aa auaadMtuoiFukuda* Mitsunori and Yasuda Takao of Rab27 regulation of through independently size signaling cell Rap–ezrin epithelial renal controls Slp2-a ARTICLE RESEARCH ß eevd2 pi 03 cetd3 coe 2013 October 30 Accepted 2013; April 27 Received ([email protected].) correspondence for *Author Japan. 980-8578, Tohoku Miyagi Sciences, Sendai, Life Aoba-ku, of Aobayama, School University, Developmental Graduate of Neurosciences, Department and Mechanisms, Biology Trafficking Membrane of Laboratory certain in membrane plasma C2A the promotes to its vesicles Slp2-a through secretory Similarly, of 2004). membrane docking through Fukuda, plasma and membrane (Kuroda the domain and in plasma SHD the phospholipids through the and melanosomes with the melanosomes to on In Rab27 mature with 2002). anchoring interaction to al., their et localized Kuroda promotes is 2006; Slp2-a the (Fukuda, for melanocytes, Rab27 molecule effector GTPase an as small characterized 2001), originally Mikoshiba, was and whose it Fukuda and 2001; and domain al., C2A et family, the (Fukuda (SHD) domain) (named domain C2B domains protein homology C2 tandem Slp C-terminal N-terminal synaptotagmin-like and an a of is the consist Sytl2) members as of known also member (Slp2-a; 2 protein Synaptotagmin-like INTRODUCTION Rap1GAP, disease, kidney Slp2-a Polycystic Ezrin, size, Cell WORDS: KEY Slp2-a of cells. ablation II MDCK Functional of size cells. and the in the signaling increase an Rap of in inactivates resulted size Slp2-a the cells domain, size II modulates MDCK C2B cell of membrane the renal that plasma the through found of to We GAPs control Rap1 domain. recruiting C2B the by uncharacterized in previously a Slp2-a a through of Rab27- and a function demonstrate domain and independent we Rab27-binding vesicles Here domain. N-terminal Rab27-bearing C2A phospholipid-binding effector its of Rab27 through transport a organelles regulates is that (Slp2-a/Sytl2) protein 2 protein Synaptotagmin-like ABSTRACT idnsidct htSp- otosrnlcl iethrough size cell renal Rab27. of controls independently Our signaling Slp2-a spreading. Rap–ezrin of cell that regulation renal by indicate characterized findings is that disease kidney fetvl niie elsraigo l2akokoncls We miglustat, cells. Slp2-a-knockdown inhibitor, conserved. of synthase spreading evolutionarily glucosylceramide cell inhibited the target been effectively downstream with a has ezrin, Rap, of of activity proteins in the of Slp2-a Slp blockade size of Interestingly, cell the by function of mechanism the the control that for indicating thereby compensate cells, II to MDCK found was Bitesize lodsoee bratepeso fSp- n increased ( and pcy Slp2-a in of ezrin expression of activity aberrant discovered also 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,5750doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Nphp3 pcy ie oe fpolycystic of model a mice, ) Drosophila Slp 2 oan ncnrs oteCAdmi,hwvr the the however, of only domain, exhibits activity which C2A domain, binding the C2B phospholipid the to activity of contrast the binding function physiological In Rab27 and the domain. SHD both C2A require the Rab27-bearing to of docking of the thought docking and the is membrane, plasma of process the mediator to organelles generally key and is a vesicles Slp2-a findings, be these to on thought Based 2012). al., et (Ga Yasuda membrane apical tubulogenesis be the podocalyxin-containing and to Rab27-bearing to to vesicles signaling of cell and shown docking of cells promoting been regulation epithelial by the renal recently in of involved has membrane be apical Slp2-a the 2006). to localized al., et Saegusa eas l2ai loepesdi h ea uue fmice of pcy tubules in Slp2-a renal of the expression investigated ( in domain. we 2012), C2B expressed al., the et also (Yasuda through is membrane Slp2-a it plasma that Because the showed recruiting to by and GAPs signaling line) Rap–ezrin Rap1 through cell size epithelial cell renal renal regulates (a II kidney cells canine II) Madin–Darby of (MDCK morphology the controlling in morphology. a cell been in proteins a have Slp of Rab27- existence not of the function a must hypothesized conserved we i.e. 2003), cell function and evolution, SHD, effector unknown. during Rubin, epithelial N-terminal retained Rab27 remains an and domain, also of lacks Serano binding here protein maintenance mechanism 2006; Btsz Because precise in al., the et 2001), although (Pilot al., Slp2-a the to morphology similarity of et sequence high involvement shows (Fukuda that the Slp and molecular mammalian is of (Kuroda the the example unknown of remain Another Drosophila generation but so 2004). does in melanocytes, it Fukuda, involved which be of by mechanism example, to morphology an shown As elongated been morphology. Rab27- cell a has in implicate Slp2-a Slp2-a studies of several role literature, independent the determined. in be to established yet has 2004), (Ga Fukuda, and ability Kuroda 2012; binding phospholipid weak ye fsceoycls(ote l,20;Me 2008; al., et (Holt cells secretory of types zi npymc.Orfnig niaeta Slp2-a-mediated that indicate findings Our mice. pcy of activity precise in We increased and the ezrin understood. Slp2-a but poorly of al., cyst expression are 2004), aberrant et discovered diseases Wilson, renal the (Hildebrandt 2009; spreading, underlying failure Harris, mechanisms cell and kidney of Torres to renal all 2009; progression by disorders and genetic characterized growth, of autosomal are group nephronophthisis including heterogeneous which and a PKDs diseases, are and recessive (NPHP), Torres kidney autosomal 2003; and al., Cystic dominant et mutation al., 2009). Olbrich et missense 2009; (Bergmann Harris, al., gene homozygous et (NPHP3) Hildebrandt a nephrocystin-3 2008; the have in that (I614S) (PKD), disease Nphp3 nti td eivsiae h osbeivleeto Slp2- of involvement possible the investigated we study this In well been has Slp2-a of function effector Rab27 the Although pcy ie pnaeu oe fplcsi kidney polycystic of model spontaneous a mice, ) l ooou ieie(tz,teol homologue only the (Btsz), Bitesize homologue Slp ´ vzSnitbne l,2012; al., et lvez-Santisteban ´ vzSnitbne al., et lvez-Santisteban ´ nasche ´ ta. 2008; al., et 557

Journal of Cell Science is,w netgtdteivleeto l2ai ea cell II renal MDCK in control of Slp2-a rates of growth the involvement comparing by the proliferation cells investigated epithelial we renal in First, size cell a regulates Slp2-a as signaling RESULTS PKD. Rap–ezrin function, of treatment kidney Slp2-a-mediated the normal for for target discuss crucial potential is we size cell and renal of control ARTICLE RESEARCH 558 the in control to cells size Slp2-a-KD cells cell the II of in size MDCK Slp2-a greater subconfluent The of use analyses. role subsequent to subconfluent the the decided of investigate phenotype we not the was as phenotype cells, their distinguishable since but clearly 2012), control so al., than et larger Yasuda times under (see 1.5 cells) larger (approximately cells larger control were Slp2-a- significantly cells conditions 1C). the KD polarized (Fig. under than were Even larger) conditions. cells subconfluent cells times then Slp2-a-KD four Slp2-a-KD We (approximately of cells. the control morphology Intriguingly, the cells the than was Slp2-a-KD RNA the faster there investigated 1B), much Although hairpin (Fig. rate confluent 1A). growth short (Fig. became their 2012) in Slp2-a had difference al., no we using et that (Yasuda by cells (shRNA) II previously MDCK (KD) established Slp2-a-knockdown and cells xrsino he ifrn l2asalitreigRNAs interfering sh S1A,B) an Fig. small material of (Slp2-a (supplementary re-expression Slp2-a cells because II different and MDCK three in results (siRNAs) same of the expression because attributab been shRNA, have to unlikely was eut niaeta l2argltsrnleihla elsize cell epithelial renal not regulates was Slp2-a Rab27. cells these of that Slp2-a-KD together, independently Taken the indicate S1C). Rab27A and Fig. results of cells material level (supplementary control the MDCK observed control the between the difference in of the (supplementary size expression the Furthermore, on expression cells. effect no II Rab27A had S1E,F) Fig. endogenous by material or negative S1D), or of Fig. (Q78L) material knockdown (supplementary mutant Rab27A active of constitutively (T23N) either mutant a Rab27A of of ablation expression functional by findings, these with r to the Consistent Slp2-a as able the same was the and 1D, membrane, protein, Fig. plasma wild-type the in to E11A/ shown localized Slp2-a was As protein 2004). mutant, mutant Fukuda, Rab27-binding-deficient and a (Kuroda Rab27 using R32A a by S size investigated as of cell we ability described 2004), binding Rab27 Fukuda, originally the and whether Kuroda was 2006; (Fukuda, Slp2-a effector Because 1D). (Fig. SR nteSp--Dclsrsoe hmt omlcl size cell normal to them restored cells Slp2-a-KD the in ) ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal el.