oii.Tu,Cc2ifune h aneac fsal actin cells. maintained hair stable and cochlear turnover, of viability of actin and function of maintenance phosphorylated tuning the elaborate of through influenced levels structures Cdc42 enhanced Thus, by cofilin. represented downregulated turnover and AJCs, actin disrupted and microvilli abnormal including rdcdpeoye iia otoeof those to similar phenotypes produced etrfrDsaeBooyadItgaieMdcn,Fclyo Medicine, of Japan. Faculty 113-0033, Medicine, Tokyo Tokyo, Integrative of and University Biology Disease for Center yais nttt fMlclradClua isine,Uieit fTokyo, of Univeristy Biosciences, Japan. Cellular 113-0032, and Tokyo Molecular of Institute Dynamics, nvriyGaut colo imdclSine,Ngsk 5-53 Japan. 852-8523, Nagasaki Sciences, Biomedical of School Graduate University ilg,Dprmn fAaoy aut fMdcn,Uiest fMiyazaki, of University Medicine, Japan. of 889-1692, Faculty Miyazaki Anatomy, of Department Biology, oa A522 USA. 52242, IA Iowa, Japan. 606-8315, Kyoto University, Kyoto Japan. 657-8501, Kobe University, aeioUeyama Takehiko cells by hair complexes cochlear junctional in apical Cdc42 and stereocilia of Maintenance ARTICLE RESEARCH 2040 2014 January 22 Accepted 2013; September 30 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This work ` this to equally contributed authors *These 8 3 1 FRET turnover, Apical Actin cell, complex, Hair junctional Stereocilia, Deafness, Cdc42, WORDS: KEY of cells Hair (stereocilia). protrusions actin stable elaborate of highly possessing cells role hair in the Cdc42 analyze employed influences to knockout We conditional hair-cell-specific regulation epithelia. ear differentiated inner actin in Cdc42-dependent structures actin how steady-state about known little However, organization. actin is dynamic of regulator key a is Cdc42 ABSTRACT ekSrey yt rfcua nvriyo eiie yt 0-56 Japan. 602-8566, Kyoto Medicine, of University Prefectural Kyoto Surgery, Neck ilg,RKN oe6004,Japan. 650-0047, Kobe RIKEN, Biology, n Jsi oherhi cells. hair cochlear membranes in stereociliary AJCs at and activation and presence biosensor Cdc42 Cdc42-FRET a indicated expressing mice transgenic from junctional cells hair of apical imaging (FRET) transfer at energy resonance fluorescence and cells belt hair in expression actin GFP–Cdc42 Adenovirus-encoded circumferential (AJCs). complexes waving a by and hair accompanied outer depletion, thinning in and fusion than stereocilia cells as hair began inner and cells, in robust more was hair degeneration Cochlear frequencies. cell in high at resulting particularly loss maturation, hearing after progressive degenerated progressively but normally aaiSaito Naoaki aeioKoji Takehiko y Shimizu Aya uhr o orsodne([email protected] [email protected]) ([email protected]; correspondence for Authors eateto ilg,Cleeo iea rsadSine,Uiest fIowa, of University Sciences, and Arts Liberal of College Biology, of Department Biostudies, of School Graduate Signaling, Cell and Bioimaging of Laboratory Kobe Center, Research Biosignal Pharmacology, Molecular of Laboratory 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,24–02doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 1, auiNakao Kazuki , 7 ` 9 en Fritzsch Bernd , lcrnMcocp aoaoy etrfrDevelopmental for Center Laboratory, Microscope Electron 7 eateto itlg n elBooy Nagasaki Biology, Cell and Histology of Department 1, 6 aoaoyo ebaeadCytoskeleton and Membrane of Laboratory * , ` iouiSakaguchi Hirofumi , Atoh1–Cre;Cdc42 2 eateto tlrnooyHa and Otolaryngology-Head of Department Cdc42 4 4 aoaoyo nmlResources, Animal of Laboratory ohtk Hishikawa Yoshitaka , 5 iiino itceityadCell and Histochemistry of Division 8 hgnb Yonemura Shigenobu , kokoni DKcells MDCK in -knockdown Cdc42 flox/flox dltdhi cells, hair -deleted iedeveloped mice 2, ,TksiNakamura Takashi *, 5 uuuNinoyu Yuzuru , 9 au Hisa Yasuo , 00 n pcljntoa opee AC)(vnve al., et (Ivanov (AJCs) 1992), complexes al., junctional rate al., et 2005). apical et Theriot turnover (Schwander and stereocilia 2004; a in 2010) stable al., with relatively et are dynamic they (Ponti whereas highly seconds in are the measured and filaments lamellipodia actin as 2010). to tail, such Welch, topology, structures, and cell membrane some (Campellone local In overall structures as cellular alter well distinct as form rearrangement polarity, and and turnover geometry actin Dynamic INTRODUCTION 06,weesRcadRo nuelmlioi n stress and lamellipodia induce al., et RhoA Yang and 2000; Rac is al., Cdc42 et whereas effects. (Chen 2006), regulators different formation common filopodia in cells for results have hair essential activity they in their studied, Although effectors, characterized expressed 2000). been and are best al., of all not the et and are (Kalinec maintenance has GTPases, RhoA small and cells and Rho-family Rac1 hair development Cdc42, in the stereocilia. Cdc42 regarding unknown of particularly remain function AJCs the at the turnover However, 2006). actin 2011). al., of al., et (Nunes arrangement et effect cell (Collado and proper barrier mode a epithelial of the maintenance cell and the hair for as in essential mechanism such structure al., turnover function, actin et actin another (Zhang are the finding AJCs about stereocilia. such question no a reported raising have 2012), (Rzadzinska others turnover 2004), al., actin steady et their dynamic maintain treadmill-like a stereocilia through Although that state of requirement. suggested turnover has lifetime 2008), model a al., recent et is hair (Collado components and as replaced stereociliary Moreover, not length usually precisely. the are determined cells are thus, stereocilia stimulation; of of movement shape mechanical coordinated High the upon 2004). on al., depends stereocilia et cells organized (Frolenkov hair are height of stereocilia graded sensitivity however, of tip; rows with distal precise filaments the into actin-based actin at and involves parallel ends are microvilli plus of the hundreds that organized 2010; which of exquisitely al., composed Stereocilia, process as filopodia et known 2010). active are (Matsumoto al., protrusions, an motility et somatic through Schwander and function amplifier OHCs stereociliary whereas cells an sensors, hair true as outer are during IHCs roles of detection: distinct rows sound have common row share three they to single machinery, believed and a are mechanotransduction OHCs (IHCs) in and IHCs arranged cells Although and are (OHCs). hair cells hearing inner hair detecting cochlea, of epithelia the In sensory balance. specialized are which d4 sakyrgltro h ci yokltn However, cytoskeleton. actin the of regulator key a is Cdc42 cells, hair ear inner in present are AJCs and Stereocilia 1,2 khr Goto Akihiro , 2 1,2 ihyk Matsuda Michiyuki , ieoh Kassai Hidetoshi , 3 hgfm Morioka Shigefumi , 3 4 tuAiba Atsu , hr Suetsugu Shiro , Listeria 4 1,2 and , comet 6 ,

Journal of Cell Science tebyncdy()1. n 1. swl spsntldy3 day postnatal as the well as assessing CAG- E15.5 by and 13.5 (P3) (E) day embryonic at Atoh1–Cre of inactivation hair-cell-specific Cdc42 for factor transcription (bHLH) Fg A.W sdthe used We 1A). (Fig. C-ie HsadOC tP,wt atclryintense particularly with P0, at be of OHCs in stereocilia in proteins detected to and reactivity was actin-related immunoreactivity known IHCs Cdc42 against TCA-fixed 1999). fixation, al., antibodies (TCA) et several (Hayashi acid for Therefore, trichloroacetic fixation. effective using conventional used with Cdc42 tissues we ear endogenous specific a inner detect detect in not could signal to but antibodies attempted anti-Cdc42 different two We S1B). Fig. a beti h arclsof bundle cells hair hair a the is in Cdc42 reactivity this absent that expected, As was 2013). stating al., et report (Shin protein (stereocilia) a with consistent – H Fg A.N ifrne eeosre nhearing in observed were differences No at frequencies age-matched 2A). between low (Fig. the kHz to 2–4 corresponding stimuli click broadband pcfcsga nIC n Hsa 1of P1 at a OHCs showed and also IHCs Cre (supplementary in P3 against signal at Immunostaining specific OHCs S1A). and Fig. IHCs material to restricted was staining Fg 1B). (Fig. h A/I iae(IK aha n h cofilin the and al., formation pathway et AJC regulates Melendez through also 2010; (LIMK) Cdc42 al., stabilization 2011). et kinase (Matsumoto and cycle phosphorylation induction PAK/LIM filopodia the 2007), Suetsugu, promotes and Arp2/3 and (Takenawa actin the N-WASP including with component 2010), complex interaction Welch, its and (Campellone through nucleators polymerization Cdc42 actin 2008). Ridley, induces and (Heasman respectively formation, fiber ARTICLE RESEARCH iea ek rheterozygous or weeks 3 at mice 2– in hearing examined We the brainstem. auditory from upper pathway used 8-week-old auditory the the first to in test we cochlea potentials hearing evoked function, electrophysiological detects an cochlear that (ABR), assess response to brainstem order In in loss cell hair Cre;Cdc42 cochlear and loss hearing Progressive and shown) reaction not chain polymerase data transcription of reverse (RT-PCR; expression a confirmed using cochlea and examined We Atoh1–Cre the of Inactivation RESULTS and 2010) al., et Qin 2006; u o nlittermate in not but 02,Xglsann a is bevda 1. ntecochlea the in of E15.5 ( at vestibule observed first and was staining X-gal 2002), Atoh1–Cre;LacZ hs aapoienwisgt notefnto fCc2at Cdc42 of epithelia function differentiated the in loss. AJCs actin into cell and insights hair protrusions downregulated new apical cochlear provide with progressive data slowly These associated to leading was turnover, and hair in morphology, maintenance their for cells. AJCs and membranes stereociliary CAG- ee eso htCc2i oaie n tfntosat functions it and localized is Cdc42 that show we Here, flox Cdc42 flox Ce ta. 02 aa ta. 03.Teptenof pattern The 2013). al., et Jahan 2002; al., et (Chen CAT CAT drce eobnto a sesdi h ne ear inner the in assessed was recombination -directed flox/flox rngncmice transgenic flox Atoh1–Cre;Cdc42 eeinrsle nanra troii n AJC and stereocilia abnormal in resulted deletion flox -LacZ -LacZ .Cnitn ihapeiu eot(hne al., et (Chen report previous a with Consistent ). mice Atoh1–Cre;LacZ Cdc42 Atoh1–Cre Cdc42 ie(aantson.Ccla X-gal Cochlear shown). not (data mice ) eotrmc hratrrfre oas to referred (hereafter mice reporter Atoh1 b Cdc42 glcoiaeatvt frearranged of activity -galactosidase eei ne a arclsb s of use by cells hair ear inner in gene flox/flox nvivo in rmtr ai helix-loop-helix basic a promoter, flox/flox 2 flox/flox / Atoh1–Cre;Cdc42 2 ieand mice Atoh1–Cre;Cdc42 ie(upeetr material (supplementary mice Mlne ta. 2011). al., et (Melendez nsitu in iebtnti control in not but mice ie(i.1) hc