ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 2601 doi:10.1242/jcs.154898

CORRECTION PI4KIIIa is required for cortical integrity and cell polarity during Drosophila oogenesis

Julie Tan, Karen Oh, Jason Burgess, David R. Hipfner and Julie A. Brill

There was an error published in J. Cell Sci. 127, 954-966.

The synonym for l(1)3Ah21 was incorrectly identified as zw2c21 on pages 955 and 963 of this article. The correct synonym is zw2123, not to be confused with the allele generated in this study, which is PI4KIIIaD123.

Accordingly, on page 955, the sentence ‘Like PI4KIIIaD123, males hemizygous for zw2c21 or other zw2 alleles die as first instar larvae (Shannon et al., 1972).’ should read ‘Like PI4KIIIaD123, males hemizygous for other zw2 alleles die as first instar larvae (Shannon et al., 1972).’

The authors apologise to the readers for any confusion that this error might have caused.

2601 lsammrn P)(amn ta. 2009). al., et (Hammond (PM) membrane plasma PI(4,5) om1.76 oot,O,MG04 Canada. 0A4, M5G ON, Toronto, 15.9716, Room 1,Aeu e isOet otel ubc 2 R,Canada. 1R7, H2W Quebec, Montreal, Ouest, Pins des Avenue 110, Ato o orsodne([email protected]) Canada. correspondence 3J7, for H3T *Author QC, Montreal, Montreal, of University Medicine, of eeis nvriyo oot,1Kn’ olg ice oot,O,MS1A8, M5S ON, Toronto, Circle, College King’s 1 Canada. Toronto, of University Genetics, ui Tan Julie PI4KIII ARTICLE RESEARCH ß 954 2013 December 2 Accepted 2013; February 8 Received 1 PtdIns4 of majority the that lipid suggested been and has proteins it the coat PtdIns4 despite of Interestingly, adaptors, 2012). importance al., clathrin recruitment et (D’Angelo and as proteins to transfer such binding its effectors of because of cell. Golgi the the in at trafficking phosphoinositides of majority PtdIns4 the generating phosphatidylinositol in (PtdIns4 P step of 4-phosphate phosphatidylinositol conversion on to named (PtdIns) present catalyze Phosphatidylinositol ring. groups are inositol (PI4Ks) the phosphate of 4-kinases 5-positions species of and 4- combination 3-, phosphoinositide the cellular the many to seven on according effects The powerful exert processes. they of percentage lipids, small membrane a only constitute phosphoinositides Although INTRODUCTION PI4 4-phosphate, Phosphatidylinositol 4-kinase, PI WORDS: KEY PtdIns(4,5) analyze we in Here, roles (PI4KIII melanogaster understood. their well yet not cells, Drosophila to are cultured proteins and development been yeast animal effector have in recruit extensively enzymes studied that pathways Phosphoinositide-generating gatekeepers signaling acting conserved membranes. organelle in processes, as molecules and cellular signaling potent myriad as regulate Phosphoinositides ABSTRACT ebaepeoye.Tu,PI4KIII Thus, phenotypes. membrane Drosophila ucinlydsic olo PtdIns4 of pool PtdIns(4,5) distinct for functionally requirement unique a reflect exocyst effects the These PI4KIII and Sec5. recruit Moesin to subunit failure crosslinker with cytoskeletal-membrane associated for integrity the mutant cortical cells in germ defects Female exhibit polarity. cell and trafficking eurdfrpouto fpam ebaePtdIns4 membrane plasma of production for required Drosophila rga nCl ilg,TeHsia o ikCide,PCL 8 a Street, Bay 686 PGCRL, Children, Sick for Hospital The Biology, Cell in Program 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,9496doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 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David , eie tthe at resides 4 ,tefirst the ), Department PI4KIII P a and P is a , Fba ta. 00 ee l,20;Taae l,21;Xiong 2012; al., et Thapa PtdIns(4,5) 2007; Furthermore, yeast al., 2012). in polybasic et shown al., He was the et 2010; as subunits, al., to Exo70 et and directly (Fabian Sec3 binding the of by domains presumably exocytosis, ,-ipopae[PtdIns(4,5) 4,5-bisphosphate ci euaos nldn h yokltlP crosslinker cytoskeletal–PM PtdIns(4,5) the requires which including Moesin, regulators, actin ste oPtdIns(4,5) to tied PtdIns4 PM is of role the addressed have Although 2012). al., et (Hammond vitro identity PM defining in role 02 alse l,19) nfisadi amla cells, mammalian in and flies In 1999). 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F-actin- al., its et of (Fievet site occlusion binding autoinhibited relieve to phosphorylation ta. 00 ugs ta. 02 hoge l,21;M tal., et Ma 2010; al., et Khuong 2012; (Brill al., organisms et Burgess multicellular 2000; in al., functions et their address studies 2005). al., et Weixel 2012; al., et PtdIns4 PtdIns4 ucin fyatP4saeruhyprlee nmammalian in disparate paralleled Golgi, PI4KIII These roughly the 2005). where are at al., PI4Ks cells, and et yeast nucleus (Strahl of secretion the functions directs in contrast, it functions In where essential 2002). signaling. organization, al., has actin PKC1–MAPK regulates et PIK1 and it (Han where morphology Audhya dispensable PM, vacuole the is 2002; at LS6B Emr, localizes whereas STT4 and 2000), (Audhya al., et roles essential overlapping (PI4KIII PIK1 nooe.BdigyathstreP4s T4(PI4KIII STT4 PI4Ks, three has PI4KIII yeast whereas Budding endosomes. 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(Fig. transgene srqie uigoogenesis. during required is zw2 PI4KIIIa and D 123 sgg D a et-ht 3 zeste-white 123 c21 D PI4KIIIa 123 complemented white and PI4KIII ;alsoknownas a eesv ehl n eiyosmales hemizygous and lethal, recessive was zw2 PI4KIII D a oovosefc on effect obvious no has PI4KIII 123 zw6 aeseiemutation male-sterile a lmntd(ermne l,1989). al., et (Perrimon eliminated was Jd ta. 92,w tested we 1972), al., et (Judd eegnrtduigteFLP-FRT the using generated were a eas iblt a ul rescued fully was viability because w egbrn complementation neighboring two , PI4KIII D a a et-ht 2 zeste-white ou (top), locus of Schematic (A) oogenesis. for required as cDNA. bars, bars, white exons; i.1. Fig. osrc bto) lebars, Blue (bottom). construct and (middle) uli cl a:20 bar: Scale pycnotic nuclei. exhibit GLCs arrowhead). 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Journal of Cell Science EERHARTICLE RESEARCH 956 in reduced severely is pMoe egg (G), single WT a with indicates Compared (B) (arrowheads). line epithelium 2 Dashed follicular are arrow). the panels (E, of Right protrusions side (blue). F-actin a ToPro have by and GLCs rescued (red) late-stage Early are Rhodamine–phalloidin whereas blue). defects (ToPro; (arrowheads), Actin DNA canals (F) and ring chamber. red) clustered (Rhodamine–phalloidin; and F-actin arrows) for (B,C, stained chambers egg (D–F) 2. Fig. in defects morphological for basis cellular the PI4KIII understand To PI4KIII hlodnt visualiz to phalloidin vdneo odrcl irto seblw n us cell and 1E) degenerate. nurse (Fig. to In nuclei and appeared shown). pycnotic (not exhibited below) appendages GLCs dorsal late-stage (see lacked addition, but migration 1E), cell addition, (Fig. dumping border appeared In (WT; of type cells 1C,D). wild evidence follicle (Fig. in observed of 1C-D spacing chamber Fig. layer regular the egg overlying unlike irregular, the the to restricted of of being organization ( than chambers rather anterior ooplasm, egg the later the in or found 9 were stage nuclei of 50% in that fflil el H rohas.