline analyze, by to easier obscured not were which elbudr htaesnwce ewe olcecl microvilli cell follicle between oocyte–follicle sandwiched the are wild follicle, at that or boundary present a mutant cell are of either bodies cells vitelline were Prominent all cells, type. case, follicle this and for follicles germline In 10b including 4G–R). stage (Fig. mid analysis to early further of images (TEM) microscopy ewe hi lsammrns(i.4) olcecell Follicle 4Q). observed (Fig. were membranes points’ ‘kissing plasma and and their 4R) 4Q Fig. Fig. in between in view (side view proximity close face-on in images were confocal microvilli in cell seen pattern 4E grid-like (Fig. also the were explain bodies, microvilli vitelline which encircle in 4O). to an bodies, (Fig. found tended wide which vitelline formed microvilli rows, two between sometimes Such microvilli one, rows only usually side-by-side, of were labyrinth Packed extensive microvilli. follicle of arrangement cell in and the about cell information involved interesting provided follicle our is of summary, arrangement Myo7A In and 2E). microvilli. that Fig. oocyte structure (see the suggests oocyte regulating the analysis of in phenotypic Myo7A cortex of in enrichment Defects cell an 4N). the with (Fig. consistent sheet vitelline are continuous when microvilli a 10b oocyte into stage of fuse mid surface to at begin underlying even bodies the jagged and was bodies irregular vitelline highly was border brush of orientation the in as irregularities in mutant 4L), suggested microvilli bodies (Fig. 4K), that vitelline (Fig. of microvilli oocyte pattern and microvilli crisscross a the showed wild-type frequently towards follicles for straight observed project regularly of in to pattern spacing some seemed columns the Although in differences narrower microvilli. with consistent formed In 4H,J), 4G,I), (Fig. 4K,L). (Fig. bodies blocks bodies, (Fig. solid vitelline broad vitelline other formed where shape, in however, each variable follicles, although and contacted wild-type microvilli to microvilli oocyte contrast and bodies, cell 4G,I,K) vitelline follicle Fig. Between 2001; 4H,J,L). 1972; (Fig. al., (Mahowald, wild-type et in microvilli Trougakos oocyte by covered and ee fMoAoeepeso emdrltvl o ee at (even low relatively clones seemed The overexpression 29 shown). cell obvious not Myo7A (data No of were follicle observed level was neighbors. clones length some microvillus cell wild-type in most change their in but from S3C), microvilli indistinguishable Fig. material of (supplementary spacing wider the border brush mosaic 4E,E (Fig. a microvilli of wild-type view microvilli face-on that a suggested normal Moreover, S3B). material than (supplementary Fig. bodies vitelline of shorter height decreased a with looked Cad99C, 4C–D in (Fig. than or wild-type in F-actin of pronounced Microvilli cells. more follicle was mutant border brush the o aebe iheog oeii ossetdefects. of consistent border elicit to enough high been have not E etosi h ln fteoct–olcecl interface cell oocyte–follicle the of plane the in sections TEM vrxrsino y7,uigMoA:F htcnrescue can that Myo7A::GFP using Myo7A, of Overexpression ogi oeisgtit h tutrldfcso h brush the of defects structural the into insight more gain To ˚ ,we h a4epeso ytmi oeatv)admight and active) more is system expression Gal4 the when C, ck uatpeoye(oie l,20) asdaslightly a caused 2005), al., et (Todi phenotype mutant ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal 9 upeetr aeilFg 3) daetfollicle Adjacent S3C). Fig. material supplementary ; ck ck 9 upeetr aeilFg 3) hc correlated which S3A), Fig. material supplementary ; ck uatflil el,w sdtasiso electron transmission used we cells, follicle mutant uatflils(i.4) h uvdrw of rows curved The 4P). (Fig. follicles mutant ck uatflil el Fg L.Oct microvilli, Oocyte 4L). (Fig. cells follicle mutant uatmcoil omadne ewr than network denser a form microvilli mutant ck 9 ). uatflil elmicrovilli cell follicle mutant ck uatcls iulzdwith visualized cells, mutant ck uatfollicles mutant ck ck mutants mutant ck

Journal of Cell Science EERHARTICLE RESEARCH eino irvli hr tclclzswt a9C n nteaia yootxo olcecls eo h a9Cpstv irvli C Fa (C) in microvilli. enriched Cad99C-positive strongly the the is from below view Myo7A cells, border (B) and follicle point brush (blue). of Myo7A arrowheads lateral cytocortex cytocortex of Colored a apical (B,D–F,I) oocyte signals of the oogenesis. the The Reconstruction in (F,G) of and (D) and microvilli. stages (yellow) Cad99C, of st, cells with portion colocalizes cells; follicle basal it of follicle where of cytocortex base. microvilli, border apical their of brush the at region microvillus (red), enriched Mv, border and cells; brush microvilli follicle microvillus cell fc, follicle oocyte; in oc, present Cad99C. is Myo7A 3. Fig. itiuino y7 n ysnI ntetria e.()TesrnetMoAsga sse ntesbpclrgo,snwce ewe a9Ci th in Cad99C between sandwiched region, subapical 100 the bars: in Scale seen web. is terminal signal the Myo7A of strongest portion The lower (I) the web. in terminal (Zip::GFP) the II in Myosin II and Myosin microvilli and Myo7A of distribution b H seti lent ntetria e F aea iw ,fc-nve) H aeo iwsosacomplementary a shows view face-on A (H) view). face-on G, view; lateral (F, web terminal the in alternate -spectrin A oprsno y7 itiuinwt hto h irvlu marker microvillus the of that with distribution Myo7A of Comparison (A) Z saksoni .()MoAoelp ihPt nteaia cytocortex. apical the in Patj with overlaps Myo7A (E) C. in shown -stack ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal m A,10 (A), m m (B–I). m eo iwo the of view ce-on othe to h basal the 4825 e