was which 1B), (Fig. mice ieuigARwith ABR using mice Atoh1–Cre;Cdc42 nvitro in yrdzto (ISH) hybridization Atoh1–Cre Cdc42 flox/+ Oaie al., et (Otani flox/flox RAin mRNA nvivo in iea 8 at mice +/ Atoh1– 2 mice mice +/+ . ek Fg B.W ute ofre ern osin loss hearing confirmed further We stimulation Cre;Cdc42 2B). tone-burst (Fig. kHz from weeks frequencies 8–32 high 2 at An particularly loss 2A). hearing severe (Fig. in resulted weeks 8 by SPL Cdc42 (SPL); sapstv oto.Saebr 50 bar: Scale control. positive a as B hehl oprdwt hti oto ie(i.2A, (Fig. mice control in that with 35.0 compared threshold ABR t4wesi epnet ihfeunisadprogressively 2C). (Fig. and weeks frequencies 2002), 8 high by frequencies to Although all response encompass in to deteriorated detected weeks was level 4 the DPOAE to the at emitted in sound decrease is significant that low-level A OHCs canal. of a ear mechanism active detects the which by generated response, (DPOAE) emission ci rd muotiigi C-ie raso ot from Corti of organs TCA-fixed in immunostaining (red) actin A relative positive). to the assigned shows panel cell lower hair The outer mice. Cdc42 and wild-type arrow) P5 (IHC, of cell arrowheads) hair (OHC; inner cochlear in expression mRNA stereocilia. cochlear 1. Fig. ek [ weeks nete h ohe rtevsiue nldn h sensory the including vestibule, the or cochlea the either ability in swimming and gait normal lives. their throughout exhibited and impairment t2weeks, 2 At troii of stereocilia Atoh1–Cre;Cdc42 oovoscagsi rs isemrhlg eedetected were morphology tissue gross in changes obvious No 6 RAsga ee eemndb h A mg nlzr(e was (red analyzer image DAB the by determined level signal mRNA . ess23.1 versus 2.3 flox/flox Cdc42 Atoh1–Cre;Cdc42 ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal n n § § Atoh1 flox/flox ;21.7 6; Atoh1–Cre;Cdc42 ;20.0 6; mN xrsini ohe n d4 oaiainat localization Cdc42 and cochlea in expression -mRNA Atoh1–Cre;Cdc42 iewr sda otosfralsbeun studies. subsequent all for controls as used were mice flox/flox lofntosi etblrhi el Ce tal., et (Chen cells hair vestibular in functions also ieuigtedsoto rdc otoacoustic product distortion the using mice 6 6 (A) 28S . ess18.3 versus 3.2 . ess25.0 versus 4.8 iea 0 oeteasneo d4 tiigin staining Cdc42 of absence the Note P0. at mice 6 nsitu In . BSL,porsigt 70.5 to progressing SPL), dB 2.2 RAcmlmnayoioDApoewsused was probe oligo-DNA complementary rRNA flox/flox flox/flox yrdzto uprpnl detects panel) (upper hybridization m flox/flox .()Cc2(re)adfilamentous and (green) Cdc42 (B) m. ie cl as 10 bars: Scale mice. iehdn eetbebalance detectable no had mice 6 6 iehdasihl elevated slightly a had mice . Bsudpesr level pressure sound dB 2.8 . BSL respectively]; SPL, dB 4.9 m Cdc42 m. 6 flox/flox Cdc42 Atoh1– . dB 3.2 2041 and

Journal of Cell Science nhi elvaiiy hlodnsann fteflmnosactin filamentous the of in staining Phalloidin focused (F-actin) viability. we cell therefore, hair S1C); on Fig. material (supplementary weeks hpso ioii n troii ntevsiuewr normal the were and vestibule cells the in hair stereocilia of in and number kinocilia basal the of the contrast, to shapes in sensitivity and In reduced and 2D) with frequencies. IHCs (Fig. consistent high was weeks in which 4 particularly 2D,E), at weeks, (Fig. cochlea turn 8 the at of loss turn extensive middle the in cells eeocsoal eetdwtotaycagsi h spiral the in changes in vascularis any stria without or OHCs ganglion detected and IHCs occasionally of losses However, were OHCs shown). and not IHCs (data of normal development was the that suggesting mice, control splmnaymtra i.SC.Tenme fIC and in VIIa IHCs myosin of marker hair-cell-specific number Atoh1–Cre;Cdc42 the The with labeled S1C). OHCs Fig. material (supplementary EERHARTICLE RESEARCH 2042 AJCs to in localized not and was observed GFP–Cdc42(T17N;4A) membranes contrast, In was stereociliary 3A). (Fig. at fluorescence reconstructions cochlear GFP–Cdc42 confocal organotypic Intense in motif, explants. membrane-targeting the mutant Cdc42 lacking inactive TCA- an GFP–Cdc42(T17N;4A), in adenovirus-encoded or expressed lost GFP–Cdc42 we were samples, features immunostained structural fixed precise Because in functions cells. prominent has hair Cdc42 where site the identified next We AJCs cells and hair stereocilia cochlear at in Cdc42 of activation and Localization ampularis] crista and macula, of vestibular Corti, of [organ epithelia in loss cell hair and hearing progressive Slowly 2. Fig. aeilFg S1D,E). Fig. material Cdc42 eann HsadOC nec unin turn each in OHCs and IHCs remaining n * le and blue) rqec on-oiathaigls sdetected. is loss hearing sound-dominant frequency n e ice)i h idetr fccla from cochleae of turn middle the in circles) red and P § , ;* 3; Atoh1–Cre;Cdc42 .5 C g-eae PA f rqec t8 2 6ad2 H)trsod in thresholds kHz) 20 and 16 12, 8, at frequency (f2 DPOAE Age-related (C) 0.05. Atoh1–Cre;Cdc42 flox/flox P , Atoh1–Cre;Cdc42 .5 p pcl d middle. Md, apical; Ap, 0.05. and Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 flox/flox flox/flox flox/flox flox/flox iea ek a dnia ota in that to identical was weeks 2 at mice iea ek (supplementary weeks 5 at mice Cc2K,rd mice. red) (Cdc42-KO, flox/flox flox/flox Atoh1–Cre;Cdc42 iea 0ad2weeks 2 and P0 at mice Cdc42 mice. ieidctdls fhair of loss indicated mice Atoh1–Cre;Cdc42 flox/flox Cdc42 n § and ;* 4; flox/flox n Atoh1–Cre;Cdc42 § flox/flox Atoh1–Cre;Cdc42 P nml;* animals; 4 , .5 D ersnaie( Representative (D) 0.05. iea 8 at mice and flox/flox Atoh1–Cre;Cdc42 P ieso rgesv n ihfeunysuddmnn ern loss. hearing sound-dominant high-frequency and progressive a show mice , .5 B g-eae lc n oebrt(,1,2 n 2kz B hehlsin thresholds ABR kHz) 32 and 24 16, (8, tone-burst and click Age-related (B) 0.05. flox/flox flox/flox aue(rlno ta. 04 dt o shown). not (data 2004) al., structurally et and functionally (Frolenkov are mature cells 3G). were hair when (Fig. AJCs P9 and at ML141 stereocilia observed also the inhibitor at ratios Cdc42 FRET/CFP following high selective Moreover, significantly the decreased the with ratios in treatment FRET/CFP 3D–F). higher (Fig. high stereocilia was of portions These basal ratio the in FRET/CFP than portions the membranesupper basolateral Intriguingly, junctions stereociliary the 3D). cell–cell at (Fig. at apical that the than intense at membranes) higher (apicolateral was and 3D) FRET/CFP) (Fig. two- (hereafter membranes FRET: transfer using ratio The 3C). mice) energy CFP (Fig. microscopy biosensor mice fluorescence resonance excitation transgenic (Cdc42-FRET photon fluorescence P2 biosensor Cdc42 from (FRET) harvested a Corti we expressing AJCs, of and organs membranes stereociliary is examined Cdc42 at whether functioning investigate To and 3B). active (Fig. AJCs or stereocilia the eaaye h lrsrcueo raso ot,particularly Corti, of organs in of ultrastructure stereocilia, the analyzed in We cells hair cochlear of Atoh1–Cre;Cdc42 loss and degeneration Progressive HsadOC in OHCs and IHCs a among with 4C). gradient (Fig. 4G), rows length rows (Fig. three the precise row within in and and each alignment arranged in W-shape were distinct length stereocilia rows determined OHC curved gently whereas 4A). moderately few (Fig. a a weeks into 8 arranged with at were IHCs mice IHCs of control in in Stereocilia arrangement maintained regular was OHCs The and (SEM). microscopy electron iea ek.Nt h H-adbsltr B)dmnn arcl loss. cell hair (Bs)-dominant turn basal and IHC- the Note weeks. 8 at mice mice. h ellrarneetadaia ofgrto fboth of configuration apical and arrangement cellular The Cdc42 flox/flox n § A g-eae lc B hehls(BSL in SPL) (dB thresholds ABR click Age-related (A) ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal )AeaFur48palii tiigsoshi ells (white loss cell hair shows staining Alexa-Fluor-488–phalloidin 4) flox/flox iea h g f2 n ek.()Tepretgsof percentages The (E) weeks. 8 and 4 2, of age the at mice and Atoh1–Cre;Cdc42 flox/flox Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 mice flox/flox flox/flox flox/flox ie rgesv n high- and Progressive mice. ieuigscanning using mice iea 3were P3 at mice Cdc42 flox/flox n § (cont, 4;

Journal of Cell Science ) ntemdl unof turn 4K– (Fig. middle the fused often the in the were in stereocilia In stereocilia but present M). weeks, frequently rod-like 8 a more at for were fused turn IHCs except apical with 4B). weeks, were (Fig. 8 IHCs turn at fusion middle of weeks lost 6 stereocilia were number at IHCs and cochlea small most the loss of Finally, turn 4J). IHC middle (Fig. at the and in eliminated observed 4I), randomly frequently (Fig. were weeks and 4 degeneration further showed cainlICdgnrto ihprilseeclafso was weeks, fusion 2 in stereocilia at partial mice mature in with observed control fully stereocilia degeneration was in IHC of hearing those occasional when array from However, and 4F). indistinguishable (Fig. shape also as The were stereocilia same 4E). the OHCs and was (Fig. which ablated pattern, IHCs staircase were a control in kinocilia rows in which aligned were in shape, mature EERHARTICLE RESEARCH dnia otoei oto ie(upeetr aeilFig. material in (supplementary IHCs mice P8, control At in S1F). those to identical Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 flox/flox ie(i.4) IHCs 4H). (Fig. mice flox/flox flox/flox ieotie a obtained mice ie the mice, ude ihadsutdWsae rfl;hwvr no however, profile; stereocilia W-shaped 4D). scattered (Fig. disrupted observed had was a fusion OHCs stereocilia remaining with the bundles and 4B), (Fig. rnucdta hto Hsdrn hspro.OC were in OHCs period. weeks this 8 during at IHCs lost of occasionally respectively. that weeks, than 31.4%, 8 and pronounced 15.2%, 6 was 4, 2, stereocilia at fused 88.2% and with 42.8% IHCs of percentage ntemdl uno h ohe t2ad6wes oapparent No weeks. 6 and 2 at of AJCs cochlea in further the detected were of changes to turn IHCs middle using (TEM) AJCs the and in stereocilia microscopy in changes electron structural the transmission examine used We in AJCs and Cre;Cdc42 stereocilia of ultrastructure Disturbed hne eeosre nteutatutrso troii and stereocilia of ultrastructures the in observed were remarkable changes whereas S4A,B), Fig. material (supplementary weeks 2 h eeeainadls fOC eemc less much were OHCs of loss and degeneration The ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal flox/flox mice ebae arw.Saebr:10 bars: Scale stereociliary (arrow). the membranes at GFP– localized not is but Cdc42(17N;4A), GFP–Cdc42, OHCs; cochlear the of (in view lateral reconstructed Scale (arrowhead). IHC 2 a bar: in junctions at cell and apical (arrows) stereocilum individual GFP–Cdc42 an of covering localization membrane of show the panels A with top The infected (green). and adenoviruses h indicated wild-type 16 P1 for from cultured Corti mice of organ AJCs. Dissected and (A,B) the membranes at stereociliary function cell and hair localization Cdc42 3. Fig. bandfo ifrn raso ot.Scale Corti. 