(et l2aK el eetasetdwt pEGFP- with pEGFP-C1-Slp2-a transfected C1, were cells Slp2-a-KD Slp2-a-KD (Left) using cells. experiments Rescue (D) experiments). unpaired ie hw nteiae ntelf.* left. the on images the in shown sizes tie oiiefratn uliwr tie ihDAPI with 10 stained bars: were Scale Nuclei that (blue). actin. area the for measuring positive of by stained determined size was The type red). cell staining; each actin (for Texas-Red- phalloidin hours, with conjugated 24 stained for and cultured permeabilized, were fixed, (Left) cells cells. Slp2-a-KD times Slp2-a-KD and the of Control at size counted Increased were (C) at cells indicated. dishes trypsinized 10-cm and individual 0, cells. in day Slp2-a-KD seeded and were control cells of 50,000 the curve on Growth shown the (B) are of left. kilodaltons) positions (in The markers control. mass GAPDH internal molecular cells. an control was as the cells used in Slp2-a-KD was level the the in of protein 5% Slp2-a approximately cells. of Note II level cells. Slp2-a-KD MDCK the and that larger control to in leads expression Slp2-a Slp2-a (A) of Loss 1. Fig. en n s.e.m. and means ( hw nteiae ntelf.* left. the on images the in shown (blue). DAPI 10 with bars: stained Scale were Texas-Red-conjugated Nuclei with (red). stained phalloidin then for and were cultured hours, cells dishes, 24 The glass-bottomed hours. 35-mm 48 on for re-plated cultured and (green), R32A n 5 5fo he needn xeiet) ausare Values experiments). independent three from 55 t ts ( -test m n .(ih)Qatfcto ftecl sizes cell the of Quantification (Right) m. 5 0fo he independent three from 50 soetesz fSp--Dcells. Slp2-a-KD of size the estore m SR .(ih)Qatfcto ftecell the of Quantification (Right) m. et noftre feto the of effect off-target an to le N-eitn omo Slp2-a of form RNA-resistant p- srqie o oto of control for required is lp2-a rpEGFP-C1-Slp2-a or eeotie ytransient by obtained were P , .0,Dnetstest Dunnett’s 0.001, P , .0,Student’s 0.001, SR -E11A/

Journal of Cell Science lss ooou fSp- Fkd ta. 01.Frt we First, 2001). al., et (Fukuda Slp2-a has (Neumu of 2005), Slp1 al., homologue with and et interact closest (Schultess adhesion to Rap1 shown e.g. GTPase- for a been events, Rap1GAP2, (GAP) because cellular protein and basic 2001), activating to Slp2-a- al., in known et the is (Bos role Rap1 spreading of central because Rap1, mechanism a on the size play cell of of exploration control mediated our focused We Rap1GAP2 with interacts Slp2-a ARTICLE RESEARCH eune(AX fKRs(i.3,lf)(oaah and (Kobayashi left) 3A, (Fig. membrane-targeting membrane-targeted K-Ras plasma plasma of a (CAAX) test using a To by sequence size. generated cell construct we of Rap1GAP2 control hypothesis, the during functions our membrane Rap1GAP2 plasma that Slp2- the hypothesized the we at of 2D), cytoplasm (Fig. the cells in a-KD dispersed the was for Rap1GAP2 required Because is size membrane cell of plasma with the control to interaction Rap1GAP2 of Slp2-a Targeting through that size indicate cell results lacks size epithelial Rap1GAP2. cell these renal on size which together, effect cell regulates no Taken in Slp2-a-C2A, had increase 3C). 2B), (Fig. (Fig. GFP-tagged an ability in binding whereas resulted Rap1GAP2 cells 3C), II (Fig. MDCK overexpression whereas its control i.e. size, effect, in dominant-negative cell a in exhibited C2B (RGDN- increase mutant an control Slp2-a-binding-deficient D in the in resulted of RGDN As overexpression cells size. Venus-tagged cell II affect of MDCK should alone, overexpression dominant-negative a site expected, by binding interaction the the e.g. of construct, control disruption the size, in cell involved interaction actually of the the is Rap1GAP2 If and not Rap1GAP2. Slp2-a for and between required but Slp2-a also between is construct, interaction sequence the EVTKTT RGDN the that the indicating Slp2-a, (right), As 3A RGDN Slp2-a. Fig. RGDN- of the in domain a C2B from shown the and with sequence we assays RGDN) EVTKTT immunoprecipitation end, as the this designated of (RGDN- Rap1GAP2; To mutant of (amino deletion sequence. Rap1GAP2 518–687 of this mutant acids truncated with of N-terminal an 537–542 interacts generated acids also (amino Slp2-a sequence (Neumu EVTKTT Rap1GAP2) through Slp1 C-terminal with interact to its size shown cell been has affects the Rap1GAP2 interaction Because Slp2-a–Rap1GAP2 for the required of Disruption is plasma Slp2-a the that to Rap1GAP2. indicating of Slp2-a- localized membrane-localization of 2D), plasma cytoplasm also (Fig. the cells was in dispersed KD it was in it cells, expressed whereas plasma membrane, was II the alone MDCK to Rap1GAP2 control When colocalized the 2C). above, mainly with (Fig. described were Consistent membrane Rap1GAP2 assays cells. and co-immunoprecipitation II (mRFP)- Slp2-a the MDCK protein of control fluorescent results C- in red Slp2-a co-expressed protein)-tagged monomeric fluorescent tagged we yellow and C2B of Rap1GAP2, variant Rap1GAP2 the subcellular and (a the Venus to Slp2-a investigate terminal To mapped of 5). the lane was localization and bottom, Slp2-a 2B, 2A), (Fig. (Fig. in domain Rap1GAP2 both site with expected, interacted Rap1GAP2-binding As Slp2-a Rap1GAP2. possible and and a Slp1 for Slp2-a test between to interaction assays co-immunoprecipitation performed VKT i o Fg B.Smlry F-agdSlp2-a- GFP-tagged Similarly, 3B). (Fig. not did EVTKTT) D D VKTmtn,itrce ihteCBdmi of domain C2B the with interacted mutant, EVTKTT VKT i.3,lf) n hnpromdco- performed then and left), 3A, Fig. EVTKTT; le ta. 09,w netgtdwhether investigated we 2009), al., et ¨ller le ta. 09,the 2009), al., et ¨ller l2a(i.4) et eepesdVnstge tzin size. Btsz cell on Venus-tagged effect its expressed evaluated as and we way cells Slp2-a-KD same Next, and as the control and, 4B). in (Fig. Rap1GAP2, Rap1GAP2, and with Slp2-a domains that interacted Btsz C2 Btsz of form tandem expected, in short the a Slp2-a between only interaction of contains possible performed a function were for in test assays the to Co-immunoprecipitation size for cells. Btsz cell II compensate Because MDCK and could 2006). growth Btsz al., affect whether et to an Pilot shown lacks Drosophila been 2001; but have al., domains mutations C2 et tandem (Fukuda C-terminal SHD contains that Slp elsz a aebe osre cosseisfrom species across conserved been of have control the in may Slp Drosophila of size suggested restored results function the These recruiting cell cells 4C). affect Rap1GAP2 (Fig. not Slp2-a-KD the cells did II that the MDCK Btsz control in however, of Intriguingly, size Btsz size. membrane of cell plasma the normal expression to localized that was Btsz and that noteworthy is It fRpGP otepam ebaeadissubsequent its and size. membrane cell of plasma control the for to the required localized is to targeting activation clearly that was Rap1GAP2 indicate results it These of though 4A). (Fig. even the membrane no size, on plasma had effect cell construct mutant its on the expected, evaluated GAP effect As and impair cells. Slp2-a-KD 2005), completely of al., to size we et shown size, (Schultess been activity has cell mutation of in the control Rap1GAP2-N357A-CAAX, which the construct, additional in of an activity involved generated GAP the the actually to whether of is determine cells To Rap1GAP2 expression the 