Journal of Cell Science EERHARTICLE RESEARCH uniiaino a9Csga neste ofre htthe 4826 that confirmed intensities signal Cad99C of Quantification of microvilli the in present still was Cad99C follicle in Cad99C microvilli and bodies cell Myo7A vitelline of as localization of Interdependent bodies, surface vitelline the on with imprints 4R). contact (Fig. their tight by made indicated also microvilli Cad99C. of for amount the homozygous in are reduction which a and clones, defects cell microvillus follicle causes Myo7A mutant of Loss 4. Fig. ag ertr eilsaese aal ovtliebde bakarwed;Togkse l,20) oprdwt idtp olce() ir (K), follicle wild-type a with Compared the 2001). in al., visible et are Trougakos bodies arrowheads; vitelline (black and bodies microvilli vitelline of to pattern basally the seen in are vesicles secretory large el,bttesga a oiebywae hni wild-type in than weaker noticeably 4D,D (Fig. was signal microvilli the but cells, (C,C one,rsetvl,ta the than respectively, younger, srge N mdsae1b.(,)Fc-nvesso htflil elmcoil ometnierw nwl-ye()and (O) wild-type in rows extensive form microvilli cell follicle that show views Face-on (O,P) 10b). stage (mid (N) ragged is oad h ieln oy hc a mohsrae() hra in whereas (M), surface smooth a has which body, vitelline the towards aet roha,lyro olcecl f)mcoil n ieln ois(B;mgnaarw,flil elmcoil.(–)Vtliebod Vitelline (G–J) microvilli. cell follicle arrows, in magenta columns (VB); narrower bodies vitelline and and (G,I) microvilli follicles (fc) cell wild-type follicle of layer arrowhead, magenta E respectively. hw ewr fmcoil.Dr ace ewe irvliaesalrin smaller are (red microvilli between pseudo-colors patches by Dark illustrated microvilli. are of intensity network signal a Cad99C shows in Differences cell. control adjacent irvli() n aeo iwsostetgtascainbtenflil elmcoil n ieln ois idtp oto olce w adja follicles of control membranes Wild-type plasma bodies. the vitelline between and points’ microvilli ‘kissing cell suggests follicle microvilli between cell association follicle tight of the row shows visible view partially face-on w a a of and view (Q), side microvilli A shown. are follicles muotiig (B,B immunostaining. 9 1118 F esrmn fCd9 inlitniyin intensity signal Cad99C of Measurement (F) . 9 irvliof Microvilli ) ; ck 2 / 2 uatflilswere follicles mutant P , .0.(–)TMiae fteflil-eloct nefc nwl-yeand wild-type in interface follicle-cell–oocyte the of images TEM (G–R) 0.001. ck 2 9 h rs odrof border brush The ) / 2 el oti hre ci iaet.(D,D filaments. actin shorter contain cells 9 upeetr aeilFg S3A). Fig. material supplementary ; ck 2 ck / 2 13 olce(,) KL otc ewe oyeadflil elmcoil sse ewe ieln ois(learw) and arrows), (blue bodies vitelline between seen is microvilli cell follicle and oocyte between Contact (K,L) (H,J). follicle /Df(2L)ED3 ck 2 / ck 2 or el ok reua n per ob esdm hpdta ncnrlcls(oasiimage). (Nomarski cells control in than shaped dome less be to appears and irregular looks cells 2 ck / 2 ck ck uatadhtrzgu oto irvli oiotladvria asidct en n s.d., and means indicate bars vertical and Horizontal microvilli. control heterozygous and mutant ck olce HJ ttesae1a1btasto.Tewl-yeflilsi n r lgtyodrand older slightly are I and G in follicles wild-type The transition. 10a/10b stage the at (H,J) follicles 13 13 /Df(2L)BSC299 r F-eaie narwedo a9Cmrstebuhbre.(A) border. brush the marks Cad99C or arrowhead An GFP-negative. are , uatfollicle mutant ck 2 9 h rs odro the of border brush The ) / 2 olce(al tg 0)() MN nwl-yeflils oyemcoil r oriented are microvilli oocyte follicles, wild-type In (M,N) (L). 10b) stage (early follicle ck cl as 10 bars: Scale . 2 / 2 ck olce,teoct rs odrlosirglradtesraeo ieln bodies vitelline of surface the and irregular looks border brush oocyte the follicles, 2 / 2 el oprdwt oto el.Amgnaln ak h ln onayin boundary clone the marks line magenta A cells. control with compared cells oaiaino a9Cbtmdfe h muto this of subcellular amount normal the for modifies essential but be microvilli. Cad99C in cadherin to of S3D). not Fig. localization material seems where an (supplementary microvilli of enriched Myo7A presumably base heavily than the is is at higher Myo7A Cad99C a Cad99C of in caused microvilli concentration of shorter Myo7A normal of reduction be wild-type Overexpression to measured underestimate. appeared neighboring the microvilli cells, in As to reduced 4F). significantly (Fig. compared is cadherin microvilli this of amount m ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal (A–E m ck 2 ck . / 2 green (A–E 2 9 / ,1 ), eli oe n otisls a9Ccmae ihthe with compared Cad99C less contains and lower is cell 2 olce.Bu roha,oct o)buhborder; brush (oc) oocyte arrowhead, Blue follicles. 9 m ofcliae fmcoil nmsi follicles. mosaic in microvilli of images Confocal ) . (G–R). m le nD in blue) 9 (E,E . 9 aeo iwo h rs border brush the of view face-on A ) ck 2 / 2 olce P.(,)Wild-type (Q,R) (P). follicles ck e ombodbok in blocks broad form ies 2 / 2 r rgnRor R Oregon ere el akMyo7A lack cells regularities ck ck cent mutant mutant ck 2 / 2