10 of bars: organs different are G from and obtained F D, a microscope. under excitation observed two-photon were mice from Cdc42-FRET Corti P2 of organ CFP, Dissected and (D–G) YFP respectively. of variants and are YPet Turquoise-GL biosensor: FRET Cdc42 the intramolecular of representation Schematic (C) ebae(e h lutainaoei) 3D A it). above basolateral illustration the the of (see that membrane to the stereocilia from of depths, level to various surface of apical the cross-sections to show oblique is plane that image Note the arrow). the (small at membranes than basolateral arrow) the (large at membranes higher apicolateral is and (arrowheads) stereocilia the at nalOC.()Rpeettv RTCPratio FRET/CFP ( Representative images (G) OHCs. stereocilia all of portions in basal in in than higher portions is upper ratio the FRET/CFP (see The are direction illustration). OHCs vertical five in the aligned that obliquely of Note to row stereocilia. base the of the in from top OHCs sections serial five in of obtained series OHC3 a FRET/ of the ratio of showing CFP organ image an Composite in (F) OHCs1–3 Corti. and an IHCs showing of drawing overview Schematic (E) material 1. supplementary Movie in available is movie n Jsaesgiiatydcesdb ML141. by decreased * significantly stereocilia are the AJCs at and ratios FRET/CFP that (mean showing results a these shows of panel quantification 500 right-hand after The min treatment). 4 ML141 and (before ML141 inhibitor P , 0.01. m .Telwrpnl fAadBshow B and A of panels lower The m. n m § .()TeFE/F ai shighest is ratio FRET/CFP The (D) m. )soigteefc fteCdc42 the of effect the showing 3) Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 xz flox/flox flox/flox m xs images axis) 6 m Atoh1– s.e.m.) ieat mice 2043 mice m M

Journal of Cell Science h ae h etclsaeo Jswsrfldin ruffled was AJCs of shape vertical at Cre;Cdc42 fused The were stereocilia base. these to that the indicating appeared length, 5B), and in (Fig. plate normal cuticular rootlets be normal with visibly cores a penetrated actin which some contained and elevated lt Fg A.I otat h ebae ttebs of base the at membranes of the cuticular IHCs the contrast, most into apically In inserted in were rootlet stereocilia 5A). IHCs single (Fig. a control the had plate circumferential in each and Stereocilia the and 5A). located membrane of (Fig. density belt junctional borderedactin perijunctional apical were thick mice arcuate-shaped underlying control in the IHCs by 5B). (Fig. cells supporting icmeeta ci eti Hswstinrin the thinner that was seen IHCs be could in it belt Cre;Cdc42 magnification, actin high circumferential At 5C). (Fig. EERHARTICLE RESEARCH 2044 al., in et Cdc42 Nakano of 2003; localization al., subcellular been the et examined have (Ben-Yosef we AJCs First, and for 2009). model stereocilia) a primordial as an used established to we analogous AJCs, (structures and stereocilia in investigate to vitro Cdc42 and of deletion Cdc42 role of the effect the confirm further Cdc42-deleted To of model cells a hair as cochlear cells MDCK in Cdc42-KD in IHCs 5A), and (Fig. IHCs mice control of in rows adjacent IHCs) cells Cre;Cdc42 regular of supporting the row of row each to a to is contrast there In (where cells weeks. supporting 6 at AJCs in stereocilia cochlear of Degeneration 4. Fig. nraei ubrtruh4()t ek J.Teaia uno ohe t8wesrtissm Hs hc aefeunl ue troii (K–M, stereocilia fused frequently have which IHCs, some retains weeks 5 8 bars: at Scale cochlea arrow). of double turn (M, apical stereocilia The short (J). and weeks arrows) 6 (L, to long (I) and 4 arrowheads), through number in increase un(–)otie from obtained (K–M) turn tics atr ihcnitn egho troii nec o.(,)Temrhlg fIC()adOC()in (F) OHC and (E) IHC of morphology The (E,F) row. each in stereocilia of length consistent with pattern staircase ADGKM.()Bt HsadOC r eual lge napaein plane a in aligned regularly are OHCs and IHCs Both (A) (A–D,G,K–M). hrceitcWpoie D In (D) W-profile. characteristic Atoh1–Cre;Cdc42 otydsperd hra Hsaeprilydpee n aesatrdseecla C In (C) stereocilia. scattered have and depleted partially are OHCs whereas disappeared, mostly dnia ota in that to identical oe sn DKcls hc oss microvilli possess which cells, MDCK using model flox/flox flox/flox flox/flox Cdc42 flox/flox ieta ncnrlmc Fg 5D). (Fig. mice control in than mice iecmae ihta ncnrlmice control in that with compared mice iewr fe betadrpae by replaced and absent often were mice flox/flox ie troii uin(rohas sfrtosre t2wes IM h trolafso arwed)adICls (asterisks) loss IHC and (arrowheads) fusion stereoilia The (I–M) weeks. 2 at observed first is (arrowheads) fusion stereocilia mice, Cdc42 Atoh1–Cre;Cdc42 ie(o hw) G h eua ra fseeclai Hsof IHCs in stereocilia of array regular The (G) shown). (not mice flox/flox Atoh1–Cre;Cdc42 cn;ACG and A,C,G) (cont; flox/flox Atoh1–Cre;Cdc42 Hsa ek,seeclaaefwri ubradhv ottercaatrsi -rfl n precise and W-profile characteristic their lost have and number in fewer are stereocilia weeks, 8 at OHCs flox/flox Atoh1–Cre;Cdc42 iewere mice Atoh1– Atoh1– Atoh1– flox/flox in Cdc42 flox/flox mice. flox/flox irvliwr ctee n a bomlyrge,fused, that ragged, MDCK showed abnormally 6G). (Fig. had (SHIM) morphologies elongated and or microscopy scattered short were helium-ion microvilli scanning by Cdc42 of knockdown we Next, stable 2010). with al., et cells (MDCK (Qin MDCK report previous established a with consistent DKclssal xrsigGPCc2(MDCK GFP–Cdc42 expressing stably cells MDCK ettda h eljntost atal vri h ufc fthe of MDCK of surface two the number in overlie partially cells the to often neighboring junctions was that cell border the found cell at the dentated we and reduced SEM, significantly was Using microvilli 6B,C). (Fig. sh197 ute tde.Terdcdnme fmcoil n the and microvilli MDCK of in number border reduced cell The dentated studies. further omo h ncieGPCc2T7;A Fg EF.High- MDCK 6E,F). of (Fig. examination GFP–Cdc42(T17N;4A) morphological inactive an resolution the shRNA-resistant of of an not expression form but GFP–Cdc42 adenoviral-mediated of form by shRNA-resistant rescued completely oaie orcl nMDCK in correctly localized el (MDCK cells n MDCK and agtda J nMDCK in TJs at targeted oaiaino O ttecl ucin a eoee by recovered was junctions cell the at ZO1 of localization a nes tteaia ebaeadwal iha h lateral the at MDCK fluorescence high weakly of GFP–Cdc42 and membrane cysts. membrane apical the produce at intense to was Matrigel on plated eeotie sn MDCK using obtained were BDFHM iea h g fP EF,2() I,6() n weeks 8 and (J), 6 (I), 4 (H), 2 (E,F), P8 of age the at mice (B,D–F,H–M) E mgso h ra fCria h idetr AJ n h apical the and (A–J) turn middle the at Corti of organ the of images SEM ial,w xmndtefraino ih ucin Ts in (TJs) junctions tight of formation the examined we Finally, iea ek.()In (B) weeks. 8 at mice m Cdc42-KDg ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal AB;2 (A,B); m Cdc42-KD Cdc42 Cdc42-KDg Cdc42 cont flox/flox sn h otefciesRAplasmid shRNA effective most the using ) m Fg D.MDCK 6D). (Fig. ) (C–L). m el.Atog h Jmre O was ZO1 marker TJ the Although cells. flox/flox iea ek.()I h idetr fcclain cochlea of turn middle the In (H) weeks. 8 at mice ncmaio ihtecnrlMDCK control the with comparison in ) GFP–Cdc42 Hsa ek,seeclahv the have stereocilia weeks, 8 at OHCs Atoh1–Cre;Cdc42 Cdc42-KDg Cdc42-KDa Atoh1–Cre;Cdc42 cont Cdc42KD el nteecss(i.6A), (Fig. cysts these in cells el Fg A,Z1wsnot was ZO1 7A), (Fig. cells Cdc42-KDg el Fg B iia results similar 7B; (Fig. cells Cdc42-KDg el;dt o hw) The shown). not data cells; flox/flox lns(MDCK clones flox/flox el eealmost were cells iea ek,IHCs weeks, 8 at mice a sdfrall for used was Cdc42-KDg iea 8is P8 at mice GFP–Cdc42 Cdc42-KDa cells )

Journal of Cell Science 02.TepopoNWS a oaie tteaia surface apical MDCK the of membranes at apicolateral and localized was phospho-N-WASP The 2002). irvliadTso MDCK of TJs and microvilli n Js oudrtn d4 inln nteesal actin actin with stable MDCK associated these in molecules in turnover several signaling examined microvilli Cdc42 we stereocilia, understand structures, To in AJCs. dynamics and actin compromises deletion EERHARTICLE RESEARCH h itre lrsrcue nteseeclaadAC of AJCs and stereocilia the in ultrastructures Atoh1–Cre;Cdc42 disturbed The mice MDCK in signaling actin-regulatory Altered GFP– 7D). not (Fig. but Cdc42(T17N;4A) 7C) (Fig. GFP–Cdc42 shRNA-resistant introducing idn fCc2t t d4/a neatv idn (CRIB) binding Tyr interactive at phosphorylation Cdc42/Rac with its together to by region, activated Cdc42 is N-WASP of state. binding inactive the in when conformation eue ci unvri arclsof cells hair in turnover actin reduced -AP hc sadwsra agto d4,hsaclosed a has Cdc42, of target downstream a is which N-WASP, flox/flox Cdc42-KD ie oehrwt h bomlte in abnormalities the with together mice, cells. Cdc42-KD cont el,sgetdta Cdc42 that suggested cells, Atoh1–Cre;Cdc42 u o MDCK not but , 256 Cdc42-KD Sesg tal., et (Suetsugu el and cells Cdc42-KD flox/flox , wv’sann fZ1i MDCK the in Furthermore, microvilli ZO1 had in S2C,D). of insert reduction Fig. staining small filter ‘wavy’ a material a and (supplementary on bases number their plated at S2B) microvilli fused Fig. material supplementary J eeanra splmnaymtra i.SE.Thus, S2E). Fig. material (supplementary abnormal MDCK were TJs i MDCK did agtn uui MP1 n on hti a nacdin enhanced was phosphatase it that myosin found examined and substrate we (MYPT1) 1 ROCK 2010), subunit Rho-associated al., the targeting of et substrate of (Amano a phosphorylation is (ROCKs) LIMK kinases that protein Given 2007). al., et agt fPK,wsuepcel nrae nbt lnsof clones downstream both are in increased which unexpectedly 8B), was MDCK (Fig. PAKs, cofilin of and targets 8A) (Fig. LIMK el ihsal ncdw fNWS (MDCK N-WASP of knockdown stable MDCK with S2A). Fig. material cells (supplementary Matrigel on plated cells agt fCc2 n h eeso hsh-A a reduced was phospho-PAK of levels the MDCK and in Cdc42, of targets 2-ciae iae PK)aewl-nw downstream well-known are (PAKs) kinases p21-activated NWASP-KD Cdc42-KD ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal Cdc42-KD Cdc42-KD el,cnitn ihapeiu eot(Garvalov report previous a with consistent cells, i.5 itre lrsrcueo troii and stereocilia in of AJCs ultrastructure Disturbed 5. Fig. ote netdit h uiua lt arw.