4A). (Fig. restored Rap1GAP2, size cells normal wild-type Slp2-a-KD in Unlike Rap1GAP2-CAAX 2013). Fukuda, ecnaeo hi muti h oto el a 217 a was as cells expressed control cells the Slp2-a-KD in amount the ( their in of Rap2 percentage and of and amount their Materials The (i.e. Rap1 cells. activities Slp2-a-KD the active the Rap2 in in and increased were Rap1 described levels) both GTP GTP- that as of found level cells and and the Methods Rap1 Slp2-a-KD measured both we in inactivate 2011), Rap1/2 Bos, to and ability (Gloerich the Rap2 has cells Rap1GAP Slp2-a-KD in Because activated is signaling Rap hwdta hi elsz a nrae diiey(i.5B), (Fig. additively mutants increased active had the size of cell each their expressing that showed cells, cells II MDCK with control comparison in and expressed Rap1A simultaneously Fig. active were when Rap2A material Intriguingly, did. supplementary but mutant spreading, negative cell 5B; induced neither mutants (Fig. active both cells expected, in As S2B). them II expressed and MDCK Rap2A and control and (G12V Rap1A mutants of respectively) negative S17N, and active We mechanisms. constitutively different by generated Rap1 spreading activated cell renal whether induce the investigated Rap2 then and in and amount 160 S2E), its Fig. was material of percentage cells a control the as in expressed Rac1 active cells of amount Slp2-a-KD is [the Rac1 cells Slp2-a-KD that demonstrated intestinal in activated We in also Rap1. (Gloerich of Rac1 size independently of 2012) cell target al., upstream regulate et an to activating by reported cells been epithelial whereas has 2004), Rap2 al., et active (Arthur (GEFs) guanine factors Rac1 exchange localizing nucleotide in by spreading size cell cell promoting regulate by to cells HeLa reported different been by has size Rap1 cell active regulate mechanisms: to Rac1 reported modulate been Rap2 have and they Rap1 signaling, both Although S2A). Fig. material n 5 Drosophila )ad326 and 3) ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal ohumans. to Srn n ui,20) eas investigated also we 2003), Rubin, and (Serano 6 tzi h nykonhmlgeo mammalian of homologue known only the is Btsz 1 ( 51% n 5 6 ) epciey(i.5;supplementary 5A; (Fig. respectively 3), 0 ( 20% n 5 ) Fg A supplementary 5A; (Fig. 5)] 6 16% 559

Journal of Cell Science rnfce ihpeu-1RpGP gen n utrdfr4 or.Teclswr epae n3-mgasbtoe ihs utrdfr4hour along 4 intensity 10 for Fluorescence bars: cultured (blue). Scale dishes, DAPI bottom. glass-bottomed with the 35-mm at stained on shown were re-plated is Nuclei were panels) (red). cells (upper phalloidin The Texas-Red-conjugated lines hours. with white 48 stained for and cultured permeabilized and fixed, (green) pVenus-N1-Rap1GAP2 with transfected EERHARTICLE RESEARCH 560 t on shown is panels) (left lines white broken the along w intensity glass-botto cells Fluorescence 59.2 35-mm II (blue). was on MDCK DAPI re-plated Slp2-a Rap1GAP2. with were with and stained cells Rap1GAP2 Slp2-a were The were of of Nuclei hours. associations rate Colocalization fixed. 48 their (C) colocalization are then for chains. and domains The and cultured C2B light cells, hours and and and COS-7 4 C2A (red) heavy for in the pmRFP-C1-Slp2-a G domain; dishes co-expressed and immunoglobulin homology were (green) indicate Slp pVenus-N1-Rap1GAP2 Rap1GAP2 SHD, asterisks with right. The FLAG-tagged co-immunoprecipita the co-transfected A. and the on in in shown mutants Rap1GAP2-b described used is truncated the as Slp2-a mutant of Slp2-a of each analyzed Mapping mutants of T7-tagged (B) truncated activity (Bottom) left. binding The shown. the Rap1GAP2 Slp2-a. on The also of shown lines. organization are solid domain anti-FLA by kilodaltons) the with represented (in assays of are co-immunoprecipitation representation markers by Schematic mass (Top) analyzed molecular Slp2-a. were the in associations of site their positions and The Rap1GAP2. cells, beads. COS-7 of agarose in localization antibody-conjugated co-expressed membrane were plasma Rap1GAP2 the FLAG-tagged for and required a) is Slp2-a 2. Fig. 6 %( 4% n m 5 m. 5.()Pam ebaelclzto fRpGP.CnrladSp--Dclswere cells Slp2-a-KD and Control Rap1GAP2. of localization membrane Plasma (D) 25). A neato ewe l12aadRpGP.T-agdSp o Slp2- (or Slp1 T7-tagged Rap1GAP2. and Slp1/2-a between Interaction (A) ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal h broken the inassays tion eright. he inding tag G med s, ere

Journal of Cell Science hra l ftengtv uat fRpA a2 and Rap2A Rap1A, of spreading, mutants cell negative induced the cells negative using II of its of MDCK all not by control but whereas the Rac1, size of in S2C). mutant cell mutant, and Fig. active the material (G12V in of supplementary renal Rac1 Expression 5C; of Rac1 of Fig. mutants respectively; negative size of T17N, and the active involvement constitutively regulate investigated possible next We Rap2 pathways. the different and through cells Rap1 epithelial that suggesting ARTICLE RESEARCH 02,oeo h R aiypoen htbn -ci n are and F-actin bind that proteins family al., ERM et (Gloerich the ezrin of activate one to 2012), reported been has cells Rap2 Slp2-a-KD Because in by activated is 5D; size Ezrin (Fig. cell cells epithelial Slp2-a– signaling. Slp2-a-KD renal the Rap that regulates modulating the indicating interaction of S2D), Rap1GAP2 Fig. size material the supplementary decreased Rac1 elsraig ots u yohss egnrtda generated ezrin we of mutant truncated hypothesis, C-terminal of a form our and phosphomimetic T567D), a (ezrin test ezrin, ezrin of mutant To active constitutively induce 2006), spreading. Rho-GDI Hinds, and and ezrin Yang cell activated 2010; that GDP hypothesized al., Rho further et and the we (Hsu cells Rac1 sequester activity with and Rac1 II interacts bind inhibits which MDCK to (Rho-GDI), activation ability inhibitor control initiate its dissociation to through the known is Rac1 with ezrin of activated comparison Because 6A). Slp2- in (Fig. subconfluent the cells a in also showed phosphorylation a-KD analysis ezrin cells immunoblot in the Slp2-a-KD increase of clear subconfluent results The in ezrin. the activates activity that hypothesized we Rap2 in 2012), cells al., increased Slp2-a-KD al., et confluent (Yasuda in et study phosphorylation shown previous increased (Bretscher our was because form) spreading (active and ezrin 2011), cell of al., of et regulation Ross 2002; the in involved ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal xeiet) ausaemasads.e.m. and means are Values experiments). * left. ( the test on Dunnett’s images the in shown bars: Scale cytoplasm. 10 the in present mainly was GFP-Slp2-a-C2B plasma whereas the membrane, to that localized Note was above. GFP-Slp2-a-C2A described as sizes measured cell were their and (green), C1-Slp2-a-C2B with pEGFP- transfected or cell were pEGFP-C1-Slp2-a-C2A on cells pEGFP-C1, Slp2-a II of MDCK domain (Left) C2B size. the of Effect (C) ( mgso h et * the left. in the shown on sizes images cell the 10 of bars: Quantification Scale (blue). DAPI stained were with Nuclei cytoplasm. the in dispersed td.TeSp--2 idn ciiyo each of activity binding this Slp2-a-C2B in The used study. mutants point its and and mutants Rap1GAP2 deletion of domains functional of the representation Schematic (Left) Rap1GAP2. cell spreading. to Slp2-a– leads the interaction of Rap1GAP2 disruption Functional 3. Fig. oiiefratn oeta ohteRGDN the RGDN- both and that construct Note stained actin. that for was area positive type the cell measuring each by of determined size The (red). Texas-Red-conjugatedphalloidin with stained hours, then 24 and 35-mm for on cultured re-plated dishes, were glass-bottomed cells The hours. 48 RGDN- with pVenus-C1- transfected or were pVenus-C1-RGDN cell cells pVenus-C1, on II Rap1GAP2 MDCK of (Left) RGDN size. left. the the of on Effect shown (B) are mass kilodaltons) molecular (in the markers of positions The agarose beads. antibody-conjugated tag anti-T7 with assays were co-immunoprecipitation associations by their analyzed and cells, COS-7 in RGDN- or RGDN FLAG-tagged and Slp2-a-C2B (Right) T7-tagged activity. GAP N357A impairs an which represents substitution, domain GAP the within A the and sequence, the membrane-targeting after represents plasma CAAX Rap1GAP2 The of right. C-terminus the the on shown is mutant n 5 m 0fo he needn experiments). independent three from 50 .(ih)Qatfcto ftecl sizes cell the of Quantification (Right) m. D VKT(re) n utrdfor cultured and (green), EVTKTT A h l2abnigst in site Slp2-a-binding The (A) n 5 D 5fo he independent three from 45 VKTwr co-expressed were EVTKTT P D , VKTcntutwere construct EVTKTT .0,Dnetstest Dunnett’s 0.001, m P .(Right) m. , 0.001, 561