(–)Imnbotn flstsfo sN-rae 2cls(,)adqatfcto fimnbos(,) elto of Depletion (J,L). immunoblots of quantification and (I,K) cells S2 dsRNA-treated from (K,L). lysates levels of Immunoblotting (I–L) arrowheads). PI4KIII (H, cells follicle of xmnto of Examination a a PI4KIII a ihayo he sNsrdcspo eeswtotafcigttlMepoenlvl IJ.C-elto of Co-depletion (I,J). levels protein Moe total affecting without levels pMoe reduces dsRNAs three of any with srqie o ci raiainadMei activation Moesin and organization actin for required is D 123 9 slik .Mtn g hmesa ae tgsshowed stages later at chambers egg Mutant ). a Ls g hmeswr tie ihRhodamine– with stained were chambers egg GLCs, uatgrlnsdslydfcsi -ci raiainadMei activation. Moesin and organization F-actin in defects display germlines mutant and gfp PI4KIII -ci.I Tegcabr,Fatnwas F-actin chambers, egg WT In F-actin. e sNssrea oiieadngtv otos epciey cl as 50 bars: Scale respectively. controls, negative and positive as serve dsRNAs a D 123 Lsb AIsann revealed staining DAPI by GLCs PI4KIII a ececntut GH ofclmcorpso g hmessandwt nipo (green), anti-pMoe with stained chambers egg of micrographs Confocal (G,H) construct. rescue n 6,nrecell nurse 536), 6 mgiidveso h oe ra,soigteoct otx(ros n h apical the and (arrows) cortex oocyte the showing areas, boxed the of views -magnified on ln h otxo h emclsadi igcanals ring in and cells germ the early of In cortex 2A,D). the (Fig. along found Fg E.Fatnaogteoct otxwsbcldand buckled from was the observed never GLCs of were cortex appearance phenotypes control rigid F-actin in These oocyte and 2D). oocyte smooth (Fig. the the the cortex WT to across contrast along or in into disorganized, F-actin and protruded others, not 2E). often but (Fig. F-actin nuclei cell of nurse some aggregations between present was actin a PI4KIII hnoecutr(i.2) fGC ihmr hnoecutrof cluster one than more with ( GLCs ( more Of not 92% in 2B). canals, were found ring (Fig. were canals or as cluster 2C) ring one correlate, (Fig. where cluster than to single cysts a appeared into in coalesced defects the observed tightly of These was center 3D,F). F-actin the Fig. cortical towards 2C; clustered (Fig. canals cyst ring and reduced greatly n 5 3 nGC ihoecutr nlate-stage In cluster. one with GLCs in 33) rngn,idctn htte r u ols of loss to due are they that indicating transgene, ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal PI4KIII PI4KIII n 5 AF ofclscin fery AC n late-stage and (A–C) early- of sections Confocal (A–F) m m. 3 hwdcria -ci,cmae ih45% with compared F-actin, cortical showed 13) a a Lshv eue otclF-actin cortical reduced have GLCs PI4KIII Ls(,arw) u o nteaia regions apical the in not but arrows), (H, GLCs ovo D +fmlsadwr ece ythe by rescued were and females /+ a fwd D 123 and Ls otclFatnwas F-actin cortical GLCs, PI4KII PI4KIII a iteefc npMoe on effect little has a D 123 Ls F- GLCs, a PI4KIII