()Upper (B) single (arrow). a plate panels: has cuticular each the and into surface inserted rootlet apical the on Stereocilia located view). are the magnified beneath (right, layer membrane dense plasma a belt as actin observed circumferential (arrowheads) arcuate- the the with a by AJCs is bordered shaped there plate and cuticular (left) actin-rich aligned thick alternately are (SC) cells atrss r ntergtsd.Teatnbl (outlined belt actin The side. right cells the Hair on belts. are actin (asterisks) perijunctional and AJCs the ywiedt)i hne in thinner is dots) white by Cre;Cdc42 el.Saebr:1 bars: Scale cells. fIC ttemdl uno ohe t6weso age. of weeks 6 at cochlea of In turn (A) middle the at IHCs of n ue troii ae(oe) C ls-pvesof in views AJCs Close-up the (C) (upper) (lower). cells base stereocilia hair fused supporting neighboring and of and views microvilli close-up with are cells panels arrows). two (white right rootlets The with actin several arrowheads) contain (black they cores and arrows) fused (black often base are the IHCs at remaining the Lower in microvilli. stereocilia apical panels: the by supporting characterized by cells, displaced were which IHCs, some missing n etclac ncnrl u hw ag fruffling of range a shows smooth but in display controls in AJCs arcs The vertical (asterisks). and flanked cells middle, the hair in two cell by supporting a of section cross a el hwdsmlr u idr hntpsthan phenotypes milder, but similar, showed cells Atoh1–Cre;Cdc42 cells. el Fg A,weespopoyainof phosphorylation whereas 8A), (Fig. cells Cdc42 Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 flox/flox Cdc42 flox/flox ie(oe aes.Ec ae shows panel Each panels). (lower mice flox/flox cn)mc,IC n supporting and IHCs mice, (cont) m flox/flox AB;50n C;20n (D). nm 200 (C); nm 500 (A,B); m NWASP-KD uprpnl)and panels) (upper flox/flox ie D ls-pvesof views Close-up (D) mice. Atoh1–Cre;Cdc42 flox/flox Cc2K)mc are mice (Cdc42-KO) el niae that indicated cells mice. E images TEM NWASP-KD flox/flox Atoh1– 2045 hair ;

Journal of Cell Science unvrwsas eue nhi el,w vlae h actin the evaluated we of cells cells, hair hair the in in rate reduced depolymerization also was turnover omlzd(i.8) h ciaino h RhoA–ROCK fully the almost of was MDCK activation MYPT1 in The of pathway 8D). that (Fig. was cofilin and normalized of normalized phosphorylation enhanced partially the Y27632, inhibitor EERHARTICLE RESEARCH 2046 was S3D,E) (1.47 Fig. mice material control in previously (supplementary stereocilia between as of interest comparable Corti, length of of The areas 2004). organ the al., of of et culture of (Rzadzinska ends shortening explant described minus the causes the to at and F-actin, depolymerization continuous polymerization by actin stereocilia D, arrests cytochalasin inhibitor polymerization which actin an added We mice. MDCK hsh-oii ugse htCc2dlto ed oreduced to MDCK leads in deletion turnover Cdc42 actin that phospho-myosin suggested to S3A,B). phospho-cofilin Fig. antibody material an (supplementary (MLC2) and 2 RhoA chain light active to antibody h ilclzdpopoNWS n nacdlvl of levels enhanced and phospho-N-WASP dislocalized The Cdc42-KD el Fg C.Atrtetetwt h ROCK the with treatment After 8C). (Fig. cells Cdc42-KD Cdc42-KD el a ofre sn an using confirmed was cells el.T etwehrteactin the whether test To cells. Atoh1–Cre;Cdc42 6 0.03–1.85 6 0.04 flox/flox m m) troiir hreigwsls in less was shortening stereociliary 1.04 aeilFg 3) sepce,seeclaatrtetetwas (supplementary treatment in after longer D significantly stereocilia significantly expected, cytochalasin but was As slightly with but S3F). Fig. D, treatment material cytochalasin by with reduced treatment before l h he ra bevd(upeetr aeilFg S3G). Fig. material (supplementary observed areas three the all (0.35–0.58 (1.11 and upre yasuyta hwdta h ihs FRET/CFP highest the Corti that of showed was organ that result explant study This an a mice. cells in by transgenic cells supported hair biosensor hair exogenous Cdc42-FRET in of methods: from imaging GFP–Cdc42 two FRET adenovirus-encoded following and and the in of localization AJCs by and the expression cells membranes time stereociliary hair the first cochlear at the Cdc42 for of activation demonstrated we Here, DISCUSSION 6 Atoh1–Cre;Cdc42 6 0.02 0.02–1.20 ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal m m )(upeetr aeilFg 3) ..tert of rate the i.e. S3F), Fig. material (supplementary m) /2h hni oto ie(0.48–0.81 mice control in than h) m/32 6 0.04 n MDCK and MDCK cells. in microvilli shape- of and abnormalities number Reduced 6. Fig. eue irvlii MDCK in microvilli reduced 10 bars: junctions Scale cell–cell (arrowheads). the at Note GFP–Cdc42 signal. of GFP localization the show adenoviruses. insets indicated Colored the by infected and epciey cl a:10 bar: (arrowheads), Scale cyst-cells respectively. of surfaces (arrow) lateral apical and the at weak accumulation and GFP–Cdc42 strong the Note Alexa- Fluor-568–phalloidin. with counterstained and Matrigel in cultured irvli epciey cl a:50nm. 500 bar: Scale respectively. fused microvilli, and ragged lengthened, arrowhead shortened, red showing and arrows, red arrowhead, el xrsigacnrlsRA(MDCK shRNA control a with expressing compared cells microvilli fewer substantially had cl a:5 bar: Scale oe mgsotie ysann helium-ion scanning by obtained images power * GFP–Cdc42(17N;4A). not but GFP–Cdc42 adenovirus-encoded of expression opooia bomlte nmcoil in MDCK microvilli in of abnormalities range morphological the showing (SHIM) microscope ufc fMC el rw nafle insert. filter a on MDCK grown Both cells apical MDCK the of showing surface images SEM (D) established. hN s17,todfeetclones (MDCK effective different most two the (sh197), Using shRNA cells. MDCK in sh333) shRNA- canine different targeting three plasmids of effects the showing flox/flox m )ta ncnrlmc (0.99 mice control in than m) A ofcliae fMDCK of images Confocal (A) Cdc42-KDg Cdc42-KDa ie(1.46 mice Cdc42-KDg Cdc42-KDa m .()SMiae fMDCK of images SEM (E) m. Atoh1–Cre;Cdc42 el.Bakarw black arrow, Black cells. Atoh1-Cre;Cdc42 n MDCK and el rw nafle insert, filter a on grown cells n MDCK and 6 0.06–1.78 Cdc42 m Cdc42-KDg Cdc42-KDg .(,)Immunoblot (B,C) m. m .()Rcvr of Recovery (F) m. P Cdc42-KDg , s2,sh197, (sh29, .1 G High- (G) 0.01. GFP-Cdc42 m were ) flox/flox el ythe by cells flox/flox 6 /2h in h) m/32 Cdc42-KD 0.05 ellines cell 6 cont 0.02– cont mice mice ). cells m m)

Journal of Cell Science ietifuneo d4 eeindrn ci unvrin turnover by actin supported MDCK further in during levels was phospho-cofilin speculation enhanced deletion the This AJCs. Cdc42 and of stereocilia influence direct EERHARTICLE RESEARCH eue ci eoyeiainrt nteseeclaof stereocilia the in Cre;Cdc42 rate depolymerization actin reduced cells hair cochlear and in observed of localization phenotype disturbance The of cells. the AJCs structural hair and cochlear with prominent stereocilia in Cdc42, a Consistent observed was plays of microvilli. Cdc42 profiles that MDCK the activation suggesting 3D-cultured thereby in of 2012), surface role al., apical et the (Yagi at cells was Cdc42 of ratio MDCK in TJs of Disruption 7. Fig. ta. 06,admoi V stecrirpoeno hri and whirlin of protein carrier the is (Disanza XVa myosin complex and 2006), Eps8–IRSp53–Cdc42 filopodial al., promote Eps8, the et to forming known espins, Sekerkova is by 2011; Eps8 e.g. and growth al., 2011). stereocilia, al., regulatory et et loss, (Manor loss of actin Zampini 2011; whirlin of tip as and deletion Stereocilia the XVa such the myosin at by elongation. located changes, caused molecules also and degenerative are of shortening fusion, variety shortening, a showed O muotiigi ofun MDCK confluent in immunostaining ZO1 oeteasneo O tcl–eljntosi MDCK in junctions cell–cell at ZO1 of absence the Note CD ofcliae fZ1imnsann nMDCK in immunostaining ZO1 of images Confocal (C,D) o F–d4(7;A D.Ist,DP tiig cl as 20 bars: Scale staining. DAPI but Insets, (C) (D). GFP–Cdc42 expressing GFP–Cdc42(17N;4A) ZO1 cells not in of junctions presence cell–cell the at Note immunostaining adenoviruses. indicated by infected h aueseeclain stereocilia mature The Atoh1–Cre;Cdc42 flox/flox oherhi cells. hair cochlear flox/flox iemgtb osqec fthe of consequence a be might mice Cdc42-KD Atoh1–Cre;Cdc42 cont A n MDCK and (A) cells. Atoh1–Cre;Cdc42 AB ofcliae of images Confocal (A,B) Cdc42-KD Cdc42-KDg Cdc42-KDg Cdc42-KDg flox/flox el n a and cells cells. cells ´ B cells. (B) Atoh1– m flox/flox tal., et m. mice oprt ttebs fseecla(aauh ta. 2008). al., et tyrosine (Sakaguchi stereocilia protein of 2004), base to and known the are 2003) myosinVI al., and at al., PTPRQ 1999). et cooperate et al., et (Goodyear (Self (PTPRQ) VI (Kitajiri myosin Q as radixin receptor such mouse phosphatase tethering, mutant of membrane or disturbed knockout the those have other to overlapping of several considered The to in lines stereocilia. membrane up’ found of plasma base is the ‘zipping the phenotype of at tethering cytoskeleton the the actin be in the al., might in dysfunction fusion et a actin stereocilia to Alternatively, results (Sakaguchi due 1999). of al., tip and et the Self downregulation 2008; base towards actin the membrane tapered the interstereociliary the of at of shaping consequence core prevents which a fusion Stereocilia depolymerization, stereocilia. be of base the might at initiate to seemed that oii nMDCK in cofilin of 2012). al., those cell hair et hair to (Johnson in in results expressed GTPase-activating similar mutation temporarily whose a and phenotypes is and is development that (Heasman during GTPases which cells Cdc42 small ELMOD1, of to and a protein homologous is 2008), which highly RhoQ, Ridley, as GTPase such factors, small polymerization stereocilia actin their be developing other in not might Cdc42 by of and phenotype compensation the protrusions late-onset by explained actin the affects stable Alternatively, polymerization of actin development. maintenance of the reduction partial only a that suggests However, Ktjr ta. 00;hwvr h ote tutr of structure rootlet the however, 2010); Cre;Cdc42 stereocilia al., fused show et also (Kitajiri rootlet, mice, stereocilia TRIOBP-knockout the shown). at maintain not which proteins (data phospho-ERM stereocilia of and base PTPRQ the of distribution specific of 02 xii eue iooi.Teedt niaeta the that indicate data al., dynamics the These et (Flynn both filopodia. filopodial cofilin reduced lacking and exhibit neurons arrested (ADF) 2012) Moreover, factor and 2007). depolymerization actin al., cofilin et (Garvalov of inactivation that reported d4-eedn aha nseecla troii uinis in fusion the seen Stereocilia change exclude specific stereocilia. unconventional cannot another or in undefined we pathway hitherto Cdc42-dependent a However, of the involvement possibility. possible at also of this mice, particularly biosensor to reduction Cdc42-FRET supports activity, due in stereocilia stereocilia Cdc42 The