Journal of Cell Science EERHARTICLE RESEARCH 562 and/or (PP1 phosphatases protein facilitates activating by in phosphorylation Rap2A which ezrin, spreading. activated of cell to phosphorylation that leads through turn Rac1 indicate of these activation together, strongly effect Taken no spreading. results had ezrin cell As S17N, by Rap2A ezrin-T567D-mediated induced 6C). mutant, on spreading (Fig. Rap2A cell the cells the whereas inhibited T567D, of II it effect i.e. the MDCK antagonized T567D, T17N) ezrin control (Rac1 mutant in Rac1 of the mutant expected, T567D active an with ezrin together Rac1 ezrin, further or Rap2A negative to either constitutively attempt of a mutant an co-expressed we In control hypothesis, induces activation. our of ezrin support Rac1 activated size through that the suggesting spreading increased 6B), mutants, cell (Fig. Both ezrin, cells cells. (WT) II II wild-type MDCK MDCK the control in not size but cell evaluated on and effect ability, their Rho-GDI-binding possesses that (ezrin-N) nrae eaielvl aebe hw oihbtezrin inhibit to shown been have levels ceramide Increased -Dcls(i.6,) ilsa nciae zi n reduced cells. and Slp2-a-KD ezrin the of inactivated Slp2- size Miglustat of We the 6D,E). size disease) and (Fig. 1991). C activity cells ezrin Niemannn-Pick the a-KD al., on of inhibitor, 2012), Igdoura, treatment et synthase and the GlcCer (Venier Shayman for a (drug of 2010; effect miglustat inhibitor al., the investigated synthase et therefore to (GlcCer) reported (Natoli 1991). been glucosylceramide al., al., has treatment by et cells et epithelial (Shayman increased Zeidan renal arrest be 2012; of growth al., level cell ceramide et induce The Canals to 2010; and al., 2008) et (Canals PP2A) xrsinwsrltvl o Fg A o ih,oe circles), open right, top 7A, GFP–Slp2- (Fig. of low level relatively the When was 7A). expression (Fig. a cells Slp2-a II GFP-tagged MDCK control overexpressed in we plasma size, the cell from affects Slp2-a membrane of mislocalization whether investigate II To MDCK increased induces size cytoplasm cell the in Slp2-a of Mislocalization ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal ausaemasads.e.m. and means are Values experiments). independent three from ev hi.Tepstoso h molecular the of positions The chain. G heavy immunoglobulin indicate asterisks The beads. agarose antibody-conjugated tag anti-FLAG with assays co- immunoprecipitation by analyzed their were and associations cells, COS-7 in co- expressed were Rap1GAP2 FLAG-tagged Slp2-a) and (or Btsz T7-tagged Rap1GAP2. Btsz and between Interaction (B) experiments). 10 bars: the Scale in cells. membrane Slp2-a-KD plasma the were to 3A) localized Fig. also (see N357A-CAAX Rap1GAP2- and Rap1GAP2-CAAX both the Rap1GAP2, to of contrast localization In cytoplasmic 1. Fig. the of in legend described as measured was size their and (green), Rap1GAP2-N357A-CAAX pEGFP-C1- or Rap1GAP2-CAAX pEGFP-C1- pEGFP-C1, pVenus-N1-Rap1GAP2, with transfected Slp2-a-KD were (Left) cells cells. Slp2-a-KD of the size on membrane plasma the to Rap1GAP2 of control size. the cell for required of is localization Rap1GAP2 membrane Plasma 4. Fig. ie hw nteiae nteleft. the on images * the in shown sizes bars: Scale 10 cells. both Slp2-a-KD in and membrane control plasma the to localized was Btsz (green). or pVenus-C1-Btsz pVenus-C1 with size. cells transfected cell Slp2-a-KD were on and Btsz cells the Control of (Left) Effect (C) on left. shown the are kilodaltons) (in markers mass rmrts ( test * Kramer left. the the in on shown images sizes cell the of Quantification P , m .0,Suetsunpaired Student’s 0.001, .(ih)Qatfcto ftecell the of Quantification (Right) m. A feto ocbetreigof targeting forcible of Effect (A) n 5 0fo he independent three from 40 P , .0,Tukey– 0.001, t ts ( -test m .(Right) m. n 5 45

Journal of Cell Science EERHARTICLE RESEARCH fRpGP.Js sepce,we a1A2was Rap1GAP2 2–4), when (Figs expected, size as cell of Just localization the control Rap1GAP2. affect to was expected the of Rap1GAP2 was Slp2-a and in of panels). mislocalization Slp2-a spreading two involved right between cell bottom, actually interaction induced and circles; the and closed was Because cytoplasm left, top GFP–Slp2-a the 7A, overexpressed (Fig. in right, top the dispersed 7A, however, often (Fig. increased circles), GFP- As control level images). closed lower the expression the of in GFP–Slp2-a that panels two as the left same 7A, the GFP–Slp2-a-expressing (Fig. cells almost the expressing of was size cells the II and 7A, MDCK circles) (Fig. open membrane plasma left, the top to localized predominantly was it yols sarsl fSp- vrxrsinmyla to lead may size. overexpression cell Slp2-a of membrane control of the the proper in in result for plasma signaling Rap1GAP2 abnormal a required and as Slp2-a above, is of cytoplasm red Rap1GAP2 mislocalization noted and graphs, signaling, cell Slp2-a left as that of lower cells localization Because, 7B, II (Fig. plasma MDCK the alone triangles). in to Rap1GAP2 opposed Rap1GAP2 as expressed Slp2-a of circles), of often localization level closed that high was graphs, membrane a to left expressed Rap1GAP2 that 7B, similar cells of (Fig. II were MDCK localization 7B), in (Fig. Rap1GAP2 cytoplasmic observed cells of Slp2-a: II patterns of MDCK control localization in the Slp2-a with co-expressed ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal experiments). ( test nteiae ntelf.* left. the on shown images sizes the in cell the of Quantification 10 (Right) bars: Scale (green). Rac1-T17N pEGFP-C1- or pVenus-C1-Rap2A-S17N with pVenus-C1-Rap1A-S17N, transfected pEGFP-C1, were cells. cells Slp2-a-KD Slp2-a-KD of (Left) size Rap the of on inhibition signaling of Effect (D) experiments). loecn rtisue eeaesonin shown are here the used of proteins levels fluorescent expression of The legend the 1. in Fig. described as measured size was their and (red), pmStr-C1-Rap2A-G12V and (green) pVenus-C1-Rap1A-G12V with co-transfected and (green), Rap2A-S17N pVenus-C1- or pVenus-C1-Rap2A-G12V pVenus-C1-Rap1A-S17N, Rap1A-G12V, pVenus-C1- were pVenus-C1, cells with II transfected MDCK manipulating (Left) by signaling. size Rap cell of left. Modulation the (B) on (in shown markers are mass kilodaltons) molecular The the cells. of Slp2-a-KD positions the in Rac1 activated and were the Rap2 for Rap1, used assays. were pulldown that proteins the fusion show GST panels proteins, bottom G The small respectively. total panels and middle active and show an top as The used control. was internal GAPDH S2A. Fig. supplementary material in shown control are negative experiments of results The Methods. Materials and the in described as and immunoblotting assays were pulldown Rac1 by and analyzed Rap2 Slp2-a-KD Rap1, in Active signaling cells. Rap cells. of epithelial Activation renal (A) in size the cell of underlying control signaling Rap 5. Fig. unt’ et( test Dunnett’s 10 Scale bars: S2B–D. Fig. material supplementary hw nteiae ntelf.* left. the on images the in shown 10 bars: Scale (green). pEGFP-C1-Rac1-T17N or transfected pEGFP-C1-Rac1-G12V were pEGFP-C1, cells with II manipulating MDCK by (Left) size Rac1. cell of Modulation (C) experiments). independent three left. the on images * the in shown sizes P , m .0,TkyKae et( test Tukey–Kramer 0.001, .(ih)Qatfcto ftecl sizes cell the of Quantification (Right) m. n 5 0fo he independent three from 50 m .(ih)Qatfcto ftecell the of Quantification (Right) m. n 5 0fo he independent three from 70 P , .0 Dunnett’s 0.001 n 5 P 6from 46 , m 0.001, m. 563