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(Fig. cells follicle of the staged and membranes similarly PM oocyte apical the both juxtaposed along F-actin with colocalized pMoe ebae eaaigW us elnce Fg 3G–I), (Fig. nuclei cell in prominent evident nurse to was WT which contrast PI4KIII separating PM, In Thinner of micrographs. membranes 3C,D). (Fig. loss electron canals often reflected transmission cytoplasm, ring staining the clustered lectin within as near aggregates 3C,D), (Fig. concentrated lectin-positive membranes as along well staining lectin discontinuous ncnrs,early-stage 3A,B). lectin-positive 2004; (Fig. contrast, chambers, F-actin Schwarz, egg cortical In WT with and colocalized early Murthy largely In membranes 2006). 2002; al., al., et Verdier et surface cell (Dollar and intracellular membranes on glycoproteins chambers, binds egg which tomato late-stage lectin, fluorophore-conjugated and using early- visualized were in membranes integrity PM examine To us elnce Fg KL,adnn ewe tes(i.3K). (Fig. some others between between none membranes and 3K,L), thin (Fig. 3J), nuclei cell (Fig. nurse canals ring clustered ta. 04 Fg IJ.Ttllvl fMermie unchanged, remained Moe PI4KIII of (Hipfner levels that Slik Total demonstrating 2I,J). kinase as Moe (Fig. 2004) the not al., against was et directed but RNAi (GFP), as protein dramatic fluorescent green against directed splmnaymtra i.SB.Hwvr oatndefects actin in no observed However, 10B were S2B). stage were Fig. levels of material cortex pMoe Fig. the (supplementary material at experiments, reduced supplementary immunofluorescence slightly 2K,L; (Fig. In effect S1). no had PI4KII, eaiaino -ci rmteP ugse possible a suggested PM the from F-actin of Delamination oeaiewehrls of loss whether examine To a a a srqie o lsammrn nert n exocyst and integrity membrane plasma for required is D xrsinb N nefrne(Ni n eesof levels and (RNAi) interference RNA by expression 123 , PI4KIII 54%cmae omc ramn rRNAi or treatment mock to compared 25–40% Lshdvscltdmmrnsaround membranes vesiculated had GLCs P PI4KIII a PI4KII oPtdIns(4,5) to D 123 fwd PI4KIII a Ls nW tg 0 g chambers, egg 10B stage WT In GLCs. PI4KIII D g hmes(e eo) pMoe below). (see chambers egg 123 a uatoctsrsmldWT resembled oocytes mutant osntafc o rdcinor production Moe affect not does Ls Mewsgetyreduced greatly was pMoe GLCs, a PI4KIII P sNsrdcdteaon of amount the reduced dsRNAs a D 2 lobokdpo accumu- pMoe blocked also , 123 Drosophila a Lsdslydthinner, displayed GLCs Drosophila fetdoeallvl of levels overall affected P 2 PI4KIII fwd idn srequired is binding PI4Ks, uatoocytes mutant 2clswere cells S2 PI4KIII a D 123 fwd GLCs. a and ,as ooaie ihFatni h oye(i.4) hs effects These 4B). (Fig. oocyte that to whorl the large specific in a were formed F-actin times nuclei at with and some colocalized 4A,B), between (Fig. seen others (52%, were not affected but membranes mildly presence GLCs, as the late-stage scored and In were epithelium missing F-actin follicular cortical intact Thus, defects of clones. an was from cell with result follicle GLCs mutant could unmarked or only effect or of cells, indirect presence the follicle an degenerated to the due be on could normally germline also phenotype the of that somatic This cyst severe epithelium 3E,F). more (Fig. the follicular overlying with had chambers somatic encapsulates with egg also the GLCs early nuclei eight In phenotypes, cell 3D). of germ (Fig. out membrane between seven F-actin membranes; cortical of loss paralleled nihdo oyemmrns(utyadShaz 2004) Schwarz, and was (Murthy and In PM membranes the 5A,B). at (Fig. oocyte found In was on Sec5 distribution. chambers, enriched Sec5 egg examined 6–8 2005; we stage al., WT 2004), et for Murthy Schwarz, 2005; homozygous exocyst and al., et the GLCs Murthy (Beronja including Sec6 