Journal of Cell Science yolsi alcue erimn fMoAt irvliin microvilli to Myo7A of cells. Cad99C recruitment follicle the causes that tail conclusion the cytoplasmic corroborates strongly result This h muto y7 ntermiigcl oy(i.5G (Fig. body cell remaining the in Myo7A by of microvilli amount to strong the Myo7A of abnormally recruitment Cad99C Cad99C-FL, an to Similar Cad99C showed The 5G,G of GFP. (Fig. microvilli accumulation Myo7A with of of replaced enrichment and region deleted basal are region extracellular for Cad99C responsible is microvilli, the microvilli. type to of recruitment wild lengthening Myo7A of the that than to rather comparable was 5E body (Fig. cell remaining 5F the (Fig. in 10b stage at normal be to n pclctcre tsae1a(i.5E microvilli (Fig. in 10a reduced stage somewhat at cytocortex be apical in to and seemed detected S3F). Myo7A was of amount Fig. concentration Myo7A material Cad99C containing in microvilli (supplementary increase a no including core 2005), Notably, al., F-actin et lengthened (D’Alterio microvilli induced long isoform this abnormally Cad99C-FL, replaced to and similar deleted Nevertheless, is GFP. tail by cytoplasmic the Cad99C of the expressed majority the that studied which cells in we in possibilities, Myo7A these of distribution between filament distinguish Fig. actin To material longer (supplementary S3E). much microvilli lengthened the the with of Myo7A due bundles of be could association or an expression to Cad99C increased by induced of directly expense cell. the the of at rest microvilli the overlong in the drastic Myo7A to a recruited displayed 5I,I is that (Fig. Myo7A intensity cells signal follicle Myo7A 5C of in (Fig. reduction region Patj basal with and Myo7A central of colocalization reduced h togyicesdMoAsga ntemcoil was microvilli body the cell remaining in the 5C in Myo7A signal (Fig. of depletion Myo7A a by increased accompanied strongly The EERHARTICLE RESEARCH irvli nefc htbcm oepoone during pronounced more 5D,D (Fig. became 10a stage that from overlong effect the progression in an amounts high abnormally microvilli, 5C–D in (Fig. present type was wild to Myo7A length contrast entire in the microvilli, the along Myo7A of distributed expression. homogeneously of Cad99C-FL be distribution to to the response appeared in in change seen dramatic was of A full- Myo7A microvilli 2005). effects long al., of cell the excessively et Overexpression (D’Alterio causes follicle tested (Cad99C-FL) isoforms. we Cad99C in Cad99C length Cad99C, localization transgenic on various dependent Myo7A is whether microvilli determine follicle To to Myo7A recruits microvilli Cad99C cell of tail cytoplasmic The irvlio ol eadrc feto h oso Cad99C. of loss the the of to effect direct due a be be Cad99C-depleted cell could could of or size remaining signal reduced microvilli the and Myo7A in structure Patj apical impaired unaffected strongly with reduced in be colocalization The to location by appeared body. its and indicated in 5B), as represents accumulated (Fig. of cytocortex, still that pattern Myo7A apical 5A,B). surface spiky the (Fig. The apical missing was Cad99C. the microvilli lacked at that Myo7A clones cell follicle ofrhrts hshptei,w expressed we hypothesis, this test further To hsecsiercuteto y7 omcoil ih be might microvilli to Myo7A of recruitment excessive This enx nlzdtesbellrdsrbto fMoAin Myo7A of distribution subcellular the analyzed next We D D 9 9 ,F ,D x:F a soitdwt infcn eraein decrease significant a with associated was ex::GFP the in domains cadherin all which in ex::GFP, 9 9 ,I .Ti nlddteaia yootx hc showed which cytocortex, apical the included This ). 9 .W ocueta h a9Cctpamctail, cytoplasmic Cad99C the that conclude We ). D x:F nti oe(i.5H,H (Fig. zone this in ex::GFP D y:GP(i.5E–F (Fig. cyt::GFP 9 ,F 9 0 ,wihcrepnst an to corresponds which ), .Tedsrbto fMyo7A of distribution The ). 9 o1b(i.5C–C (Fig. 10b to ) 9 ,atog tappeared it although ), 9 .Ti mle that implies This ). 0 .Ised the Instead, ). 9 .Moreover, ). D 0 ,adthe and ), cyt::GFP, 9 ,I 9 9 0 ). ). ). Previous MoA:F)o h ebaebudctpamcti of tail Myo7A cytoplasmic (Cad99C full-length membrane-bound the either Cad99C or expressed encoding that (Myo7A::GFP) ovaries transgenes from lysates tissue GFP-tagged using for out test microvilli carried To were physically. to interact vivo proteins Myo7A in two of these that recruitment suggested complex Cad99C-dependent protein a The form Cad99C and Myo7A osre nCd9 oooso te net ( insects other not is of SH3- however, homologs an motif, This for Cad99C gambiae 2011). consensus in al., minimal et conserved the (Kaneko In site fitting (PTGP binding PCDH15. 1596), motif PxxP position one rather of only is contains at region Cad99C and of residues, be cytoplasmic proline region in to cytoplasmic poor the seems the PCDH15 Myo7A PCDH15, to to of with contrast bind domain SH3 to interaction The sufficient 2006). direct al., et enables (Senften Myo7A vertebrate complex a form proteins these that indicates microvilli. in of border. involved was organization brush is and Myo7A oocyte structure Myo7A that the the indicate regulating in microvilli, results when defects these cell caused together, follicle Taken also pattern in Myo7A defects of spacing to lack addition wider In showed overexpressed. and narrower a follicles, wild-type in by in observed separated occasionally than bodies were also vitelline of but of were islands cells microvilli rows be for follicle of the to Myo7A-depleted remains rows whether vertebrates described microridges Similar but from in determined. originate been 1976), epithelia microvilli Wassersug, certain cell have follicle and of Sperry surface patterns (e.g. the vitelline whorl on surround and microridges Similar between the meander that bodies. are in microvilli rows pronounced cell in follicle less apical that organized discovered was the We cells, of Myo7A. of follicle center absence the individual towards of the microvilli surface from of results which length that border, the increasing brush Moreover, microvilli the misoriented. of often produced appearance were dome-like and Myo7A shorter the be that lacking in to brush appeared cells epithelium found apical can follicular Follicle the we secretory ovary. of cells the Here, features of epithelial diversity. structural border organization, several functional of and affected structure large Myo7A surface their a of apical terms reflecting in the differ dramatically on Protrusions DISCUSSION nlssidctsta y7 n a9Cfr protein a form indirectly. Myo7A Cad99C or to binding directly and region either cytoplasmic Cad99C Myo7A the with that that complex, biochemical our indicates panel; summary, found In center analysis S4C). 