Journal of Cell Science -Dclswr rae ihmgutt(10 miglustat with treated were cells a-KD EERHARTICLE RESEARCH PPta scaatrzdb utcsi ypatckidneys, dysplastic 564 multicystic by human of characterized spontaneous type is A (adolescent) late-onset al., that 2004). the NPHP Wilson, et of 2009; (pcy) (Hildebrandt model Harris, mouse kidney and the Torres a cell- 2009; in of renal and possibility disorders signaling the mouse Slp2-a-mediated spreading-related investigated of in we dysfunction in experiments between expressed renal 2012), al., link of (a et is set (Yasuda cells rows) final and two the II top 1) 8B, MDCK Fig. (Fig. tubules of line; proximal size cell the epithelial regulates mice Slp2-a cyst pcy renal of Because the kidney in the activation of ezrin epithelium and expression Slp2-a Increased (10 miglustat with treated were cells Slp2-a-KD miglustat. to response in cells Slp2-a-KD of size Decreased (E) Quantification * left. (Right) the pmStr-N1). on with images together the pEGFP-C1 in (or shown sizes 10 pmStr-N1-Ezrin-T567D cell bars: with together Scale pEGFP-C1-Rac1-T17N 1. o or Fig. Modulation pVenus-C1-Rap2A-S17N, (B) of and left. legend pEGFP-N1-Ezrin-N, ( the or the on test pVenus-N1-Ezrin-T567D, shown pVenus-N1-Ezrin-WT, in Dunnett’s are pVenus-C1, described kilodaltons) with (in as transfected markers were measured mass cells. cells was molecular epithelial II the renal MDCK of in (Left) positions size ezrin. The manipulating cell indicated. antibodies of the control with the immunoblotting underlying by signaling Ezrin 6. Fig. h 5m ls-otmddse n utrdwt ilsa (10 miglustat with cultured and dishes glass-bottomed 35-mm the hi ie eemaue rgt.* (right). measured were sizes their n 5 0fo he needn xeiet) C ciaino zi yRpsgaig Lf)MC Iclswr rnfce ihpEGFP-C1, with transfected were cells II MDCK (Left) signaling. Rap by ezrin of Activation (C) experiments). independent three from 30 P , .0,Dnetsts ( test Dunnett’s 0.001, m P Mor50 , .0 unt’ et( test Dunnett’s 0.001 m )fr2 or n avse.Cl yae eeaaye yimnbotn ihteatbde indicated. antibodies the with immunoblotting by analyzed were lysates Cell harvested. and hours 24 for M) n 5 0fo he needn xeiet) ausaemasads.e.m. and means are Values experiments). independent three from 70 m n Mor50 5 m .(ih)Qatfcto ftecl ie hw nteiae ntelf.* left. the on images the in shown sizes cell the of Quantification (Right) m. 0fo he needn xeiet) D niiino zi ciiyb ilsa.Slp2- miglustat. by activity ezrin of Inhibition (D) experiments). independent three from 30 m )fr2 or.Teclswr tie ihTxsRdcnuae hlodnand phalloidin Texas-Red-conjugated with stained were cells The hours. 24 for M) eeso te l aiypoen eent reverse A not. were proteins expression expression Slp2-a the family that that showed Slp but analysis mice, mice (RT)-PCR other pcy pcy transcriptase clearly the of was and of level WT kidneys expression is levels Slp2-a the of Slp2-a the in kidneys that higher where found the we tubules, in 8C), the compared (Fig. level then proximal we expression When renal mice. Slp2-a WT the the in panel) expressed to cyst bottom normally the renal in addition 8A). the arrowheads (Fig. in 8B, in (Fig. expressed kidneys al., mice be large pcy et to of very epithelium found (Bergmann have was 2003), defects Slp2-a al., Intriguingly, heart et congenital Olbrich 2008; and inversus situs A ciaino zi nSp--Dcls h ellstswr analyzed were lysates cell The cells. Slp2-a-KD in ezrin of Activation (A) ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal m Mor50 m )fr4 or n hnr-ltdon re-plated then and hours 48 for M) P elsz by size cell f , 0.001, hi size their fthe of

Journal of Cell Science EERHARTICLE RESEARCH o ytepnini h iny fpymc Fg E.As 8E). (Fig. mice pcy of responsible kidneys signaling the cell in the expansion for highly cyst investigate searched is To for causes we epithelium. mice it cyst possibility, pcy renal 7A), in this their Fig. Slp2-a in of signaling 1C; amount Fig. abnormal increased the spreading; that of overexpression cell possible control and the knockdown induce both for (i.e. required Slp2-a size is cell Slp2-a II MDCK of 8D). of amount (Fig. correct unaltered but was the level, Rap1GAP2 Because expression and mRNA Rap1GAP1 the Slp1, at of increased that also was mice pcy in oe w aesi h etbos.Itiunl,w on that found we Intriguingly, blots). left 8E, the (Fig. in 2009) Harris, panels and two of Torres cAMP- kidneys 2003; lower the al., the et mice in (Gattone vasopressin and mice circulating pcy pcy blots), of level of left activated increased was the the pathway kidneys epidermal by (PKA)–CREB in A the kinase panels of protein in three dependent expression upper (EGFR) was 8E, increased receptor (Fig. pathway the factor (MAPK) of growth kinase because protein activated 2006), Harris, mitogen-activated and Torres 2006; the al., et (Omori reported previously ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal wti 1.5 membrane (within plasma the near Slp2-a Scale 10 (blue). bars: DAPI with stained were Nuclei (red). phalloidin conjugated Texas-Red- with and stained hours, then 24 for cultured dishes, glass-bottomed were 35-mm cells on The re-plated hours. 48 for cultured and (green), or pEGFP-C1-Slp2-a pEGFP-C1 with transfected cells II were MDCK (Bottom) cells. II in MDCK Slp2-a of Overexpression (A) spreading. cell to cytoplasm leads the in Rap1GAP2 Slp2-a and of Mislocalization 7. Fig. DKI el;oe ice and circles open cells; II MDCK GFP-expressing control Red triangles, size. cell against plotted was intensity fluorescence (Top Total size. right) cell against plotted ratios were the and intensity, fluorescence total to membrane plasma the near of intensity ratio fluorescence the as quantified membrane were plasma the near signals fluorescence The software. ImageJ using quantified was membrane) ebaeadddntafc elsize. cell affect not did plasma and the membrane at present mainly it was triangles), (red cells II MDCK in control expressed was when alone However, Rap1GAP2 cells. II MDCK in patterns localization similar exhibited Rap1GAP2 and Slp2-a (Left) above. described as were determined sizes cell and locations their and (red), pmStr-N1-Rap1GAP2 and with (green) transfected pEGFP-C1-Slp2-a were cells II MDCK (Right) Slp2-a. with Rap1GAP2 was , GFP of localization membrane than plasma less (the and respectively 30% 30%, than more was a Slp2- proportion plasma-membrane-localized the of which in cells, II MDCK Slp2-a-overexpressing circles, closed 0) B oepeso of Co-expression (B) 30%). m .(o et oaiainof Localization left) (Top m. m rmteplasma the from m 565

Journal of Cell Science EERHARTICLE RESEARCH nioy(e;G ntekdeso Tadpymc.Pern(-R)wslclzdt h rs odr(pclmmrn)o h pteilclso h re 50 566 the bars: p of Scale the cells respectively. in epithelial epithelium membrane, the cyst basolateral renal of the the membrane) of to (apical membrane localized border basolateral ezrin the brush and to the localized cyst to a also the localized was and indicate P-ezrin was signals. (green) EGFR–MAPK, (middle). arrowheads (P-ERM) nonspecific antibody the mice and P-ezrin indicates of pcy anti-Slp2-a asterisks mice. and Activity blots with The (top) pcy (E) right-hand and WT left. (bottom). and the F), the WT the in both (red; on of Rap in asterisk shown antibody Slp2-a, kidneys tubules is The anti-ezrin Slp1, the proximal (kb) mice. of and in with markers pcy analysis (green) G) immunoblotting weight RT-PCR and by (red; antibody molecular WT (D) analyzed the antibody anti-P-ERM left. of were of with mice the kidneys size staining pcy on The the bars: and Immunohistochemical shown mice. WT in (F,G) Scale are pcy of pathways mice. kidneys and kilodaltons) WT ezrin–PI3K/AKT of pcy (in of lysates and and markers kidneys The WT cAMP–PKA mass the mice. of molecular in pcy kidneys the mRNA and WT Rap1GAP2 the of of and positions in kidneys The (green) the mice. indicated. in antibody pcy members antibodies anti-claudin-2 of family the and Slp kidneys the (red) the of antibody in Expression anti-Slp2-a activity (C) with ezrin staining and Immunohistochemical expression (B) Slp2-a Increased 8. Fig. A iny fW n c ie cl a:1cm. 1 bar: Scale mice. pcy and WT of Kidneys (A) ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal m m. nti-P-ERM ymice cy 50 1GAP1 m nal m.