in and addition, Sec5 seen membrane subunits also affect that are mutations PM of late disintegration to to mature fail to 3F) Fig. able (e.g. GLCs are survive. early affected GLCs severely cell whereas mutant stages, border that early hypothesize of we affected 4B), evidence (Fig. mildly below). and GLCs late-stage membranes see some in of 4C,D; migration (Fig. presence the phenotypes Given distinct showed chambers PI4KIII in 6D). prominent (Fig. more were PI4KII structures These PI4KII 4A,B). (Fig. GLCs specific a from result defects PM PI4KIII observed for requirement the whether test To membranes PI4KIII nadto,mmrnstriae ihntectpamin cytoplasm the within terminated membranes PI4KIII addition, In Ls(i.5)wsn togrta nltrlflil cell follicle lateral on than in exocyst. stronger the the interface no 5C), cell that was (Fig. indicating oocyte–follicle membranes, WT 5F) the (Fig. to at GLCs contrast Sec5 In nurse of F- and 5B,E). oocyte cortical level (Fig. the retained both membranes on that reduced cell was chambers Sec5 egg 5D,E). (Fig. affected actin mildly in even Fg AF.Lapnt aidi ie ihlre v structures Lva larger with size, with in versa varied cells, vice puncta and Lva germ discrete puncta 6A,F). decorated lectin (Fig. of with Lva overlapping cytoplasm chambers, partially egg the structures WT in (Lva). earlier lamp or puncta Lava 7 marker stage cis-Golgi In the for immunostained were eeaie g hmesin chambers egg examined we nert uigognssd o eur w rPI4KII. PM in or affected Fwd and not normal require organization was not appeared localization actin do F-actin Sec5 oogenesis and that Additionally, during intact indicating integrity was 4C,D), PM (Fig. the Overall, eentvsbei aesaeW g hmesor chambers egg WT that late-stage 4C,D) in (Fig. visible cytoplasm not the were in structures lectin-positive large late-stage PI4KII eas ako otclFatn lseigo igcnl and canals ring of clustering F-actin, cortical of lack Because ofrhrdfn hs nrclua ebaedefects, membrane intracellular these define further To a , a a g hmesta a tews omllci staining lectin normal otherwise had that chambers egg uat splmnaymtra i.S) oee,in However, S3). Fig. material (supplementary mutants than fwd D D ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal 123 123 fwd and fwd Lsand GLCs Ls(i.3,) oso otclF-actin cortical of Loss 3K,L). (Fig. GLCs and PI4KIII PI4KII PI4KIII uat,wt ag ucavsbei early in visible puncta large with mutants, PI4KII aedfeeta fet ncellular on effects differential have a a a D fwd ragnrlrqieetfrPtdIns4 for requirement general a or 123 Ls as GLCs, PI4KIII g hmes us el showed cells nurse chambers, egg and Ls hslclzto a lost, was localization this GLCs, fwd PI4KII a Lsfi orcuto retain or recruit to fail GLCs fwd and and uategchambers egg mutant PI4KII PI4KII 0 fLva of ,40% ulmutants. null PI4KIII uategg mutant PI4KIII fwd n 558). a D 957 123 P or a ,

Journal of Cell Science ewr TN,ti a niaefamnaino h G.Lva tomato TGN. the As in of fragmentation trans-Golgi 3). puncta indicate the box may at this proteins 6C, (TGN), glycosylated network (Fig. bind to puncta not predicted did is lectin but lectin to, small proximity close with, in be overlap to appeared puncta Lva small itnusal n crda eaaeuis(i.6,bx3; box Lva of 6D, microscopy numbers (Fig. average the confocal units Overall, bodies). of separate Lva three as level as were scored puncta scored Lva the discrete and clusters, at some distinguishable Within 1). resolved inset 6D, that be puncta (Fig. Lva several