6B Fig. (Fig. Cad99C, material Cad99C We supplementary Myo7A::GFP precipitated experiment, mCD8::GFP reciprocal S1A; not a S4A,B). In but panel). center Fig. 6A, Fig. (Fig. or Myo7A material proteins material Cad99C these of These for supplementary presence supplementary specific immunoprecipitated panels). were the that (Myo7A, for antibodies left using analyzed Cad99C, 6A,B, antibodies (Fig. were precipitates respectively mCD8::GFP, GFP-specific Cad99C they 5H where S2C). Fig. cells, (see follicle microvilli to in localized expressed specifically were proteins uulc-muorcptto fCd9 n Myo7A and Cad99C of co-immunoprecipitation Mutual ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal neatos oimnpeiiain(oI)experiments (co-IP) co-immunoprecipitation interactions, D D , x:F u o C8:F neatdwt Myo7A with interacted mCD8::GFP not but ex::GFP protein control the and Myo7A::GFP ex::GFP, psmellifera Apis vitro in D xeiet hwdta alprinof portion tail a that showed experiments x:F) epciey h transgenic The respectively. ex::GFP), , rblu castaneum Tribolium 9 upeetr aeilFig. material supplementary ; nvivo in n stherefore is and ) Drosophila novaries. in Anopheles 4827

Journal of Cell Science EERHARTICLE RESEARCH 4828 (A,A shown. are Cad99C of loss the of effects The (A,B) microvilli. clones. cell cell follicle follicle mutant to arrows, Myo7A yellow recruits cells; tail cytoplasmic follicle Cad99C The 5. Fig. htrfet omlmcoilspten B y7 sse pclt aji F-oiiewl-yeclsbtntin not but cells wild-type GFP-positive in Patj to apical seen is Myo7A (B) pattern. microvillus normal a reflects that D:GP r eeeyrdcdcmae ihCd9-oiiemcoil.MoAi rsn nteaia ieof side apical the on present is Myo7A microvilli. Cad99C-positive with compared reduced severely are CD8::GFP, vrxrs a9C(a9CF) hc r akdb ako D nCadetpclclzto fCd9 ln h hl lsammrn nD hwa show i D, decrease a in by membrane accompanied plasma is whole and (C) the 10b along stage (C Cad99C late cytoplasm to of remaining (D) localization early ectopic the from and in increases effect Myo7A C This microvilli. of in elongated CD2 the to of Myo7A lack of recruitment by dramatic marked are which (Cad99C-FL), Cad99C overexpress osntcueardsrbto fMoAfo h elbd oteeogtdmcoil tery(E,E early at microvilli elongated the to (F body microvilli of cell portion the basal from the Myo7A in concentrated of redistribution a cause not does rngnsa ai otesga najcn idtp el.Dt hwtemean the Cad99C-FL-overexpres show Data of region cells. wild-type central adjacent and in basal signal the the in to reduced ratio strongly (I is a staining cells. as Myo7 (wt) transgenes wild-type (I) of shown. that is cytoplasm to compared body cell main the in concentration Cad99C (G,G deleted). D x:F cuuae ntebslrgo fmcoil,i otatt noeosCd9.(I,I Cad99C. endogenous to contrast in microvilli, of region basal the in accumulates ex::GFP 9 )Cad99C D xcue togercmn fMoAa h pclpl fflil el n eraei h eann elbd.(H,H body. cell remaining the in decrease a and cells follicle of pole apical the at Myo7A of enrichment strong causes ex 9 ,D 9 h rp hw y7 inlitniyi h aa n eta eino olceclsta xrs different express that cells follicle of region central and basal the in intensity signal Myo7A shows graph The ) 9 ,icuigtePt-oiieaia yootx(C cytocortex apical Patj-positive the including ), 9 n ooaie ihPt nteaia yootx(F cytocortex apical the in Patj with colocalizes and ) ht rohas h rs odr learwed,teaia yootxof cytocortex apical the arrowheads, blue border; brush the arrowheads, White 6 .. *** s.d.; P 0 ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal .(E–F ). , .0.Saebr:10 bars: Scale 0.001. 9 0 rlt tg 0 (F–F 10b stage late or ) 0 .(G–H ). xrsino Cad99C of Expression ) 9 h fetof effect The ) 9 xrsino Cad99C of Expression ) 9 irvliof Microvilli ) Cad99C Cad99C m Cad99C m. 2 2 0 / 2 .Smlrt idtp el,MoAis Myo7A cells, wild-type to Similar ). Cad99C D / 2 el u ak h pk distribution spiky the lacks but cells y:GP(yolsi aldeleted) tail (cytoplasmic cyt::GFP rngnso Myo7A on transgenes el.(C–D cells. D 21-5 x:F cdei domains (cadherin ex::GFP uatcls aee by labeled cells, mutant 9 el that Cells ) 9 ) h amount the n igcells sing Cad99C

Journal of Cell Science htteitrcinbtenCd9 n y7 smdae ya by no mediated is is with possibility Myo7A has Another and interact Cad99C Myo7A. between also of interaction could domains the FERM that Cad99C that two the site 2002). of binding one domain-binding different al., a FERM have et apparent might direct Myo7A (Velichkova a of specificity domain suggests However, SH3 site SH3-binding the domain. apparent no that the has SH3 and also Myo7A that vertebrate protein an of Keap1 domain for SH3 the between anchor interaction unlikely an ARTICLE RESEARCH rti B nlstswscnimdo h aebo rgtpanels). (right blot same the Cad99C on and confirmed (A) was Myo7A lysates of in presence (B) GFP- The protein re-probed of panels). were (IP) (left Blots immunoprecipitation proteins A. show tagged in to control antibodies and positive GFP-specific A,B, a with in as control used negative was middle a Myo7A::GFP in as (arrowheads served Myo7A::GFP mCD8::GFP Cad99C with panels). with oprecipitates oprecipitates Cad99C Myo7A (B) (A) and Myo7A. and Cad99C complex. of protein Cad99C–Myo7A 6. Fig. the which in complex, indirectly, protein