Journal of Cell Science i.SB,idctn htteatvt fRp n a2is Rap2 Rap1 and/or and by signaling Rac membrane Rap1 signaling, plasma Rap of the of activation to activity material Because proximity GAPs. the supplementary close in that 7; in spreading Fig. regulated indicating cell 1C; S1B), by (Fig. induced Fig. cytoplasm) cells either Slp2- the epithelial of in Slp2-a renal mislocalization Rap1GAP2 (i.e. of and overexpression a plasma by the Dysregulation to or them in 2C,D). knockdown anchors Slp2-a expressed (Fig. it are that that Fig. which and membrane 8D), of material found (Fig. both kidneys 2A), (supplementary We mouse (Fig. Rap1GAP2 Rap1GAP1 Slp2-a. and both S3A) and link with guanine functional signaling and a interacts specific showing (Gloerich in Rap by succeeded GAPs have respectively between controlled and we and is (GEFs) activation, 2011), Bos, factors signaling ezrin exchange Rap through nucleotide 6). and 5, (Figs activation Rac1 from species across Rap conserved Slp-mediated been membrane have (i.e. Drosophila Slp1 to function plasma 3), appears 2, recruiting the signaling) not (Figs Rap1GAP2 Slp2-a to of the domain but C2B Rap1GAP2 and the with recruiting interaction 1; Slp2-a, its (Figs by through epithelial Rab27 size S1). renal controls of cell S1C,G,H), Fig. Fig. independent material renal is (supplementary of material control which the in size, supplementary Slp2-a cell of function study epithelial present novel the a Rab27 In discovered the determined. we than been never other had functions function additional 2006), effector have (Fukuda, they SHD whether N-terminal but the the through to docking of membrane their movement plasma and/or the organelles regulate and to vesicles reported Rab27-bearing been have members family zi n l2ai h iny fW n c ie(i.8F,G). (Fig. mice pcy mice. and WT pcy of activated of kidneys of the localization of kidneys in the Slp2-a kidneys compare the and the to ezrin proceeded in in we increased signaling mice, was pcy cell phosphorylation involved result of is ezrin a signaling Because control ezrin as the that activated suggesting was in blots), 1999) right al., 8E, (PI3K)– et (Fig. 3-kinase (Gautreau of ezrin–phosphoinositide pathway kidneys the the AKT that in and increased mice, clearly pcy was ezrin of phosphorylation ARTICLE RESEARCH ntepam ebae(aze l,20;Fkd,20;Kuroda 2002; Fukuda, Ga C2 2001; 2004; the al., Fukuda, et and whereas (Catz phospholipids membrane 2002), with plasma al., interact the in domain, et C2A (Kuroda C- the Rab27 especially and domains, of SHD with form N-terminal interacts active specifically an SHD an have The domains. members C2 Kuroda all tandem 2001; terminal and Mikoshiba, 2002), and and al., Fukuda humans 2001; et in al., (Slp1–5) et members (Fukuda five mice of consists family Slp The DISCUSSION excessive this to consequently and leads 2012), cells, spreading. Slp2-a epithelial al., cell cyst of renal to et in excess ezrin Yasuda an of activation 2003; that also basolateral that al., suggested the was shown finding et to Slp2-a previously mislocalized (Pujuguet had was where membrane ezrin others panels), activated and 8F,G, excessively (Fig. we bottom the epithelium Because to the cyst expressed. localized mice, renal in be pcy the to arrowheads the found of In also was membrane 2012). was Slp2-a basolateral where ezrin al., 2013), activated et tubules al., however, (apical (Yasuda proximal et border renal expressed Hatano brush the 2002; abundantly of al., the cells et to epithelial (Bretscher pcy localized and the WT of and the both membrane) activated in ezrin was the of mice most that showed results The a1adRp oriaeyrglt elsraigthrough spreading cell regulate coordinately Rap2 and Rap1 ohmn Fg 4B,C). (Fig. humans to ´ vzSnitbne l,21) l fteSlp the of All 2012). al., et lvez-Santisteban ciae yCE hog K hshrlto.Actually, phosphorylation. PKA through (http://natural.salk.edu/ CREB was Database transcription by mRNA Slp2-a Gene CREB- that activated search possible Target a candidate highly is by signaling it a gene CREB CREB/), Slp2-a Because activated mouse the the the 8C). in in of (Fig. these identified increased was mice is site to binding pcy expression Slp2-a of addition that kidneys found In we 8E). pathways, Harris, (Fig. and Torres 2006; the Harris, 2009) and to excess (Torres contribute level PKD to increased and of reported an pathogenesis been pathway to have vasopressin, due circulating EGFR–MAPK pathway of cAMP–PKA the the 1:1000. the including of and of pathways, activation races 1:400 signaling all activation between cell in be excess several and of to of worldwide cause estimated Dysfunctions leading prevalence occurs a a It and with disease. disease renal cystic most end-stage the renal is inherited PKD PKD. common and signaling Rap–ezrin these Slp2-a-mediated of adhesion. activation cell–cell disrupting that by and possible spreading (supplementary highly cell cell–cell 2010) is cells facilitates signals the it Slp2-a-KD al., S4A), at the Fig. et in material E-cadherin observed Tsygankova of was 2003; junctions accumulation al., reduced al., et because et by (Lozano Pujuguet adhesion junctions cell–cell cell–cell 2008; impair the at to E-cadherin shown destabilizing been has signaling, ezrin niie ytgnssi os oeso K ( be by PKD by should increased is It cells mice) epithelial renal 2010). of pcy of level al., ceramide and the et that models effectively noted (jck (Natoli mechanisms NPHP inhibitor unknown mouse and largely mice) synthase in knockout GlcCer conditional GlcCer cystogenesis of blockade a that inhibited and with kidneys PKD increased accumulation mouse were and levels recently al. GM3 human et ganglioside in Natoli and resolved. GlcCer be that are to nocturia reported patients needs PKD that polyurea, in issue agents thirst, important such the an (e.g. with treatment effects of has polydipsia) al., side and et level aquaretic (Nielsen the localization channel cAMP membrane water 2002), apical the its intracellular of However, and expression al., 2012). aquaporin-2 in decreasing al., et of cell et effect (Gattone decrease undesirable Torres reduce patients the 2009; PKD they Harris, of because that and kidneys Torres cystic shown 2003; the have in level growth cAMP right). intracellular S5, Fig. kidneys material the (supplementary in mice junctions pcy cell–cell by of the spreading cell at ultimately causes E-cadherin Rac1 activated signaling destabilizing and the that RapGEF and signaling Rac1 cAMP-induced activate Rap–ezrin of Slp2-a-mediated that 1992; probable enhancement of is al., it impairment S4B), et Fig. both phenotype Rocco material similar (supplementary 2000; a mice observed pcy al., also 2000; in we et al., because et Okada and 2011), (Charron 2011; Wilson, patients destabilized al., NPHP be and et to PKD Marciano of known membrane kidneys are enhanced Because the E-cadherin, be in PKD. also e.g. of must proteins, pathogenesis signaling polarity the Rap to Fig. cell 2011), contribute material al., and induced (supplementary et cAMP-GEFs cells (Ross epithelial of S3D) (Li renal activation kidney in Because the spreading in cAMP-dependent 2008). expressed and al., be activates Rap2, to et (supplementary and shown also Rap1 been tandem have both in cAMP-GEFs activate cAMP that increased (cAMP-GEFs) S3C). cells RapGEFs the dose- Fig. of was S3B), material size Fig. cells cAMP- material the a II (supplementary and with forskolin MDCK stimulation reagent, to control elevating response in the increased in dependently expression Slp2-a eas rsn vdnefraln ewe maretof impairment between link a for evidence present also We rciia tde fsvrlaet htdces the decrease that agents several of studies Preclinical ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal Pkd1 567