not of clusters be could with to associated appeared still many often but were lectin, structures largest The 6D,E). (Fig. ( WT in 8.5% EERHARTICLE RESEARCH 958 22.4% 2); irregularly box 6C, either partially (Fig. elongated were that or puncta puncta 1) box ( Lva Lectin 6C, (Fig. larger 6C,E). shaped (Fig. with WT overlapped than smaller contrast, 6A, In (Fig. structures 1–3). lectin-positive boxes larger compare with associated being hmessoe biu,ytdsic,dfcsi Golgi in defects distinct, yet In obvious, morphology. showed chambers n 4)of 5241) PI4KII fwd n 8)ad78 in 7.8% and 5188) ucawr bomlysae oprdwith compared shaped abnormally were puncta fwd eesihl agradmr aidi size in varied more and larger slightly were uat,Lapnt eesignificantly were puncta Lva mutants, fwd PI4KII and ( n PI4KII 0.Mn fthe of Many 590). uategg mutant fetGlimrhlg n h anri hc cis-Golgi which in manner the TGN. the and with associate morphology Golgi affect (114.0 n ucain puncta og opooyi o rsl fetdi h bec of absence the in affected cis- that grossly suggest not puncta Lva PI4KIII is assess of morphology to number other and the Golgi difficult Although shape as puncta. it size, Lva and and normal made breakdown, 3J lectin (Fig. between clustering association PM well specific Membrane as region of shown). this not in consequence concentrated cyst were secondary the organelles may of This a center 6B). the (Fig. be to membranes lectin-positive localized clustered were the puncta near Lva Golgi of most than boxes localization WT, perinuclear 6B, the rather in (Fig. to WT contrast units in in However, as 1–3). puncta, individual partially lectin cluster center adjacent as the with from overlapped appeared away located puncta and Lva clusters. 6E), (Fig. (118.7 WT number and shape size, – g hmesec) hs eut suggest results These each). chambers egg 52–3 ncnrs,in contrast, In .)egcabr eesmlrt T(124.7 WT to similar were chambers egg 61.4) a nblt oass oto h etnpnt rcue a precludes puncta lectin the of most assess to inability , ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal fwd PI4KIII (112.3 hn(,,bakarwed)o o(,bto two of nuclei bottom between (K, PM no nuclei) or but arrowheads) arrowheads), black (H,I, (K,L, WT thin in each nucleus separating cell PM nurse robust egg a 7 show Stage chambers insets). compare arrows; (J, i.3 ebaeitgiyi eetv in defective is PI4KIII integrity Membrane 3. Fig. nesi r anfe 3 magnified B,D,F. are in D enlarged in are Insets A,C,E in areas Boxed (blue). ToPro and (red) Rhodamine–phalloidin (green), lectin stained fluorescein–tomato chambers with egg early-stage of sections c olcecl;m iohnra ,nrecell canal(s). nurse ring n, rc, mitochondria; nucleus; chamber. m, egg cell; same follicle the fc, of is region I different in a Inset from arrowheads). white (K,L, cytoplasm in membranes a in canals ring WT of in cluster canal a and ring (G) single a showing chambers egg and (G–I) WT of bars: Scale (F). 50 degenerate follicle to and appear chamber cells egg the are throughout membrane found of aggregates egg affected (E,F), severely chambers In ring arrowheads). of (D, cluster canals the around localizes lectin-positive membrane remaining the arrows, whereas (D, insets), F-actin cortical along decreased and staining PM lectin the reduced show contrast, (C–F) In GLCs arrows). with (B, colocalizes F-actin and cortical PM lectin the (A,B), marks WT staining In contrast. and brightness 75 mean 617.5; a m D .(–)Tasiso lcrnmicrographs electron Transmission (G–L) m. 123 a emieclones. germline Ls v ucawr fsimilar of were puncta Lva GLCs, 87prGLC, per 628.7 PI4KIII PI4KIII 6 a emnt ihnthe within terminate a ... and s.e.m.) Ls(–) tg 5 Stage (J–L). GLCs 6 AF Confocal (A–F) PI4KIII n dutdfor adjusted and n )t hs in those to 53) fwd PI4KIII a and Ls Thin GLCs. PI4KIII 6 PI4KII PI4KII a 58.8; GLC a