of actin portions of of upper Intense 2011). tip alteration deficiency. the the al., at Cdc42 by the stabilization et explained and at partly polymerization (Manor complex be might XVa–whirlin–Eps8 stereocilia stereocilia myosin of the tip form to Eps8 soitdwt mardatntroe loocr in occurs actin also steady-state turnover actin the Cre impaired in loss with hearing is progressive associated Late-onset stereocilia. Cdc42 mature in of turnover involvement dominant eeoig(2 n aue(9 aantson tgsin stages shown) not in data phenotype stereocilia (P9; observed late-onset the mature mice, biosensor and Cdc42-FRET of (P2) shaping developing in function excluded. Cdc42 be unidentified cannot bases an stereocilia of presence the osb 5(erne l,21) hs h omnpeoyeof phenotype common the in c Thus, observed 2010). disruption stereocilia al., progressive et late-onset (Perrin P5 by both loss of deletion whereas age, of atnsnl ncotand knockout single -actin efudicesdlvl fpopoyae (inactivated) phosphorylated of levels increased found We lhuhCc2atvt nseeclawscnimdi both in confirmed was stereocilia in activity Cdc42 Although -mediated ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal Atoh1–Cre;Cdc42 flox/flox Atoh1–Cre;Cdc42 b -or Cdc42 troii a nhne Fg B.Finally, 5B). (Fig. unchanged was stereocilia Cdc42-KD c atnsnl ncothi el t6weeks 6 at cells hair knockout single -actin dfcetnuosdslyincreased display neurons -deficient el,cnitn ihasuythat study a with consistent cells, flox/flox b flox/flox and - Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 Atoh1–Cre;Cdc42 ieehbtdmaintenance exhibited mice c iesget htthe that suggests mice atnlast stereocilia to leads -actin flox/flox flox/flox flox/flox b atnor -actin Atoh1– Atoh1– 2047 mice mice mice

Journal of Cell Science iesae(2h repatcluea 5 iea hc the which at in time appeared yet a not P5, had at cells culture Atoh1–Cre;Cdc42 hair limited explant of the or to abnormality due morphological h) be (32 al., might et scale effect (Zhang time occurs mild turnover the actin Alternatively, rapid 2012). which in stereocilia, of xeto ftedsa oto.Terdcdlnt eetdin detected length reduced the with The (0.48–0.81 stereocilia, portion. stereocilia in distal cytochalasin-D-treated rate the turnover of actin exception slow the to attributed iia otoeof those to similar MDCK EERHARTICLE RESEARCH 2048 addition, In of S3C). antagonism MDCK Fig. Cdc42-mediated although material the (supplementary control status signaling polymerization to RhoA actin mechanism present the feedback sensing be for A might RhoA 2006). to al., F-actin In et from (Chen (BDNF)- 8D). factor neurons (Fig. neurotrophic stimulated signaling brain-derived in RhoA Y27632 ROCK suppresses enhanced of Cdc42 upstream with of results, by these recovery with treatment moderate accordance activity the upon (2) cofilin and phospho-cofilin S3A) regulates Fig. (supplementary levels following RhoA material active Cdc42 the of increase on an (1) based that observations: pathway, RhoA–ROCK found the antagonizing we cells, of stereocilia the drastically, in not Cre;Cdc42 rate but significantly, depolymerization a actin fact, of In reduced presence disturbances. the structural to confirmed of leads we which downregulation turnover, actin cause actin might net stable both and actin or or in factors polymerization factors polymerization depolymerization tuned actin actin of elaborately deletion between thus, protrusions; be balance should The (Shin depolymerization 2013). proteins stereociliary al., as detected and et are ADF Cdc42 which (e.g. of (e.g. factors both depolymerization factors cofilin), actin the polymerization and requires N-WASP) actin and protrusions of actin action of coordinated maintenance and/or formation h ag fterpre egh(0.3–0.5 length reported the of range the rti iae(RK n OK.UigMDCK Using Cdc42-binding ROCKs. and kinase-related PAKs, (MRCK) dystrophy including kinase pathways, protein myotonic competitive several LIMKs, by regulated n 0.35–0.58 and ouaino oii hshrlto scmlxadtightly and complex is phosphorylation cofilin of Modulation NWASP-KD flox/flox m min Cdc42-KD ie h bec fadatcefc ih be might effect drastic a of absence The mice. flox/flox el a essvr hntp hnthose than phenotype severe less a had cells Atoh1–Cre;Cdc42 mice. el xiie hntpsi microvilli in phenotypes exhibited cells Atoh1–Cre;Cdc42 flox/flox m )o h itlportion distal the of m) flox/flox m ncnrlmice control in m ie a within was mice) stereocilia, Cdc42-KD Atoh1– niae ydsubdZ1lclzto nMDCK in localization ZO1 disturbed by indicated mardaia oaiaino PCi ohIHCs both not in (data OHCs aPKC and of S4C–F) of showed Fig. shown) localization analysis Suzuki material 2011; immunohistological (supplementary apical Takeichi, Our and 2006). the impaired (Ishiuchi at Ohno, cells regulator polarity and of well-known and domain a proteins is apical PAR of (aPKC) Cdc42, organ between PKC the complex in atypical The AJCs 3E). of hair-cell– level (Fig. the Corti not at connection but cellular hair-cell) hair-cell–supporting-cell (only heterologous fMDCK of htosre nMDCK in observed that ta. 06 oofadGn,20) h ultrastructural The 2009). Geny, in and AJCs (Otani Popoff reports of previous 2006; of disturbances results al., the with et compatible is which ci eto Jsi oherhi el.Tedsubneof disturbance The weeks cells. hair 2 cochlear circumferential at in the in not AJCs of AJCs but of maintenance 5D) belt long-term (Fig. also actin the is age Cdc42 in that of suggesting involved S4B), weeks Fig. material 6 (supplementary at observed ih fethi elmrhlg n iblt in viability and morphology barrier cell epithelial the hair Cre;Cdc42 of the affect Dysfunction stereocilia AJCs. in including might and cells, bases, involved hair and in be roots domains apical also of might disturbance complex Cdc42–PARs–aPKC d4-eitdatnplmrzto inln mechanisms signaling polymerization actin Cdc42-mediated odere l,20;Muue l,20) Stereocilia 2003). al., et Mburu 2006; 2003; al., et (Gong of al., in weeks loss 12 degeneration of extensive et age show the to by Goodyear OHCs known PTPRQ, and are IHCs e.g. whirlin, both stereocilia, and deficiency in XVa instance, functions For myosin specific death. with cell molecules hair of to lead ultimately might S3C). Fig. material (supplementary hnhi ells n J irpinwr o observed not this were supporting disruption further AJC S4A,B), and Fig. possibility. loss material cell (supplementary hair when serya 1–6adhi ells sams xlsvl limited exclusively almost is loss cell hair deaf 9. and severely P15–16 claudin are as and early lines 14 as mouse claudin claudin-mutant proteins TJ because the However, in mutations with mice essetdsuto fseeclaysrcueadfunction and structure stereociliary of disruption Persistent ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal rmMDCK MYPT1 from and knockdown. cofilin Cdc42 of following phosphorylation Enhanced 8. Fig. MDCK hshrlto fcflnadMP1 epciey ihdose- with respectively, effects. enhanced MYPT1, dependent the and inhibits cofilin completely of almost phosphorylation and moderately h 10 hra hshrlto fLM,cflnadMP1aeall are MYPT1 MDCK and in reduced cofilin increased is LIMK, Phospho-PAK1/2 of LIMK (C). phosphorylation and MYPT1 whereas PAK1/2 and of (B) forms cofilin phosphorylated (A), and total detect to Atoh1–Cre;Cdc42 flox/flox Cdc42-KD Cdc42 Atoh1–Cre;Cdc42 Cdc42-KDg Atoh1–Cre;Cdc42 ie iia ota rvosyrpre in reported previously that to similar mice, ncdw mardAC nMC el,as cells, MDCK in AJCs impaired knockdown Cdc42-KDa el,sgetn htteeaeother are there that suggesting cells, el ihY73 tidctdcnetainfor concentration indicated at Y27632 with cells Cdc42-KD Cdc42-KD a,MDCK (a), Atoh1–Cre;Cdc42 flox/flox flox/flox el.()Tetetof Treatment (D) cells. el,poal eas fthe of because probably cells, flox/flox iewsmr idthan mild more was mice AC muolt flysates of Immunoblots (A–C) Cdc42-KDg ie ugsigta the that suggesting mice, iebgna weeks 2 at began mice g n MDCK and (g) flox/flox Cdc42-KD iewere mice cont Atoh1– cells, cells

Journal of Cell Science TTGCTG-3 8 o 6)cnuae hlodnadscnayatbde eefrom were antibodies secondary and Invitrogen. phalloidin 568)-conjugated Alexa-Fluor- respectively. (or Chemicals, 488 Pure Wako and ROCK, of MERCK inhibitor from selective a were Y27632, and 2010) GADPH al., et (Surviladze for and Cdc42 inhibitor selective GFP a ML141, International). Bioscience); (MBL antibody (Proteus monoclonal VIIa myosin (Sigma-Aldrich); eoye yPRuigtefloigpie ar:for pairs: primer following the using 5 PCR by genotyped oiatls suiu to is unique it is 2009), loss IHC- al., Finally, barrier. epithelial dominant et the of Nakano dysfunction by caused 2003; solely the al., that et unlikely (Ben-Yosef OHCs to ARTICLE RESEARCH Human Plasmids (Millipore); monoclonal PKC (1A12; Cre monoclonal Invitrogen); ZO1 Biosciences); and ECM (Try256; phospho-N-WASP phospho-MYPT1(Thr850), unless WASP, N- Biosciences); (BD monoclonal Cdc42 (polyclonal obtained Technology); Signaling (all Cell LIMK, antibodies from phospho-MLC2 used and phospho-PAK1/2(Ser192/204), MLC2 MYPT1, monoclonal, were PAK1/2, antibodies cofilin(D3F9) phospho-cofilin(Ser3)(77G2), phospho-LIMK(Thr508/508), Cdc42, specific indicated): following The chemicals and Antibodies Animal Use University and Kobe Care the Animal to Institutional according Regulations. the out Experimentation by carried and approved Committee was study This Animals METHODS ovrf hte bomlclaini ietyrltdt arcell hair to related directly is viability. ciliation abnormal and whether is S3C) verify Fig. research to in material with (supplementary to associated Further stereocilia signaling(s) of resulted loss. leading Cdc42 maintenance the cell further ultimately additional explore which hair to maturation, required with cochlear cells, after progressive stereocilia, AJCs slowly hair in in cochlear disturbances abnormalities in morphological turnover ordered IHCs. rigorously in actin that more than OHCs a in of stereocilia of maintenance p55 length actin-regulatory and the protein alignment might for erythrocyte which by 2010), required al., as et compensation be (Mburu gelsolin such and 2006) is al., OHCs, et (Mburu to phenotype specific IHC molecules predominance the the for explanation simple of A disorders 2012). genetic al., and et agents etiologies(Schacht ototoxic various damage, noise to aging, OHCs as than such vulnerable less are IHCs general, iaai 97 n d4-RTboesrmc Gt ta. 03 are 2013) al., et described. (Goto previously mice as biosensor Cdc42-FRET and 1997) Miyazaki, Cre obtain Cdc42 5 uloie 9–1 rmAG 5 ATG, from Three 197–215 (OrigoEngine). nucleotides pSUPER(neo) for using sequences made target were 2006). sequence al., et target (Ueyama membrane- mutation a AASAA] Cdc42 into in and changed