Journal of Cell Science GCGTTCTTCAGAGA-3 GTAGCTTA-3 a2Ast 1-aetre ien.1 5 1: no. canine/human site against target (19-base siRNAs AATA-3 1 Three site Invitrogen. Rab27A by synthesized were ie3(agtst o :5 3: no. site (target 3 site euigtesz fSp--Dcls(i.6,) ae together, Taken 6D,E). (Fig. cells al., Slp2-a-KD of of et and size pathway (Shayman ezrin–PI3K/AKT the the reducing arrest miglustat inactivating inhibitor of growth synthase effect GlcCer cell the the has that induce found to also We and 1991). Zeidan 2012; 2008) al., et al., Canals 2010; et phosphatases al., et protein (Canals PP2A) activating and/or (PP1 by to shown phosphorylation been have ezrin levels ceramide inhibit increased Shayman that 2010; and 1991) al., al., et et (Natoli treatment inhibitor synthase GlcCer ARTICLE RESEARCH 568 RNAi stealth (Imai Three previously anti-Rab27A 2004). described al., as and Anti- et prepared were anti-Slp5 MA). antibodies (Concord, anti-Slp4-a, polyclonal rabbit Fitzgerald anti-Slp3-a, from anti-Slp2-a, was Anti-EGFR Slp1, CA). antibody (Carlsbad, polyclonal Invitrogen sheep from Anti-claudin-2 were goat MA). antibodies Alexa-Fluor-488/594-conjugated (Danvers, the secondary and Technology antibody antibody polyclonal Signaling polyclonal rabbit Cell rabbit monoclonal from antibody, (Thr308) were mouse anti-phospho-AKT polyclonal polyclonal and (Thr202/Tyr204) rabbit antibody, (ERK1/2) (ERK1/2) rabbit MAPK anti-phospho-p44/42 Transduction Anti-phospho-ezrin(Thr567)/radixin anti-p44/42 BD (anti-phospho-ERM) antibody, from KY). Abcam were (Thr564)/moesin(Thr558) (Lexington, from antibody anti-E- anti-CREB and monoclonal were Laboratories antibody and mouse monoclonal antibody mouse cadherin antibody, Anti-AKT polyclonal MA). polyclonal (Cambridge, monoclonal rabbit mouse rabbit Anti-ezrin (phospho-Ser133) Santa anti-CREB CA). from Cruz, was antibody, (Santa antibody Biotechnology polyclonal Cruz MO). rabbit Louis, anti-GST (St. Sigma-Aldrich HRP-conjugated from monoclonal tag mouse were HRP- tag agarose anti-FLAG anti-FLAG antibody-conjugated and HRP-conjugated antibody and Japan). monoclonal were mouse (Nagoya, (M2) antibody (Darmstadt, antibody, MBL polyclonal from rabbit polyclonal Novagen obtained protein fluorescent rabbit anti-red Biosciences conjugated antibody, anti-GFP monoclonal Merck mouse HRP-conjugated anti-GAPDH from HRP-conjugated Germany). purchased agarose antibody-conjugated monoclonal were mouse tag anti-T7 monoclonal and mouse antibody tag anti-T7 (HRP)-conjugated peroxidase Horseradish Materials METHODS a AND provide MATERIALS Rap–ezrin PKD. may of Slp2-a-mediated size treatment cell the the for epithelial target Hence, renal spreading novel cell controls PKD. renal Rap–ezrin that the in signaling increased with occurs associated to the be that due to and likely phenotype signaling, is spreading signaling, Slp2-a-mediated signaling cell of Rap–ezrin a level exhibit increased cells proper epithelial the In renal of Slp2-a. of size absence function cell effector epithelial the Rab27 renal known of their the control of that the independently discovered for and crucial are domain interactions Slp2-a-C2B the drugs for new interactors for patients. search PKD to treat necessary to be signaling ezrin will (or antagonize it that ezrin and PKD, Thus, therapeutic for promising right). target a be S5, should Fig. metabolism) glycosphingolipid material Hinds, and (supplementary Yang 2010; al., 2006) et (Hsu regulates Rho-GDI through an ezrin activity be because Rac1 should patients, 6C) PKD curing Fig. to 5D; approach (Fig. effective cells epithelial renal slowing of in means growth a cell as Rac1 inactivating that indicate findings these tre ien.2 5 2: no. site 5 (target 2 1: no. site (target nsmay ehv dniidRp Asa novel as GAPs Rap1 identified have we summary, In 9 ,st 1-aetre ien.2 5 2: no. site target (19-base 2 site ), 9 n ie3(9bs agtst o :5 3: no. site target (19-base 3 site and ) 9 9 -GAAGAAGCTTCAGGTAGCAGCTGAA-3 -GAGACTTTGGCAATCTGGAAGTAAA-3 9 9 eesnhszdb ipnGn Material Gene Nippon by synthesized were ) -CCCTGCCTCTCAGAATGCTGTTGAT-3 TM iNsaantcnn l2ast 1 site Slp2-a canine against siRNAs 9 -CCAGTGTACTTTACC- 9 -GGAGAGGTTTC- 9 -CGACA- 9 9 ,site ), ,and ), 9 ) iny eeprhsdfo LAJpnIc Tko aa)and Japan) (Tokyo, Inc. mouse Japan respectively. pcy Japan), CLEA and (Saga, from kidneys Co. KYUDO mouse purchased DBA/2JJcl were Japan). kidneys (Toyama, Ltd Co., ta. 99:5 (Fukuda previously 1999): described having oligonucleotides as al., of (underlined) pairs site et following enzyme the restriction with a PCR by Novagen) fern n 5 and ezrin, of CCTTCATCTGCTG-3 AGATCTGCCACCATGAAGGCCCAGGCCCGG-3 ATCCTCGCACAGTGGGGGCATCCCTGGC-3 GTCGACTTAGTGACCCGCACCAGAGC-3 CCATGCGCGAGTACAAAGTGGT-3 and Bio), (Clontech-Takara a or from cDNA human amplified was adult testis 2004). Btsz Marathon-Ready and Rap1GAP2, from described Fukuda, brain amplified and and mouse as were Rap1GAP1 and ezrin Kuroda Rap2A, essentially mouse Slp2-a Rap1A, 2002; and prepared human mouse Kuroda, encoding were of cDNAs and mutants cDNAs (Fukuda their harboring previously vectors and expression Rab27A pEF- tag and/or pmRFP-C1 T7 Japan), Shiga, Bio, (Clontech-Takara pEGFP-C1 construction Plasmid rget f 3,47 1 n 1 p epciey h rmr were amplify primers The to respectively. designed bp, Slp2-a, 511 were Slp1, and 715 pairs mouse 447, as 532, The primer of, performed 2012). fragments Rap1GAP2 were al., and analyses et Rap1GAP1, RT-PCR (Yasuda the previously and described preparation RNA analysis RT-PCR Total and preparation RNA Total containing DMEM in grown were cells al. et The Yasuda cells in 2012)]. 2 II 800 al., no. MDCK line et [cell Slp2-a-knockdown previously (Yasuda stable described and as established cells were II MDCK Control by interference lines RNA cell Slp2-a-knockdown of Establishment detect (to 10 hours with 48 treated also for or were then cells activity) 1 CREB Slp2-a-KD and or expression). detect hours Slp2-a (to nM 16 minutes 500 for 5 for DMEM nM, Industries) serum-free 100 in with cultured using treated were by cells cells COS-7 instructions. II manufacturer’s or MDCK the cells to II (100 according (Invitrogen) MDCK streptomycin 2000 into and Lipofectamine U/ml) transfected were (100 G Plasmids penicillin ml). containing serum, Japan) bovine Osaka, fetal Industries, 10% Eagle’s Chemical modified Pure Dulbecco’s Wako in cultured (DMEM; were medium cells COS-7 and treatment cells II drug MDCK and transfection culture, Cell 2011). al., T567D, et (Tamura ezrin previously described T17N, as Rac1 shRNAtargetedtocanineSlp1(21-basetargetsite:5 techniques PCR G12V, two-step Rac1 or RGDN- S17N, S17N, and Rap1A N357A, expression G12V, Rap2A Rap1GAP2 Rap1A tag encoding G12V, pEF-FLAG cDNAs the resulting 1999). Rap2A al., The into et ligated. subcloned (Fukuda were was vector fragments cDNA two appropriate ezrin the the full-length with then digested and was enzymes fragment restriction each cDNA, ezrin full-length the GTCGACCTATGGCAAATTGGCTCGAATGC-3 CCATGGCCTCGAACTCGTCAATGCG-3 AGCTGGGGCTCTCGCTGCTCGG-3 TGTTACATGCAG-3 GAAGCGCA-3 CATGCCCAAGCCAATCAACG-3 5 rnfce noMC Iclsb sn ioetmn RNAiMax Lipofectamine using by instructions. manufacturer’s cells the to II according MDCK (Invitrogen) into transfected hours. 24 for (Sigma-Aldrich) miglustat vector 2.0-U6 pSilencer-neo the using shRNA. expresses by which TX), 2004), Austin, (Ambion, Fukuda, and (Kuroda AGACCACAGTGAAGAA-3 a1A1 5 Rap1GAP1, 9 - GTCGACCTAGAGCAGCAGACATGATT-3 m /lG1 Ivtoe) tat iNsaantcnn l2awere Slp2-a canine against siRNAs Stealth (Invitrogen). G418 g/ml ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal 9 9 - - 9 GGATCCATGGAAGCCAAAGTGGCCGCT-3 AGATCTATGTTTGGCCGGAAGCGCAG-3 9 n 5 and - GGATCCATGCGTGAGTACAAGCTAGT-3 9 9 o a2,5 Rap2A, for o h -emnldmi fern 5 ezrin, of domain N-terminal the for Drosophila 9 -CTAACAGCCCAGCTGGGGCAT-3 9 a osrce sdsrbdpreviously described as constructed was ) D VKTwr rdcdb conventional by produced were EVTKTT 9 m n 5 and osoi Wk ueChemical Pure (Wako forskolin M DAlbay(ec Biosciences (Merck library cDNA 9 9 o GN 5 RGDN, for n 5 and 9 - GGATCCATGAGCGGCCG- 9 9 - GGTACCCGGGCCTGGG- o h -emnldomain C-terminal the for 9 9 - o a1A2 5 Rap1GAP2, for GTCGACCTATTGTA- 9 9 o a1,5 Rap1A, for 9 n 5 and o tz oobtain To Btsz. for 9 9 - AGATCTCCAC- n 5 and 9 - GTCGACCT- m Mor50 Drosophila 9 9 9 - -GCGCA- 9 GGTAC- 9 - n 5 and GGAT- n 5 and 9 9 9 - GG- and m m for M g/ 9 9 9 - - -