or directly a interacts, Myo7A. of Cad99C with of part region Myo7A are cytoplasmic that conclude Cad99C we self- together, recruitment and Taken permit the Cad99C. to by acting releasing Myo7A microvilli, additional of in an present for Alternatively, be position. could favorable this factor at not Myo7A of because is inhibition membrane that lateral organization the is at possibility Cad99C F-actin to One bind co- Myo7A. to of of failed not the Myo7A recruitment did surface the overexpression obvious to heavy to an apical to contrast cause localize owing the ectopically In membrane at that plasma 2005). lateral molecules Cad99C Cad99C al., and cells, et follicle Myo7A D’Alterio of 2005, dependency (Schlichting al., Cad99C with of interact et could sites that PDZ-domain-binding molecule predicted harmonin-like the a as such linker, AB Co-immunoprecipitation (A,B) D ex::GFP, oatnflmns(age l,20;Ueie l,2009). al., microvillus et the Umeki at 2009; molecules al., Myo7A which et activated molecule, (Yang of this binding filaments Clustering of strong consequently actin self-inhibition and to the activity ATPase contributes of Myo7A initiate investigate of would release tail to the the interesting to be Cad99C to of will binding 1995), It the al., F-actin. whether is et that with a (Hasson Myo7A of associated cells autoinhibited) Such not (or hair pool cells. monomeric for represent follicle large reported might and of been the also cytoplasm has from non-cortical pool the derived in overexpressed Myo7A was microvilli. Cad99C in in another co-dependent one are of proteins levels both normal that maintaining and microvilli, to Myo7A of Myo7A Cad99C, loss apical have full-length an not with in did cadherin together to 2005), findings, These al., similar effect. extracellular et this (D’Alterio which, microvilli contrast, membrane-bound overlong Cad99C, By caused cell. of the the of domains regions of at other microvilli in into expression Myo7A Myo7A of of of portion recruitment expense strong the cytoplasmic a to membrane-anchored led as cytoplasmic Cad99C, the well as the of Cad99C, and full-length expression of Myo7A Overexpression between Cad99C. of tie tail close the corroborate oto t yolsi al(Cad99C tail cytoplasmic its of most directly Myo7A- be of could misalignment Cad99C and Schlichting microvilli. of height microvilli. depleted 2005; reduced amount the al., length reduced in for et the the responsible 2006), (D’Alterio supporting Cad99C al., microvilli in that et of of Cad99C indicate stability of amount findings and function our the the the yet, Considering Although from modulates understood endocytosed. dissociation not Myo7A that is possible Myo7A is Cad99C is mechanism before for It membrane. occurs positive molecules the Myo7A two not from these of or were co-transport to rate. against Cad99C arguing follicle turnover 5D,G), of contained (Fig. its region that subapical slowing the cells membrane, in vesicles plasma stabilize cytoplasmic might Notably, the Myo7A core, in actin microvillus Cad99C the to Cad99C it it to although binding fixing se, by and per Alternatively, efficiency. transport its Cad99C increase lacking for might that necessary microvilli indicates not This in is distributed. Cad99C. normally Myo7A the present be of to still raising seemed transport and was microvilli, the Myo7A Cad99C in in reduced, involved Cad99C is Albeit of Myo7A that amount possibility the in reduction interaction the actin the the that for to Myo7A microvilli. propose of of proposed we affinity core increased together, been facilitates (Sakai Taken Cad99C complex with transporting has 2011). MyRip–Rab27a possibly al., the mechanism with et manner, Myo7A processive similar of interaction a along A move in promote can cargo. core Myo7A might that actin so Cad99C the formation with dimer and interaction activation through membrane akdaymdltr feto h yolsi ein Here, have region. might cytoplasmic the protein of Cad99C cytoplasmic effect high truncated modulatory abnormally of any the the masked of independently that integrity level however, microvilli expression structural speculate, control We of interactions. can outgrowth Cad99C full-length extracellular and of of the absence that domains suggesting the 2005), cadherin al., in et even (D’Alterio Cad99C microvilli long abnormally uho h y7 htwsrcutdt irvliwhen microvilli to recruited was that Myo7A the of Much strongly studies interaction genetic our from data The epeiul eotdta a9Cpoenta smissing is that protein Cad99C that reported previously We marked a found we Myo7A, of absence the in Interestingly, Cad99C ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal uat,sgetta a9Ci rca o recruiting for crucial is Cad99C that suggest mutants, D y)wsal oinduce to able was cyt) 4829

Journal of Cell Science ta. 05 aantson.W ocueta y7 and the Myo7A in that functions protrusions. conclude independent apical We and of shown). regulation co-dependent not have data Cad99C 2005; al., et n ho 02 eefudin found were the 2012) with Chao, for and characteristic interfere hairs are wing not that multiple (Nu defects does Neither denticle eggshell. larval Myo7A functional nor By of a 2008). of al., loss formation et that Elalayli 2006; (D’Alterio indicating al., collapse from et eggs and Schlichting contrast, dehydrate 2005; al., to et eggs causing defective in the formation, that finding border the is brush this with Consistent cells. follicle olceclswr osdrbymr eeeta in than severe more considerably were cells follicle lnsi h olclreihlu eeidcdb FLP/FRT-mediated by induced using were recombination, epithelium mitotic follicular the in clones ae nteasneo F-aacradrie