aa) was (T17N) 183–187 C-terminal (KKSRR, dominant-negative motif was polybasic a [the Cdc42(T17N;4A) targeting-defective introducing pEGFP(C1). (Agilent by into kit made subcloned Mutagenesis and Site-Directed Technologies) Lightning into QuickChange cloned using and Clontech) ta. 2012). al., et 9 9 -GCATACCTGGAAAATGCTTC-3 -TACTGCTATGACTGAAAACCTC-3 h hr ari N sRA xrsinpamd otiiga containing plasmids expression (shRNA) RNA hairpin short The nsummary, In Mtie l,20)mc eebccosdwith backcrossed were mice 2005) al., et (Matei flox/flox Atoh1–Cre Cdc42 9 for ; Atoh1–Cre;Cdc42 ognrt xeietlaias fsrn were Offspring animals. experimental generate to a mlfe yPRuigcN MCpnlII; panel (MTC cDNA using PCR by amplified was f Cdc42 Cdc42 ;Cdc42 C2,SnaCu) ctltdtblnmonoclonal tubulin acetylated Cruz); Santa (C-20, Cdc42 Cdc42 flox Cdc42 flox/flox ,5 eeincue h oneuainof downregulation the caused deletion dfcetpeoye r rmrl or primarily are phenotypes -deficient ncdw K)tse ees17[canine sh197 were tested (KD) knockdown 9 pUC118 -ATCGGTCACTGTTCTACTTTG-3 flox flox/ CAG- has ie hc eebccosdto backcrossed were which mice, Atoh1–Cre;Cdc42 + 9 rgn of progeny -GATTACGACCGCTGAGTTA-3 flox l uain eeintroduced were mutations All . loxP 9 n 5 and CAT 9 . stsfakn xn2(Aizawa 2 exon flanking -sites flox 9 -CCAGTGAAACAGCA- -LacZ Cdc42 flox/flox flox/flox ie(aa and (Sakai mice Cdc42 Atoh1–Cre and ie In mice. flox/flox Atoh1– 9 and to 9 , , lcn i ietcagswti h agtn eune(lowercase sequence targeting the within changes 5 letters; silent six by made were placing plasmids GFP–Cdc42(T17N;4A) and GFP–Cdc42 resistant 3 w agtsqecsfrNWS ncdw etdwr sh1396 were tested knockdown 5 N-WASP 1396–1416, for nucleotides (canine sequences target Two eelblda hi 5 their at labeled were GGGATCTGAAGGCTGTCAAGTATGTGGAGTGCTCTGC-3 rvosyetbihd(i ta. 00] h9(5 sh29 2010)], al., et GTTGGTAA-3 (Qin established previously 3–7 nmueCc2 5 nucleotides (coding Cdc42; antisense mouse AGCCTTCAGATCCCGCGCCAGC-3 DNA 2012) al., 45-base in et mice. 432–476 (Choijookhuu wild-type previously P5 described using as performed was ISH situ In CsCl by the of obtained titer final was was The solution solution dialysis. cells virus by virus followed HEK293-FT centrifugation high-titer density HD of buoyant final FuGENE using transfection The plasmid by (Promega). destination PacI-digested vector made with (Invitrogen) was entry initial The solution (Invitrogen). pDONR221 technology the adenovirus Gateway using the and obtained constructs pAd/ were into pDONR221 (Invitrogen) the vector in cloned destination GFP–Cdc42(T17N;4A) CMV/V5-DEST and and GFP–Cdc42 GFP, were PCR (Invitrogen). sh197, to by resistant amplified GFP–Cdc42(T17N;4A), and GFP–Cdc42 GFP– GFP, GFP, of GFP–N-WASP expression and for Cdc42 solutions adenovirus of Preparation tfeunyo f–2wr lctduigtopiaytn tml,f1 stimuli, tone primary two using elicited were 2f1–f2 DPOAE combined software. of USA) USA) frequency IL, Research; IL, at (Etymotic v2.40 Acoustics; II CUBeDIS (Mimosa with System Diagnostic Auditory hearing, assess To measurements DPOAE (Olympus). and microscope ABR camera light DP26 a a using with by Zeiss) Carl photographed II; were X- (Axioplan stainings Chemicals). H&E Pure (Muto and solution using gal (H&E) stained Eosin and 5- and de-paraffinized, and hematoxylin slides, wax Myer glass paraffin al., on in collected et embedded were (Kassai were sections described histology previously for as Samples ears, 2008). inner detect dissected to and used embryos was staining X-gal stainings H&E and X-gal etdws1,8ad1 o oto,ad1,1 n 0for 10 and 14 12, and control, for 10 and 8 Cre; 10, was a tested in resulting level intensity by pattern. sound intensity lowest wave sound the ABR the recording recognizable decreasing and Hearing steps by dB averaged. defined 10 filter were were band-pass stimuli SPL) Hz 500 (dB 50–5000 from Processing thresholds using waveforms by ABR Signal Hz and 40,000 Real-time settings, at were ms waveforms 3 12.8 BioSigRP ABR for a System USA). recorded using FL, with Technologies, TDT performed canal (Tucker–Davis was with Systems ear recording testing together an ABR the software through to tube. software conducted acoustic 8, SigGenRp at and using placed plastic stimuli the generated speaker burst were to were condenser tone kHz or anterior EC1 32 Click electrodes just or respectively. 24, ear, vertex, needle the 16, subjected at earth the and ear to other and body posterior of just ground heating a subcutaneously mg/kg on 50 Reference, placed and with injection pad. anesthetized intraperitoneal by were pentobarbital weight Mice respectively. weeks, PAswr esrdb omrilisrmnainHearID instrumentation commercial by measured were DPOAEs ubro etder a 3 6 2 ,1,2 o oto iead16, and the mice control and ears for group, 28 both 14, for each 6, 40 in in 22, 28, brainstem 16, 8, tested auditory 13, 31, or were by was 12, ears tested animals unilaterally tested were of four weeks number either least 8 and At measurement 6 separately. 5, (ABR) 4, 3, response 2, of age the 9 n h45(5 sh1405 and ) o PA,maueet eetse iaeal.Tenme fears of number The bilaterally. tested were measurements DPOAE, For Cdc42 hybridization ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal 9 -GcTTAaGgCCatTaAGTTA-3 flox/flox 9 n h3 (5 sh333 and ) iea h g f2 n ek,respectively. weeks, 8 and 4 2, of age the at mice Atoh1–Cre; , 9 GTCTT ATGGAAGTAATG-3 TA -GGTGCAT 10 Atoh1–Cre; 9 edwt digoxigenin-11-dUTP. with -end 11 PFU/ml. Cdc42 9 -GCAGAGCACTCCACATACTTGAC- 9 Cdc42 -GCTTGTTGGGACCCAAATTGA-3). 9 b -GGAATTGTGGGTGCATTAATG- flox/flox glcoiaeepeso nwhole in expression -galactosidase 9 flox/flox n es (5 sense and ) 9 ). n itraecnrlmc at mice control littermate and iea ,3 ,5 n 8 and 6 5, 4, 3, 2, at mice 9 -GCGATGGTGCC- 9 -GCTGGCGCG- 9 .sh197- ). 9 probes ) Atoh1– 2049 m TM m-

Journal of Cell Science fec auaotie rmthree from obtained macula was which occupied, layer, mostly each cells cell myosin-VIIa-positive hair antibody where of per VIIa hair area images the myosin vestibular confocal as with of identified Five stained evaluation iodide. were propyl For maculae and sample. for utricular normalize the to fixed cells, of pillar cells, ten skewing the including or and field The a shrinking respectively, counted. as was defined turns area were was unit basal area interests per unit cells and of hair middle fields myosin-VIIa-positive of apical, visual number the Three phalloidin. from with selected and VIIa myosin isce ise eefxdb %prfradhd PA n01M 0.1 in After 1999). al., (PFA) et paraformaldehyde (Hayashi solution TCA 10% 4% or 7.4) by (pH buffer phosphate fixed were tissues Dissected amplitude. floor noise Immunohistochemistry of 16 subtraction 12, after 8, plotted of and frequencies f2 kHz ear at 20 the respectively, measured into and was inserted SPL amplitude was DPOAE dB probe and 55 USA) canal IL, and Acoustics; 65 (Mimosa ER-10C of an levels pressure f2/f1 sound with with f2, and ARTICLE RESEARCH h xlnso 5ogn fCriwr utrdfr6h n hnthe then 2050 and h, 6 for 0.02 cultured containing were medium Corti with of replaced was organs medium P5 of explants The Corti of of culture explant organs organotypic using a experiments D Cytochalasin under counterstained observed buffer, phosphate and M microscope. phalloidin, 0.1 confocal in Alexa-Fluor-568-conjugated PFA 4% with as with infection prepared the were infection, ( cultures adenoviral medium For explant 2008). al., Corti 2 et (Sakaguchi of described organs previously infection adenoviral organotypic and cochlea P2 of culture explant Organotypic 2.5% PFA, 2% in OsO 1% fixed with dissected postfixed were and were epithelia buffer Corti same of tissues Organ the PB. in M ear 0.1 in (GA) inner glutaraldehyde dissected Freshly TEM and SEM three from OHCs and IHCs of counting hair cell vestibular For of evaluation and OHCs, cells and IHCs of Counting 25 at h 2 for antibody primary fixed with (PBS-0.3T), incubated X-100 were tissues Triton 0.3% containing PBS with permeabilization iah -10eeto irsoea 0kV. 80 at on microscope examined and electron grids H-7100 copper Hitachi on placed Germany), Microsystems, Leica (thickness sections eodr nioyo hlodnfr1ha 25 at h 1 568)-conjugated for phalloidin (or Alexa-Fluor-488 or antibody by secondary followed BSA, fat-free 0.5% and ysnVI-oiieclspriaewsanalyzed. was image per cells myosin-VIIa-positive ojgtdpalii n AI(nirgn.Imnsannswere Immunostainings Zeiss). (Carl (Invitrogen). microscope confocal Alexa-Fluor- LSM700 DAPI a with under observed and counterstained After phalloidin and buffer. conjugated phosphate antibody, Alexa-Fluor- by M primary secondary followed with BSA, 0.1 fat-free conjugated incubated 0.5% in were with PBS-0.03T PFA cells in antibody fixed 0.45- 4% PBS-0.3T, or by with Biosciences) fixed permeabilization (BD were Matrigel insert with filter glass glass-bottomed the slide on cells dish, MDCK coverslip. a with (Invitrogen) anti-fade ape eeebde nEo 1 ei fe post-fixation after analysis, resin TEM 812 60 For Epon at 2010). polymerized in al., Japan), embedded et (Okenshoji, resolution (Notte were higher much SEM has samples which conventional S-2380 Zeiss), helium- Carl than Hitachi scanning (SHIM; MDCK PLUS a ORION microscope of SMT on visualization ion an Precise using examined performed kV. Dried was and 10 Japan). microvilli at stubs Koki, microscope (Hitachi on electron dryer scanning mounted critical-point were HCP-2 acetate, a samples isoamyl in by followed dried series, ethanol and an in dehydrated were tissues Cre; Cre; m ftehg-ie dnvrlslto a de oteculture the to added was solution adenoviral high-titer the of l Cdc42 Cdc42 xvivo ex 5 flox/flox flox/flox .0 utmpatcertp(imtro m tahdto attached mm) 3 of (diameter tip ear plastic custom A 1.20. a )fr2h h xlnswr ie –0dy after days 4–10 fixed were explants The h. 2 for 1) day iea ek,fxdogn fCriwr tie for stained were Corti of organs fixed weeks, 2 at mice iewr ae,adtenme fnce in nuclei of number the and taken, were mice , 0n)wr u na lrmcooe(EM-UC7; ultramicrotome an on cut were nm) 70 ˚ vrih n ultra-thin and overnight C Cdc42 ˚ 4 ,admutdi Prolong in mounted and C, nH in Cdc42 flox/flox 2 o .FrSEM, For h. 2 for O m yohlsnD. cytochalasin M flox/flox ˚ CinPBS-0.03T m and polyester m and Atoh1– Atoh1– olwdb muoltigfr2ha 25 at h 2 for immunoblotting by followed Hs hc aelne troii hnOC,i h pcltrsat turns apical Alexa-Fluor-488- microscope. the with in confocal OHCs, stained a than stereocilia h, under longer 32 0–45 observed have after which and IHCs, fixed phalloidin, were conjugated tissues The MDCK Hsi w raso ot ( Corti of organs two in IHCs lcrprto NP2;NP EEC. t)adG1 selection G418 and MDCK Ltd) Wako). Co., mg/ml; GENE (0.5 NEPA (NEPA21; electroporation ebvt’ -5mdu Ivtoe) hnatce