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K. Reedquist, and J. Rooij, de L., J. Bos, uua M. A. Fukuda, Wandinger-Ness, and R. Bacallao, S., Nakamura, J., A. Charron, etakMgm iaafrtcnclassac n ebr fteFukuda the of members and discussions. assistance valuable technical for for Laboratory Aizawa Megumi thank We Acknowledgements for tested were Data unpaired s.e.m. Student’s and using means by as differences expressed significant are statistically The data Invitrogen). The kit; analyses fluorescence Statistical labeling confocal IgG a rabbit (Zenon with kit examined 594 labeling microscope. were direct directly or a sections was using 488 antibody and immunostained 594 Slp2-a Slp2-a Fluor or the 488 for Alexa Fluor P-ERM, anti-ezrin stain Alexa and with double Slp2-a labeled antibody, To for anti-E-cadherin or antibody. anti-claudin-2 with claudin-2 anti-P-ERM stained antibody, and were antibody, samples anti-Slp2-a The previously 2012). antibody, described al., as et performed (Yasuda was staining Immunohistochemical analysis Immunohistochemical rtce,A,Ewrs .adFhn .G. R. Fehon, and K. Edwards, A., Bretscher, Bru M., Fliegauf, A. C., J. Bergmann, Cooper, and A. L. Quilliam, T., W. Arthur, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134056/-/DC1 online available material Supplementary material Supplementary M.F.]. to of Technology 24657125 and and Sports, 24370077 Culture, numbers of Education, [grant Promotion Scientific of Japan for the Ministry Grants-in-Aid for the and T.Y.]; Society from to Japan Research 242766 the number by [grant part KAKENHI in Science supported was work This Funding manuscript. M.F. the manuscript. wrote the and wrote project and the experiments supervised the performed and designed T.Y. contributions Author interests. competing no declare authors The interests Competing az .D,Jhsn .L n air .M. B. Babior, and L. J. Johnson, D., S. Catz, cieRc)adttllsts(oa a rttlRc)wr nlzdby analyzed antibody. were anti-Rac1 or Rac1) or Rap total antibody (active or anti-Rap Rap beads with (total the immunoblotting lysates three by total beads trapped and the proteins Rac1) buffer, washing active lysis After the Rac). with (for times GST–PAK1-PBD or Rap) (for 30 with coupled aas . ekn,R . od,P,Herna P., Roddy, W., R. Jenkins, D., Canals, unt’ eto h ue–rmrts sidctdi h eedof legend the in of indicated level as significant. probability test A Tukey–Kramer figure. the each or test Dunnett’s aas . od,P n ann .A. Y. Hannun, and P. Roddy, D., Canals, fnprcsi- ucincncueebyncltaiy Meckel-Gruber-like lethality, dysplasia. renal-hepatic-pancreatic embryonic Genet. and cause inversus, situs can syndrome, function nephrocystin-3 of 2 oano yattgi I. synaptotagmin of the with domain comparison C2A machine: phospholipid-binding calcium-dependent atypical dominant cells. autosomal disease in kidney trafficking polycystic polarized and cytoarchitecture Compromised e models. new Kra A., Kispert, I., Schmedding, B., Schermer, J., factors. exchange nucleotide guanine 167 Rac localizing by spreading JFC1. uloiebnigcpct n h Taeatvt ftePIP the of activity ATPase the and capacity nucleotide-binding phosphatase. myosin/ERM inln tteotrpam membrane. sphingolipid plasma probing outer phosphorylation: the A. ERM at Y. on signaling Hannun, 1-phosphate and sphingosine M. L. cortex. cell the at integrators 32485. eitscrmd-nue R rti ehshrlto:anovel (PIP 5-biphosphate a 4, dephosphorylation: phosphatidylinositol of protein independent ERM mechanism ceramide-induced mediates 111-122. , rc al cd c.USA Sci. Acad. Natl. Proc. 82 ora fCl cec 21)17 5–7 doi:10.1242/jcs.134056 557–570 127, (2014) Science Cell of Journal 959-970. , 20) h 2 oano yattgi-iepoen3(l3 san is (Slp3) 3 protein synaptotagmin-like of domain C2A The (2002). a.Rv o.Cl Biol. Cell Mol. Rev. Nat. m fGTaoea eaiecnrl GST–RalGDS control, negative a as alone GST of g .Bo.Chem. Biol. J. a.Rv o.Cl Biol. Cell Mol. Rev. Nat. he .O,Fak . lrc,H,Kirschner, H., Olbrich, V., Frank, O., N. chle, ¨ 21) ifrnilefcso eaieand ceramide of effects Differential (2010). .Cl Biol. Cell J. ice.J. Biochem. 98 11230-11235. , P 2 , 369-377. , .5wscniee statistically considered was 0.05 287 20) a1sgaln:ahrn to adhering signalling: Rap1 (2001). 149 21) rti hshts 1 phosphatase Protein (2012). 20) R rtisadmerlin: and proteins ERM (2002). ne-obco .J,Obeid, J., M. ´ndez-Corbacho, 366 10145-10155. , 20) hrceiaino the of Characterization (2001). .Bo.Chem. Biol. J. 111-124. , 681-687. , nln .e al. et B. ¨nzlin, 20) a1pooe cell promotes Rap1 (2004). 3 586-599. , 3 bnigprotein -binding 285 m .Hum. J. Am. 20) Loss (2008). .Cl Biol. Cell J. ,32476- (2000). 2 t and ) -test, 569 a

Journal of Cell Science aoi .A,Sih .A,Rgr,K . ag . oantk,S,Budman, S., Komarnitsky, B., Wang, A., K. Rogers, A., L. Smith, A., T. Natoli, uua . ags,C n iohb,K. Mikoshiba, and C. Saegusa, M., Fukuda, K. Mikoshiba, and E. Kanno, M., Fukuda, K. Mikoshiba, and M. Fukuda, S. T. Kuroda, and M. Fukuda, acao .K,Baea,P . e,C . pvk . atun .J., D. Eastburn, N., Me Spivak, Z., C. Lee, R., P. Brakeman, K., M. M. D. V. Braga, Marciano, and G. U. Knaus, K., Smolarczyk, M., A. M. Frasa, E., Lozano, uua M. Fukuda, ARTICLE RESEARCH 570 E. V. Torres, and C. P. Harris, X., Wang, 2nd, H., V. Gattone, Ga i . oig,I .M,Za,J,Pie .S,d er .adDe,P .T. M. P. Deen, and E. Heer, de S., L. Price, J., Zhao, M., B. I. Konings, Y., Li, M. Fukuda, and H. M. Kobayashi, Fukuda, and H. Shimomura, T., Nashida, S., Yoshie, A., Imai, s,Y . i,W . o,Y . u .S,Su,C . hn .L,W,Y Y., Y. Wu, L., C. Chen, T., C. Shun, S., Y. Pu, T., Y. Hou, L., W. Lin, H., Y. M., Hsu, Arico, A., Santoro, T., Daniele, S., Booth, G., Bossi, E., Kanno, O., E. Holt, Otto, and M. Attanasio, F., Hildebrandt, K., Miyamoto, M., Matsubara, K., Mukaisho, H., Segawa, E., J., F. Fujii, R., Zwartkruis, T., Hatano, Koorman, J., M. Vliem, P., J. Klooster, ten M., Gloerich, L. J. Bos, M. and M. Arpin, Gloerich, and D. Louvard, P., Poullet, A., Gautreau, uoa .S,Fkd,M,Aia .adMksia K. Mikoshiba, and H. Ariga, M., Fukuda, S., T. Kuroda, M. Fukuda, and S. T. Kuroda, le-atsea,M,RdiuzFaiel,A . rat .M., D. Bryant, E., A. Rodriguez-Fraticelli, M., ´lvez-Santisteban, ´nasche . eek,A,Bknv .O,Dcosi .R,Hso,H tal. et H. Husson, R., W. Dackowski, O., N. Bukanov, A., Belenky, Y., aiyo -emnltp admC proteins. C2 tandem with C-terminal-type Commun. interacts of that protein family linker novel actin. and a Va/VIIa, domains-c), myosin Rab27, C2 lacking homologue abr,N,Mhau,N,Cut . ai,J,Fshr .e al. and cells et T secretion. cytotoxic A. granule in Fischer, cytotoxic Rab27a and with J., to associates Garin, participates Slp2a E. of M., isoform K. Court, identified N., newly Mostov, Mahlaoui, N., I., Lambert, Hofmann, 3rd, M., glomerulogenesis. G. and Beaudoin, F. L. M., Reichardt, small D. the by Bryant, adhesion E-cadherin of disruption the Rac. for GTPase required is PAK (2008). oto h omto fasnl pclmmrn oani pteilcells. epithelial in al. domain membrane et apical Biol. single K. Cell a Young, of formation Ban N., the control Spivak, T., A., Yasuda, Datta, S., Vergarajauregui, domain. homology Slp the Commun. of Res. identification Biophys. 3: Biochem. and 2 proteins synaptotagmin-like V, III, synaptotagmins of formation heterodimer X. and and homo- VI, for essential is motif Rab27 a of Trans. composed Soc. machinery docking novel 20) ea xrsino xhnepoendrcl ciae ycM (Epac) cAMP by 2. activated directly and protein 1 exchange of expression Renal (2008). cells. acinar parotid rat from Sci. release Cell amylase J. regulates Rab27B GTPase small carcinoma cell ARHGEF7. renal factor exchange of nucleotide Pathol. potential guanine Rac1 metastatic recruiting the through enhances complex molecular hn .Y,Ce,T .adJu .S. the T. Jou, from and secretion H. to T. Chen, contribute Y., and J. Chen, CTL of membrane synapse. plasma immunological M. the G. Griffiths, and to M. 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