nyeast- on raised and GFP-balancer 21–22 a at plates of agar absence juice apple the supplemented on based hc nrae uvvlrtsdrn is n eodinstar. second and first during rates survival increased which Df(2L)BSC299 the and nterqieeto hs w rtisfrmicrovillus for proteins two differences these and of similarities requirement reveals the cells follicle in in Cad99C microvilli. in cadherin between this of formation function Cad99C the of complex to tail cytoplasmic contributes observed the that indicates the Myo7A, and Cad99C with of together structure. amount normal normal of This, microvilli a produce achieve to and to microvilli important in Cad99C is Myo7A that show we ARTICLE RESEARCH 4830 alleles null follows: as were study this and in used strains The Drosophila METHODS AND MATERIALS in defects border brush requirements The independent genes. two some these process. least Myo7A for treadmilling at of uneven actin suggesting phenotypes the differences, the to the Alternatively, related Notably, be stereocilia 2007). could of al., base distribution interact that the to et at possible seems receptors (Michalski is which transmembrane It Myo7A, several mammalian protrusion. microvillus with basal to a the similar in of 2006), factors region additional portion al., with apical interacts portion et in Myo7A basal the the the Schlichting in distributed in to concentration 2005; than that higher homogenously much contrast al., a be showed et Myo7A In example, to (D’Alterio co-dependence. appears microvilli for not which their are microvilli Cad99C, curious, within considering Myo7A and is identical, Cad99C of It distributions morphogenesis. iusy 97.TeFPu astewsatvtdb n11–35-minute an by 37 activated at was shock cassette heat FLPout The 1997). Zipursky, the by induced was and 2005) al., et UAS-Cad99C constructs UAS-Cad99C transgenic the of Expression n vre eedsetd2dy ae.Cnrlgntpswr Oregon were genotypes Control later. days and 2 R dissected were ovaries and lns(uadRbn 1993), Rubin, and (Xu clones 1993). Rubin, Df(2L)ED3 a nue yto2hu etsok t37 at shocks heat 2-hour two by induced was CD8::GFP h elcoe eea olw:(1) follows: as were clones cell The oprsno h itiuinadfnto fMoAand Myo7A of function and distribution the of comparison A sli-ohr ta. 94 ihr ta. 04 Bejsovec 2004; al., et Kiehart 1984; al., et ¨sslein-Volhard Cad99C w 1118 ck o oiiemrig(e n u,19) Recombination 1999). Luo, and (Lee marking positive for le eerie t25 at raised were Flies . noeigdeletions uncovering 21-8 and tan n genetics and strains D D ubi-nlsGFP ex::GFP cyt::GFP Co ta. 02.T generate To 2012). al., et (Cook ˚ DAtroe l,20)and 2005) al., et (D’Alterio .Fmlswr –-asoda h ieo etshock heat of time the at old 0–1-days were Females C. UAS-mCD8::GFP ck 13 Act5c Cad99C /Df(2L)BSC299 ck #3ad1;sebelow), see 17; and (#13 #81 n 817 DAtroe l,2005), al., et (D’Alterio 18-127) and (#18-10 uatfmlsddntclas ( collapse not did females mutant . a mlydfrngtv aeigo cell of labeling negative for employed was mCD2 hsFLP1 uat nefrdwt eggshell with interfered mutants Df(2L)ED3 TubP-Gal4 . Cad99C LeadLo 99 nflil cells follicle in 1999) Luo, and (Lee ˚ Gal4 C. Cad99C and is ntrlra eeselected were larvae instar first UAS-Cad99C ck Lotcset Pgoiand (Pignoni cassette FLPout FRT40A 13 ck , and Rdre l,20)and 2004) al., et (Ryder and 13 ˚ TubP-Gal80 nautfmls (2) females. adult in C uat (Schlichting mutants ˚ n o humidity, low and C Keate l,2004), al., et (Kiehart ck Cad99C ck UAS-ck::GFP or uatflies, mutant #2adT)and T6) and (#T2 Cad99C FRT82B uat show mutants ck uatcell mutant Cad99C ck and n mutants X and (Xu 5 mutant mutant 390), (Todi UAS- ck 21-5 13 / a9C(rnmmrn n yolsi ein)adte3 of the acids and amino regions) 354 cytoplasmic C-terminal and the (transmembrane for Cad99C sequence (3) CA); View, Mountain CTTGTACAGCTCGTCC-3 ta. 07 aah ta. 02 a sdt rv xrsino the of oogenesis. expression throughout drive chambers to egg used of was epithelium 2002) follicular al., et Hayashi UAS 2007; al., et oan –) hs etdswr xrse n xrce sGST as was Specificity anti-Myo7A-GP6) extracted rabbits. anti-Cad99C-Rb3, and and pigs (anti-Cad99C-Rb1, guinea antisera expressed immunize of to were antibodies used and (cadherin peptides produce proteins Cad99C fusion and of These to Myo7A 505–751 IQ used 5–6). acids the against amino peptide domains of of antibodies the consisted (portion Cad99C and Myo7A of against domain), of production 873–1065 Myth4 the acids adjacent amino for of used consisted peptide antibodies Cad99C-specific The and Myo7A of of Generation generation The procedures. 1993). standard Perrimon, followed flies and transgenic (Brand vector transformation Kpn 5 primers – the peptide (2) signal the 2005); including Cad99C, of Not acids amino 102 N-terminal Cad99C Cad99C Cad99C eoee fe aho he osctv etiuainsteps centrifugation consecutive was lysate three tissue of the 19,000 of at each supernatant 0.3 minutes (5 EDTA, The after mM PMSF). 1 recovered mM on NP-40, 1 PBS 1% leupeptin, in NaCl, dissected mM 300 were 150 in females homogenized well-fed pairs and 160 2-day-old ice genotype, from each ovaries for of experiments, co-immunoprecipitation analysis For blot western and Co-immunoprecipitation (1:250; anti-Patj rabbit anti- Canada), rabbit and ON, 2000) al., Toronto, et Tanentzapf Abcam, (1:500; anti- mouse anti- 1:2000), GFP (GP6, rabbit anti-Myo7A 2005), pig al., guinea 1:1000), et (Rb1, D’Alterio Cad99C 1:3000; polyclonal (GP5, follows: anti-Cad99C as pig were guinea immunostaining tissue for antibodies Primary imaging and markers Cellular S4A,B). 