n35m Cell- 3.5-mm (150 on dishes attached in coated then dissected were (Invitrogen), Tak mice medium biosensor L-15 Cdc42-FRET P2 Leibovitz’s from Corti of Organs imaging FRET n %Tio -0.Ttlcl yae eecnrfgda 12,000 NEPA21 4 at centrifuged at were protease lysates using of min cell presence 20 Total X-100. the cells Triton tesque), in 1% (Nakalai cocktail and sonication inhibitor by homogenizing phosphatase MDCK protein 2006) in cocktail, al., inhibitor lysed into et were (Ueyama cells buffer transfected immunoblotting, For electroporator. were Plasmids assay pull-down and Immunoblotting 2 Then, h. 24 for Inc.) Coning 0.45- a on grown (1 suspended were cells MDCK trypsinized cysts, produce cells To MDCK of culture Three-dimensional CO 5% a in pyruvate, 37 sodium mM at 1 EMEM nonessential incubator and in 0.1% L-Gln, (Wako) grown humidified 3% acids were (GIBCO), FBS Center) amino 10% BioResource with (RIKEN supplemented cells MDCK culture Cell eetdwt h hAAtvto sa i NwatBiosciences) (NewEast Kit Assay was Activation RhoA Active RhoA BSA. the fat-free with 0.5% detected containing PBS-0.03T in diluted ot ( Corti r tbekokoncl ie rnfce ihepypUE(e)or pSUPER(neo) target empty to with sh197 transfected expressing lines pSUPER(neo) cell knockdown stable are otae nttl nlsswspromduig1011IC nten in IHCs ImageJ 120–151 NIH using ( using performed Corti measured was of was analysis organs stereocilia total, highest In the analyzed. software. were damaged. of plane a severely length on often spread and The appropriately were mounted, stereocilia were turns with tissues IHCs basal the only when or compressed middle slightly the were Coverslips in cells hair as fCri( Corti of lcdo negtwl ls ld LbTkI;NN)cae with coated NUNC) II; (Lab-Tek 250 slide of glass suspension eight-well 40 A an Matrigel. on 2% placed containing medium culture mgn,Ws hse,P)adIai otae(ipaeAG, (Bitplane Software Imaris and PA) Chester, FRET/CFP (Universal West software CA). MetaMorph with View, Imaging, HP analyzed incubation and Mountain DeepSee acquired BX61WI/FV1000 Tai were Physics, an images Mai (Spectra a a in 60 Laser to using Ti:sapphire connected maintained with imaged Japan) Olympus, were equipped and (LUMPlanFLN; microscope Samples Japan) Hit, upright previously 2013). (Tokai described al., as chamber two- performed et under was imaging (Goto microscopy FRET excitation FBS. 10% photon with supplemented DMEM/F-12 fe iain ape eeosre ySM h ecnaeof using percentage calculated was The samples. microvilli independent SEM. of four from number by cells reduced days. 200 4 observed a additional with an were cells for MDCK cultured samples were cells fixation, and h, After 2 for medium culture to used are blue to red display from colors intensity-modulated eight ratio. the FRET/CFP which the by in represent mode, represented (IMD) and Switzerland), target 02.Fxdclscutrtie ihAlexa-Fluor-568-conjugated microscope. laser with confocal a counterstained under observed were cells phalloidin Fixed 2012). ogo irvlio h pclsrae 1 surface, apical the on microvilli grow To m fplmrzdMtie,adicbtdfr7dy Yg tal., et (Yagi days 7 for incubated and Matrigel, polymerized of l ˚ 45–90 , N-WASP Atoh1–Cre; cont ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal Atoh1–Cre; MDCK , ˚ ˚ ,adtespraat eesbetdt SDS-PAGE to subjected were supernatants the and C, n 90–135 and . Cdc42 m Cdc42 oyse itrisr 1-mdaee Transwell; diameter (12-mm insert filter polyester m Cdc42-KDa Cdc42 flox/flox flox/flox m ˚ g/ flox/flox MDCK , rmteae eeue nti experiment, this in used were apex the from Cdc42 GFP-Cdc42 m n 07 Hsi iet i rasof organs six to five in IHCs 60–77 and ) m o yohlsnDtetet n 32–33 and treatment, D cytochalasin for ) ;B isine) n anandin maintained and Biosciences), BD l; ˚ .Coa ie eeotie by obtained were lines Clonal C. faeoiu ouinwsaddt the to added was solution adenovirus of l ihu treatment. without ) flox/flox Cdc42-KDg el tbyepesGFP–Cdc42; express stably cells 6 Cdc42 n 12 Hsi w organs two in IHCs 21–29 and ) ae-meso objective water-immersion ˚ sn rmr antibody primary using C n MDCK and 6 rs19 o h45 to sh1405) (or sh1396 or 10 4 DKclswere cells MDCK NWASP-KD 6 10 4 m m)in /ml) was l g lines for 2

Journal of Cell Science ln,K . ell . ekrhn . ao,S,Thrvc . urz S., Dupraz, S., Tahirovic, S., Jacob, D., Neukirchen, F., Hellal, C., K. A., Flynn, Steffen, E., Frittoli, S., Gerboth, M., Hertzog, S., Mantoani, A., Disanza, Corwin, and M. L. Igbani, C., Askew, W., Baker, R., B. Thiede, S., M. Collado, T. J. Corwin, and Z. Hu, C., J. Burns, S., T. M. Collado, Koji, and Y. Hishikawa, D., Endo, T., Nishino, Y., C. Sato, P. N., Letourneau, Choijookhuu, and R. J. Bamburg, E., A. Shaw, S., Gehler, J., T. Chen, W., N. Segil, P. and Y. Marks, H. Zoghbi, C., E., J. Wu, Johnson, P., M., Chen, Lopez, X., Mao, C., M. Parrini, L., Ma, D. F., M. Chen, Welch, and G. K. Campellone, Kawamoto, D., E. Hughes, L., T. Saunders, A., I. Belyantseva, T., Ben-Yosef, K. Kaibuchi, and M. Nakayama, M., Amano, l aaaepeetda h mean the as presented are data All analysis (HRP) Statistical Healthcare). peroxidase (GE system horseradish detection to detected ECL were the conjugated using antibodies antibody specifically primary antibody secondary bound monoclonal with The the RhoA. using active assays recognizing pull-down on based ARTICLE RESEARCH iaa . aaa . uui . iua . asi . aaa T., Harada, H., Kassai, T., Iimura, D., Suzuki, A., Yamada, R., Aizawa, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.143602/-/DC1 online available material Supplementary release. material immediate for Supplementary PMC in Deposited H.S.]. to 23592491 JSPS by number and T.U.); [grant (to Foundation KAKENHI Science Takeda Live the ‘Fluorescence by Areas N.S.); Innovative on (to imaging’, KAKENHI MEXT by supported was work This Funding and analyzed manuscript. N.S. the and wrote Hisa and Y. data H.S., obtained T.U., interpreted data microscope. analyzed electronic S.Y. turnover. an performed actin using planned, about the S.S. experiments provided and the B.F. H.S. discussed and ISH. and performed A.A. T.K. data H.K., and analyzed Hishikawa K.N., Y. and mice. animals. experiments biosensor performed performed FRET Y.N. M.M. using and and obtained A.S. A.G. S.M., T.N., experiments. H.S., of T.U., project. most the planned N.S. and T.U. contributions Author interests. competing no declare authors The interests Competing ttsia nlsswr efre sn rs . otae(GraphPad); software 5.0 Prism P using performed and were performed analyses Statistical was Bonferroni’s ANOVA by two-way or followed ANOVA one-way groups, two Student’s two-tailed unpaired using , eitdatnrtord lwdrcsnuiefraini h eeoigbrain. developing the al. in et formation E. neurite A. directs Neuron Shaw, flow C., retrograde Gurniak, actin K., mediated B. actin-bundling Garvalov, S., synergic Stern, the by mediated complex. al. is Eps8-IRSp53 et Cdc42 the P. P. by of Fiore, activity shape Di cell F., Milanesi, of J., Regulation H. Kreienkamp, K., sensory Berhoerster, into directly organs. convert balance to mammalian cells in supporting cells for hair capacity the with correlated T. J. 465-471. research. regeneration cell hair receptor exchanger-3 estrogen sodium/hydrogen via mice. of expression retinal regulation (NHE3) and Estrogen-dependent ADF/cofilin (2012). factor. of neurotrophic derived regulation brain the by 103-114. in filopodia cone participates growth Cdc42 (2006). determination. primordium fate sensory cell the of hair establishment from the Uncoupling development: ear inner not but al. development et early viability. T. and cell polymerization for Kirchhausen, actin J., PIP(2)-induced for D. required Kwiatkowski, L., Davidson, assembly. actin of control degeneration. R. cell E. hair Wilcox, cochlear J., to D. due Gardner, Genet. deaf K., are Halsey, DFNB29, A., deafness L. Beyer, al. M., et C. Itallie, Van K., 554. euao ftectseeo n elpolarity. cell and cytoskeleton the during of death regulator cell programmed interdigital and development. al. chondrogenesis limb et for K. Nakao, required T., Tachikawa, is G., Yamamoto, M., Tsukasaki, .5wscniee ttsial significant. statistically considered was 0.05 21) h otaa cuuaino ucinlEcdei sinversely is E-cadherin junctional of accumulation postnatal The (2011). itce.Cl Biol. Cell Histochem. 20) lui 4koku ie oe o uooa recessive autosomal for model a mice, knockout 14 Claudin (2003). 12 76 2049-2061. , 1091-1107. , ur Biol. Curr. eh Dev. Mech. a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 10 137 othoc post 758-765. , 129 575-587. , ur pn tlrno.Ha ekSurg. Neck Head Otolaryngol. Opin. Curr. Development 38-50. , 6 a.Cl Biol. Cell Nat. t 21) ulao rsrc:cellular race: arms nucleator A (2010). ts.Frcmaioso oethan more of comparisons For -test. eto ariegopdifferences. group pairwise of test ...Togop eecompared were groups Two s.e.m. .Neurosci. J. b 21) h-iaeRC:Akey A Rho-kinase/ROCK: (2010). yokltn(Hoboken) Cytoskeleton npoia oo fpregnant of colon proximal in 129 2495-2505. , 11 20) h oeo ah in Math1 of role The (2002). 20) eetavne in advances Recent (2008). 8 237-251. , 1337-1347. , 31 11855-11866. , 21) ADF/cofilin- (2012). 20) d4 is Cdc42 (2000). .Neurobiol. J. 21) Cdc42 (2012). u.Mol. Hum. 67 (2006). 