4A; Fig. (Fig. immunostaining S1A–C; tissue Fig. and material analysis supplementary blot western by confirmed yaespraatwsicbtdwt 50 was yield with protein incubated total was The supernatant ml. 2 lysate to adjusted was eegnrtdwt es S50sann ae ofclmicroscope confocal laser scanning 40 LSM510 Laboratories)using Zeiss (Vector a Vectashield with generated in Fluorescent were mounted to Canada). preparations conjugated ON, from Burlington, was images Laboratories, lectin or Vector 488 Potato (1:10; Fluor Technologies). FITC Alexa Life to (1:20; conjugated phalloidin Rhodamine with visualized was F-actin The Canada). trap Fluor ON, gene Burlington, GFP Technologies, Alexa (Life 555 Fluor PA), Alexa or Westgrove, 488, Laboratories, Cy5 ImmunoResearch Cy3, to (Jackson conjugated were (1:400) antibodies Secondary 1994). Kiehart, ojgtdt antcbas( beads magnetic to conjugated rcse sn db htso-S n lutao-S (Adobe Illustrator-CS6 and Photoshop-CS6 Canada). ON, were Ottawa, Adobe Figures Systems, temperature. room using at performed processed was imaging All Canada). abtat-F 140;Cotc aoaois.HRP-conjugated Laboratories). Clontech (1:4000; and 1:4000) (GP6, anti-GFP VIIA anti-Myosin pig rabbit guinea anti- 1:3000), rabbit polyclonal (Rb3, were Cad99C isolated antibodies Primary SDS-PAGE. were by separated Biotec). immunocomplexes (Miltenyi manufacturer The (3 the ice. of instructions on the minutes to 30 according for CA) h eoye o iceitywr sfollows: as were biochemistry for genotypes The o etr ltaayi,poen rmoaintsu lysates tissue ovarian from proteins analysis, blot western For m I rgeto the of fragment I ,udltdlst)adfo muorcpttos(25 immunoprecipitations from and lysate) undiluted l, –Pst -transgenes 6 D D ora fCl cec 21)17 8143 doi:10.1242/jcs.099242 4821–4832 127, (2014) Science Cell of Journal rgeto a of fragment I D x:F ak l xrclua ahrndmiso Cad99C. of domains cadherin extracellular all lacks ex::GFP ex::GFP 14 n 63 and /1.4, ex eGFP :: 9 GFP -GAATTCTGCAGTCGACGGTAC-3 zip::GFP ck::GFP ossso:()april5 partial a (1) of: consists transgene – 6 Cad99C g 14Pa-p betvs(alZis oot,ON, Toronto, Zeiss, (Carl objectives Plan-Apo /1.4 Pst a sdt eetMoi I(ai ta. 2010). al., et (David II Myosin detect to used was t4 at , I– Cad99C Cad99C 9 Pvu ˚ m namcoetiue n h volume the and microcentrifuge) a in C rmpGPN Cotc Laboratories, (Clontech pEGFP-N2 from DA.pATwsue saPelement P a as used was pUAST cDNA). flssbfe 5 MTi-C H8, pH Tris-HCl mM (50 buffer lysis of l rget C-mlfe yusing by PCR-amplified fragment, I m AS itniBoe,SnDiego, San Biotec, Miltenyi MACS; D DAcntut(’lei tal., et (D’Alterio construct cDNA ex::GFP b H seti 110;Toa and Thomas (1:1000; -spectrin 9 m T lssqec o the for sequence plus UTR fat-F antibodies anti-GFP of l and m poii,10 aprotinin, M mCD8::GFP 9 tj-Gal4 , n 5 and g h tissue The mg. 6 9 9 T ( UTR (Tanentzapf -CGATCG- m )were l) nthe in Pvu m M I–

Journal of Cell Science S.1- at CS6. taken were Images analyzed. were 6 images (10–40) multiple and viewed HT-7700 eight Hitachi controland six a For with Canada). analyzed ON, Etobicoke, High-Technologies, and (Hitachi citrate, TEM the lead and contrasted of acetate were center uranyl respectively) with the interface, the follicle-cell–oocyte through the to sections or (longitudinal perpendicular follicle microvilli or of parallel axis Canada), length ON, Concord, Microsystems, neighboring pcfe uclua rao uatadcnrlclsuigImageJ between using a cells for immunosignals control determined in and signal of was Cad99C mutant determine value To of intensity (1) pixel (NIH). area the relative subcellular cells, specified the different genotypically compare To intensity immunosignal Baie of Measurements Sciences, detection. Life signal for Healthcare used (GE were Canada) system QC, visualization Laboratories)d’Urfe ECL ImmunoResearch the Jackson and (1:1500; antibodies secondary ARTICLE RESEARCH n ..pandadpromdeprmns ..sprie h rjc and project the supervised D.G. manuscript. X.C. experiments. the experiments. performed wrote analyzed and and planned performed A.D. designed, and D.G. and R.-H.S.L. C.G., contributions Author interests. competing no declare authors The interests Competing support. departmental technical our for and facilities reagents, animal for and CT) imaging and Haven, Japan) New (Kyoto, University, Center (Yale Research University,FlyTrap Genetic Ulrich (Indiana Drosophila PA), Center Kyoto College, Stock IN), State Bloomington, Drosophila of State, Bloomington University (Penn the (The Thomas and Eberl Graham Tepass Daniel IA), thank City, We Iowa for manuscript. Canada) Iowa, Toronto, the of on (University Harris comments on Tony advice helpful and for Tepass Canada) Ulrich Toronto, thank of We (University TEM. Tepass Ulrich Angeles, and Los CA) of Jacobs Angeles, (University Roger Los Laski Ng and Frank Mark Canada), assistance, Hamilton, and technical University, Hwang (McMaster for Shang-Hsien Canada) Yeung, Toronto, Au of Maggie (University to grateful are We Acknowledgements 2.5% in OsO in hour, 1% 1 in acrolein for and 2% (Caco-buffer) overnight in 7.4 Caco-buffer ice and pH in on buffer paraformaldehyde glutaraldehyde fixed cacodylate medium, 2% mM Schneider’s 75 glutaraldehyde, cold in 2.5% dissected were Ovaries microscopy electron Transmission Cad99C five clone, to each two For in analyzed. measured were was follicles signal stage-10 in clones cell ape eeicbtdi rs EMoengt one nfresh in mounted overnight, hours, 4 65 AEEM at for fresh hardened oxide and in araldite-502/ AEEM propylene incubated and ON, of were oxide evaporation Burlington, samples propylene After Cedarlane, of overnight. (AEEM; mixture Canada) medium each 1:1 periods embedding a 15-minute Embed-812 two in for incubated treated oxide series, propylene and ethanol and chemicals an ethanol Oakville, in and 100% dehydrated