545- , 16 66 , , ot,J,I,Hl,R,MVy .M,Rmcada . rfi,B n o,D. Joy, and B. Griffin, R., Ramachandra, M., S. McVey, R., Hill, IV, J., Notte, aao . i,S . i,H . anmn .D,Zag . mt,R J., R. Smith, Y., Zhang, D., J. Sanneman, M., H. Kim, H., S. Kim, Y., Nakano, ono,K . og-us,C .adGgo,L H. L. Gagnon, and M. C. Longo-Guess, R., K. B. Johnson, Fritzsch, and J. Kersigo, N., Pan, I., Jahan, ae,V,Pue,S,Kig . oic,D,Bie,K . ors . eg F., Feng, K., Morris, W., K. Beisel, D., Rowitch, S., Kaing, G. S., Scita, Pauley, P., V., P. Matei, Fiore, Di H., Lin, L., Andrade, M., Grati, A., Disanza, U., Manor, R., Stepanyan, J., R. Goodyear, A., I. Belyantseva, T., Sakamoto, S., Kitajiri, Ito, T., Nakagawa, T., Katsuno, H., Sasaki, M., Hata, K., Fukumoto, S., Kitajiri, M. Watanabe, M., Sakahara, K., Nakao, M., Fukaya, G. T., Terashima, Kalinec, H., Kassai, and R. Urrutia, M., Zhang, F., Kalinec, avlv .K,Fyn .C,Nuice,D,My,L,Tuc,N,W,X., Wu, N., Teusch, L., Meyn, D., Neukirchen, C., K. Flynn, K., J. B. A. Garvalov, Griffith, and B. T. Friedman, A., I. Belyantseva, I., G. Frolenkov, eedz . rg,M n hn,Y. Zheng, and M. Y. Grogg, Kikkawa, J., S., Townsend, Melendez, A., Parker, H., Hilton, R., M. Romero, P., Mburu, Brown, and H. Yonekawa, R., Romero, S., Townsend, Y., Kikkawa, P., Mburu, vnv .I,Hn,D,Ueh . urt .adPro,C A. C. Parkos, and A. Nusrat, M., Utech, D., Hunt, I., A. Ivanov, M. Takeichi, and T. Ishiuchi, M., Iwasaki, S. Tsukita, and K., T. Matsui, S., Yonemura, K., Ito, Hayashi, E., Nakasyo, Y., Kamioka, K., Sumiyama, A., A., Oganesian, W., Goto, Marcotti, B., M. Wright, K., P. Legan, J., R. Kohrman, Goodyear, Y., Raphael, L., Beyer, J., Macdonald, J., I. Karolyi, W., T. Gong, ue,F . oe,L . i,H . ais . zvd,R . o,A. Gow, B., R. Azevedo, C., Davies, W., H. Lin, N., L. Lopez, D., F. Nunes, br,P,Msah,M,Vrl,A,Wi,D,E-mau,A,Hle .H., R. Holme, A., El-Amraoui, D., Weil, A., F. Varela, Kalinec, M., Mustapha, and P., M. Mburu, Mueller, A., Maricle, R., Kitani, N., Matsumoto, esa,S .adRde,A J. A. Ridley, and J. S. Heasman, 21) ifato mgn naHe a in imaging Diffraction (2010). e1000610. -ae o emaiiybriri seta o hearing. for essential is barrier permeability Ba ion and A. R. 9-based Nessler, P., Wangemann, C., D. Marcus, el:mlclrce o arcl types. cell hair for cues molecular cells: oe,K,Le .adFizc,B. Fritzsch, and J. Lee, K., Jones, Eps8. protein actin-regulatory cell the on hair depends whirlin of al. B. et Kachar, rootlets M. and resilient Z. Ahmed, forms S., hearing. TRIOBP Riazuddin, for essential S., protein stereocilia Riazuddin, Actin-bundling E., J. (2010). Bird, I., Fujiwara, stereocilia. cochlear of degeneration 166 S. progressive Tsukita, with and associated S. Tsukita, J., formation. axonal 267. commissural acetylcholine. of crossing by midline for motility A. cell Aiba, and hair outer cochlear Chem. of Biol. J. regulation the mediate oii uigteetbiheto ernlpolarity. F. neuronal of Bradke, establishment and the R. during J. cofilin Bamburg, C., Brakebusch, cells. hair ear inner of 5 morphogenesis the into insights Genetic euaigmmainphysiology. mammalian regulating stereocilia. cell hair D. of complex S. Brown, and development. D. S. 428. ifrnilrlsfratnplmrzto n ysnI oo nasml of assembly in motor II complex. myosin junctional a apical and epithelial polymerization the actin for roles Differential phosphorylation. ROCK aPKC-mediated Biol. Cell through Nat. constriction apical epithelial carboxyl-terminal tissues. their and with cells cultured proteins in (ERM) phosphorylated threonine ezrin/radixin/moesin Rac1. and of A detection kinase protein via cells crest-derived Neurosci. M. neural J. enteric Matsuda, of and migration H. Enomoto, al. of et F. maturation D. the bundles. Bowen-Pope, for hair A., required is R. cochlear phosphatase Seifert, developing lipid J., inositol C. receptor-like Kros, A J., (2003). C. Booth, A., S. Coats, mice. 2 shaker and 317-328. normal I. in M. expression Lomax, and gene A. S. Camper, C., D. 13129. opsto fatgtjnto ihahrn ucinfeatures. junction adherens with junction tight 119 a of B. composition Kachar, and microscope. stereocilia. cell signaling GTPase hair small in link dynamics (Elmod1) cytoskeleton gene actin 1 to containing domain ELMO mouse nwiln D oanmlcl novdi troii lnain cause elongation, stereocilia DFNB31. in with families involved and molecule mouse whirler domain the al. PDZ in et deafness S. a R. Coimbra, whirlin, S., Blanchard, in E., R. Hardisty, A., Rump, cell hair outer cochlear of regulation the in motility. depolymerization exit. actin of cycle role cell Pivotal hair earlier to related are mice 633-650. null 1 Neurog in noterfntosfo nvv studies. vivo in from functions their into 1158. 489-498. , 4819-4827. , 559-570. , 20) hri opee ihp5a h troii i uighi cell hair during tip stereocilia the at p55 with complexes Whirlin (2006). ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal ipy.J. Biophys. 33 irs.Microanal. Microsc. 20) a1i otclpoeto ern sslcieyrequired selectively is neurons projection cortical in Rac1 (2008). rc al cd c.USA Sci. Acad. Natl. Proc. 13 275 4901-4912. , 860-866. , 21) euaino troii eghb ysnXaand XVa myosin by length stereocilia of Regulation (2011). 28000-28005. , 20) itntsboanognzto n molecular and organization subdomain Distinct (2006). 99 21) eslnpasarl nteatnpolymerization actin the in role a plays Gelsolin (2010). 2067-2076. , 21) ilnadPr oprtvl regulate cooperatively Par3 and Willin (2011). 20) amla h Tae:nwinsights new GTPases: Rho Mammalian (2008). LSONE PLoS Cell 16 20) aii eiinycue deafness causes deficiency Radixin (2004). .Neurosci. J. .Bo.Chem. Biol. J. 21) DFadedtei regulate 3 endothelin and GDNF (2013). 599-603. , 20) mle ne a esr epithelia sensory ear inner Smaller (2005). 141 20) g-eae hne ncochlear in changes Age-related (2006). o.Bo.Cell Biol. Mol. + a.Rv o.Cl Biol. Cell Mol. Rev. Nat. er Res. Hear. 786-798. , 103 21) inln oeo d4 in Cdc42 of role Signaling (2011). o emsann transmission scanning beam ion 5 e11627. , 10973-10978. , 21) eodgnrlzdhair generalized Beyond (2013). 23 .Asc e.Otolaryngol. Res. Assoc. J. 286 9208-9219. , 19) Immunofluorescence(1999). 297 u.J Neurosci. J. Eur. 2375-2381. , ni B. ´nfi, 20) d4 regulates Cdc42 (2007). ur Biol. Curr. LSONE PLoS .Neurosci. J. 16 21) uain fthe of Mutations (2012). 20) h GTPases Rho (2000). 30-41. , .Cl Sci. Cell J. 2636-2650. , a.Genet. Nat. 20) claudin- A (2009). LSGenet. PLoS a.Rv Genet. Rev. Nat. e.Dyn. Dev. 20) Defects (2003). 21 9 7 690-701. , .Cl Biol. Cell J. .Cl Sci. Cell J. 167-172. , e36074. , 27 112 13117- , 34 28 (2004). (2005). (2010). 1149- , 2051 421- , 257- , 234 5 7 , , ,

Journal of Cell Science ef . oe . oead .G,Jnis .A,Arhm .B n Steel, and B. K. Avraham, A., N. Jenkins, G., N. Copeland, T., Sobe, T., Self, P. L. Mu and Rybak, B. Kachar, M., and Schwander, E. A. Talaska, J., Schacht, J. Miyazaki, and K. Sakai, oof .R n ey B. Geny, and R. M. Popoff, Danuser, and M. C. Waterman-Storer, L., S. Gupton, M., Machacek, A., Ponti, M. J. Ervasti, and M. J. K. Takeichi, Sonnemann, J., B. and Perrin, S. Aono, T., Ichii, T., Otani, ARTICLE RESEARCH 2052 Sekerkova B. Kachar, and S. N. Gov, D., Bowen-Pope, M., Naoz, J., Tokita, H., B. Sakaguchi, Kachar, and P. G. Riordan, C., Davies, E., M. Schneider, K., A. Rzadzinska, G. I. Macara, and Y. Hao, H., W. Meisen, Y., Qin, Biol. P. K. prevention. hearing. of its and transgene loss cre hearing (Hoboken) the antibiotics: of aminoglycoside irrespective oocytes mature in transmission. activity recombinase udigpoen ntemrhgnssadsaiiaino arcl stereocilia cell mice. hair jerker of congenic stabilization CBA/CaJ and in morphogenesis revealed the in proteins bundling transport. VI-based myosin of implication stereocilia: of Cytoskeleton end Motil. Cell proximal the receptor phosphatase at tyrosine Q protein of compartmentalization Dynamic (2008). functional stereocilia maintain self-renewal. myosins and and treadmill architecture molecular actin An (2004). toxins. 812. from lessons junctions, intercellular in Science G. Stereocilia for required but development cell hair maintenance. auditory for dispensable each cells. epithelial simple of 135-146. configuration junctional the regulates eurdfrplrzdsideoinaindrn pteilcs formation. cyst epithelial during orientation Biol. spindle polarized for required 20) w itntatnntok rv h rtuino irtn cells. migrating of protrusion the drive networks actin distinct Two (2004). 214 189 19) oeo ysnV ntedfeetaino oherhi cells. hair cochlear of differentiation the in VI myosin of Role (1999). ,G,Rctr .P n ate,J R. J. Bartles, and P. C. Richter, G., ´, 305 331-341. , 661-669. , 295 .Cl Biol. Cell J. 1782-1786. , ice.Bohs e.Commun. Res. Biophys. Biochem. LSGenet. PLoS 1837-1850. , 190 65 20) utfctdrl fRo a,Cc2adRas and Cdc42 Rac, Rho, of role Multifaceted (2009). 528-538. , 9-20. , 19) rngncmueln htrtisCre retains that line mouse transgenic A (1997). 6 e1001158. , .Cl Biol. Cell J. lr U. ller, ¨ 21) eiwsre:Tecl biology cell The series: Review (2010). 164 LSGenet. PLoS ici.Bohs Acta Biophys. Biochim. 887-897. , 21) oe fteepnactin- espin the of Roles (2011). 21) ua d4 E,is GEF, Cdc42 a Tuba, (2010). (2010). 237 20) d4 E Tuba GEF Cdc42 (2006). 318-324. , 21) ipai and Cisplatin (2012). b 7 atnand -actin e1002032. , .Cl Biol. Cell J. nt Rec. Anat. 1788 c atnare -actin .Cell J. 797- , Dev. 175 , hn,D . iza . ern .J,Raznk,A . oztk .C., J. Poczatek, K., A. Rzadzinska, J., B. Perrin, V., Piazza, S., D. Zhang, Ru V., Zampini, Y. Zheng, and L. Wang, L., Yang, E. Kiyokawa, and M. Matsuda, S., Yagi, uvlde . alr . tos,J . ooa . ru . aa,V., Salas, O., Ursu, C., Bologa, J., J. Strouse, A., Waller, K. Z., Mikoshiba, T., Surviladze, Yamamoto, T., Tezuka, H., Miki, M., Hattori, S., M., Suetsugu, J. Pagana, N., A. Tauscher, Z., Metlagel, A., Hassan, F., J. Krey, B., J. Shin, eaa . est .adLt,T L. T. Leto, and M. Geiszt, T., Ueyama, A. D. Portnoy, and G. L. Tilney, J., T. Mitchison, A., J. Theriot, aeaa .adSesg,S. Suetsugu, and T. Takenawa, S. Ohno, and A. Suzuki, ag . rse,H . rat,J . oe,D .adLcee .P. C. Lechene, and P. D. Corey, M., J. Ervasti, M., mammalian H. Prosser, of M., maturation al. Wang, et functional N. and Offenhauser, length C., cells. M. bundle hair Holley, auditory hair M., regulates C. Hackney, Eps8 H., induction, Xiong, fibroblasts. J., filopodia embryonic mouse in primary Cdc42 in Cell of Biol. proliferation Mol. involvement and critical migration, directed the affirms Cdc42GAP maintenance. structure cyst for essential is Rep. cells EMBO MDCK of membrane apical the 2160-2174. 21) ut-stp mgn assetoer eel lwpoenturnover protein slow stereocilia. reveals hair-cell spectrometry in mass imaging Multi-isotope (2012). akno,J . hlis .K,Rmr,E,WnigrNs,A tal. et A. Wandinger-Ness, E., Romero, K., G. Phillips, F., J. Parkinson, extension. neurite for essential is T. phosphorylation Takenawa, and bundle. al. hair et H. vestibular Zhao, chick J., the K. Spinelli, of D., architecture E. Jeffery, E., N. Sherman, fmliopnn o1 n o3bsdNDHoxidases. NADPH Nox3-based and Nox1- multicomponent of fatnbsdmtlt fitaellrLsei ooyoee qastert of rate the equals monocytogenes Listeria polymerization. intracellular actin of motility actin-based of cytoskeleton. the to membrane the connecting Sci. Cell J. In GTPase. Cdc42 Program Information. of Libraries Biotechnology inhibitor Molecular NIH selective the and From potent A (2010). ora fCl cec 21)17 0025 doi:10.1242/jcs.143602 2040–2052 127, (2014) Science Cell of Journal 119 tgr . ono,S . rn,C,Fres .N,Waldhaus, N., D. Furness, C., Franz, L., S. Johnson, L., ttiger, ¨ 13 17 979-987. , 237-243. , 4675-4685. , LSBiol. PLoS Nature Nature 20) utie ciaino -APthrough N-WASP of activation Sustained (2002). 20) h A-PCsse:lsosi polarity. in lessons system: PAR-aPKC The (2006). 357 481 9 e1001048. , 257-260. , 520-524. , 20) h APWV rti network: protein WASP-WAVE The (2007). 20) novmn fRc nactivation in Rac1 of Involvement (2006). 20) eetreigo d4 and Cdc42 of targeting Gene (2006). 21) upeso fRc ciiyat activity Rac1 of Suppression (2012). ehsa D ainlCne for Center National MD: Bethesda, . a.Rv o.Cl Biol. Cell Mol. Rev. Nat. a.Neurosci. Nat. e.Cell Dev. 3 o.Cl.Biol. Cell. Mol. 21) Molecular (2013). 645-658. , 19) h rate The (1992). 16 rb Reports Probe 365-374. , 8 37-48. , (2011). 26 ,

Journal of Cell Science