Sigma-Aldrich, with fixatives then and were grade Samples PA Canada). (EM Warrington, ON, hour Inc., 1 Polysciences, for from Caco-buffer in sorbitol otae aJla A a sdfrsaitclaayi pie two- (paired analysis statistical was for (GraphPad cell used 4 wild-type was Prism neighboring CA) value). tailed a Jolla, wild-type mutant to La of each percentage intensity Software, For the signal cells. (as in wild-type determined clone ratio neighboring mutant the of per measured number cell, were equal cells an eight evaluated cases, plus were most clones In cell microvillus genotype. eight the per to seven of transgenes, area Cad99C the expressing a to corresponded in which region cells, all in analyzed ihnteflil.Mgaino etiea el n hcns fthe of thickness and 1970). (King, cells follicles stage centripetal to sections used of ultra-thin were of epithelium Migration position follicular the follicle. and follicle the the of within age the for control a as 4-1K otatadbihns eeajse nAoePhotoshop Adobe in adjusted were brightness and Contrast 14K-110K. 0-mtikscin LiaE C lrmcooe Leica ultramicrotome, UC6 EM (Leica sections 100-nm-thick t ts)adgnrto fgraphs. of generation and -test) m -hc etos tie ihtliiemtyeebu eeused were blue toluidine/methylene with stained sections, m-thick ck uatflils(al omdsae1b,svrlscin were sections several 10b), stage mid to (early follicles mutant ck ck +/+ uatcl.()T eemn y7 inli cells in signal Myo7A determine To (2) cell. mutant or ck +/ 2 ˚ o 2hours. 12 for C oto el.A rao qa iewas size equal of area An cells. control ck 2 / 2 el n neulnme of number equal an and cells ck uatcls 13 cells, mutant 4 n 0.1% and ck mutant idr .adHio .L. Boe S. Haigo, and D. Bilder, T. A. Chao, and A. Bejsovec, A., Houdusse, S., Hoos, S., Nouaille, P., J. Hardelin, V., Michel, A., Bahloul, and G. C. Wright, S., K. Pawlowski, Y., H. Kwon, L., C. S., Murcia, N., K. K. Alagramam, Pawlowski, G., C. Wright, J., Zahorsky-Reeves, N., K. D., Weil, Alagramam, N., K. Alagramam, J., Reiners, Y., Kikkawa, V., Michel, A., Adato, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.099242/-/DC1 online available material Supplementary material Supplementary of Health Council of Research Institute Engineering D.G.). Canadian and (to the Sciences Canada from Natural grants the operating and by Research; funded was work This Funding ai,D . ihia .adHri,T J. T. Harris, and A. Tishkina, J., J., D. C. David, Arana, A., M. Li, S., M. Hwang, W., M. Yeung, E., D., R. D. Tran, McConnell, C., D’Alterio, E., N. Grega-Larson, E., Jr, M. A., Deal, D. A., Shifrin, W., R. S. Coburn, Crawley, A., J. Deal, J., S. Christensen, K., R. Cook, N. Perrimon, and H. A. Brand, eo,R . clthy .I n rtce,A. Bretscher, T. and Nicolson, I. and A. McClatchey, C. G., Petit, R. R., Fehon, Geisler, P., Haffter, J., G. Rauch, S., Ernest, llyi . al .D,Fkor,M,Niwne,H,Elsn .T,Hn Z., Han, T., T. Ellison, H., Neiswender, M., Fakhouri, D., J. Hall, M., C. Elalayli, Petit, and A. Bahloul, A., El-Amraoui, G. L. Tilney, and J. D. DeRosier, asn . enzla,M . atsSch,J,Cry .P n Mooseker, and P. D. Corey, J., Santos-Sacchi, B., M. A. Heintzelman, J. T., Hasson, Spudich, and S. Sivaramakrishnan, D., Stafford, Finan, R., A., J. M. Sellers, Hartman, J., Fordham, K., Choi, N., M. Billington, Ashburner, J., and Haithcock, S. McGill, J., Roote, M., Shelton, D., Gubb, B. Kachar, and J. M. A. Grati, Griffith, and B. T. Friedman, A., I. Belyantseva, I., G. Frolenkov, amnnadcdei 3 he se eepout htcoeaet shape to cooperate that products al. gene et I J. Usher Reiners, three S., 23, Shorte, cadherin R., and K. harmonin Fath, I., Perfettini, S., egg. Drosophila the from perspectives formation. denticle Drosophila in interact signaling and complex ternary a form genes, phospholipids. I membrane type with syndrome C. Usher by Petit, encoded and P. England, gene. protocadherin novel a Pcdh15, of mutation waltzer. Ames P. R. mutation Woychik, mouse the of allele new a 148 P. in R. ear Woychik, inner and the L. Stubbs, C., L. Erway, proteins. 1 type syndrome Usher C. 356. of Petit, network and the A. El-Amraoui, in U., Wolfrum, H., Yonekawa, eaoatrCd9,teotoou fhmnUhrcdei PCDH15, cadherin Usher human microvilli. of of al. length et orthologue the M. regulates the Chase, P., Cad99C, Sharma, protocadherin-based M., melanogaster Kubesh, by K., V. Mulligan, driven al. et assembly A. B. border Millis, T., K. adhesion. brush Nam, Y., intermicrovillar Q. Intestinal Zheng, Y., Zheng, (2014). S., Mao, E., the A. Benesh, of subdivision and R. coverage K. genome. extensive Cook, melanogaster provide and Drosophila to C. 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Journal of Cell Science ao,U n ahr B. Kachar, and U. Manor, P. M. Kambysellis, and P. A. Mahowald, B. Kachar, P. and A. Mahowald, E. M. Schneider, W., H. Lin, Lefe ye,E,Bos . sbre,M,B M., Ashburner, F., Blows, E., Ryder, A. Bradley, and P. K. Steel, K., A. Rzadzinska, M., H. Prosser, L. S. Zipursky, and F. Pignoni, H. G. Thomas, and D. M. Phillips, e,T n u,L. Luo, and T. Lee, izdi,S,Blatea .A,Gee .P,Le . nzyuin .A., A. Indzhykulian, K., Lee, P., A. and F. Giese, D. A., Dolan, I. A., Belyantseva, L. S., Riazuddin, Beyer, A., G. Dootz, N., K. Kobayashi, Y., Raphael, Nu A. Guichet, and C. J. Olivo-Marin, N., Chenouard, E., Nicolas, J. M. Tyska, and E. R. McConnell, R., L. Nambiar, C. Howe, J., D. Fishkind, A., K. Conzelman, M., E. Bonder, S., M. Mooseker, Ku Milligan, M., E. Wilson-Kubalek, J., Tokita, H., Sakaguchi, P., S. Kazmierczak, S. Li, and S. S. Sidhu, T., Kaneko, ihlk,N,Mce,V,Bhol . Lefe A., Bahloul, V., Michel, N., Michalski, nu,A n kb,M. Ikebe, and A. Inoue, L. Cooley, and M. 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