Journal of Cell Science oee,poietmmrn grgtsaefudi h us cell nurse the 50 in bar: found Scale are cytoplasm. aggregates membrane prominent to However, contrast In (blue). ToPro PI4KIII and (red) Alexa-Fluor-488–phalloidin (green), h oye(ros.Bre el,dse ice.(,)Cnoa etosof sections Confocal in (C,D) F-actin circles. with dashed colocalizes fwd cells, membrane Border of (arrows). whorl oocyte a the and arrowheads) (B, GLCs ebae ga;aeaeitniytknfo oiin niae yylo asi n )adars h oyepseirflil elmembranes and cell (C) follicle WT oocyte-posterior for the E) across and and cells B E) follicle in and to bars B adjacent red in membranes by bars cell indicated yellow nurse positions by along from 50 indicated and taken bars: PM positions intensity Scale oocyte from average arrowheads). the taken (red; arrows, at intensity compare enriched average (E, is (gray; membranes Sec5 membranes WT, cell In follicle B,E. lateral in in enlarged are In A,D arrows). in (B, areas Boxed (red). phalloidin in missing or disrupted are membranes WT cell with nurse Compared arrowheads), (A, (blue). ToPro and (red) Rhodamine–phalloidin a and (A) chamber mutants. PI4K other EERHARTICLE RESEARCH i.5 osof Loss 5. Fig. 4. Fig. C and (C) a PI4KIII Ls(B), GLCs PI4KII a PI4KIII PI4KIII emiecoe xii ebaedfcsntse in seen not defects membrane exhibit clones germline D uategcabr tie ihTexas-Red–lectin with stained chambers egg mutant (D) fwd PI4KIII a AB ofclmcorpso aesaeW egg WT late-stage a of micrographs Confocal (A,B) a Ls e5a hs ebae sls,ie h e5sga ttenrecl–olcecl nefc ssmlrt that to similar is interface nurse-cell–follicle-cell the at signal Sec5 the i.e. lost, is membranes these at Sec5 GLCs, and irpsSc localization. Sec5 disrupts m a m. L B tie ihfursenlci (green), fluorescein–lectin with stained (B) GLC PI4KII us elmmrnsaeintact. are membranes cell nurse ABDE ofclmcorpso g hmessandwt niSc gen n Rhodamine– and (green) anti-Sec5 with stained chambers egg of micrographs Confocal (A,B,D,E) PI4KIII a PtdIns(4,5) PI4KIII us elnce nteopam niaigta titration that indicating exhibited ooplasm, chambers the egg in PtdIns(4,5) the nuclei of of cell 50% nurse However, chambers egg developing 7A,B). WT (Fig. in F-actin cortical with colocalized enue ottaePtdIns(4,5) titrate to used been h M iia oPtdIns4 to PtdIns4 Similar PM. reduced the showed P that GLCs PtdIns4 the PtdIns4 of the of PtdIns4 examined level a the exhibit we in decrease this, a for account PtdIns4 To membranes. PtdIns4 of loss organization. ttePM. the at n nrn aas(i.7) ncontrast, In 7C). (Fig. canals ring in and ci ai a 05 fW Fg 7H; (Fig. WT of 70.5% PI4KIII was ratio actin hntpsb irto,w sdanti-PtdIns4 used we titration, by phenotypes 7B). (Fig. g chambers, egg PtdIns4 reduced showed ee bqiosepeso fPLC of expression ubiquitous level Because PI4KIII of effect the about conclusion h Madi igcnl nW Fg F.PtdIns(4,5)P 7F). (Fig. WT in canals ring in and PM the fPtdIns4 of yokltn eraoe that reasoned we cytoskeleton, PI4KIII xmndPLC examined Fg G 69 fegchambers, egg of 46.9% 7G; (Fig. in reduced also m .1,idctn that indicating ,0.01), oass Mpoponstd eeswtoteliciting without levels phosphoinositide PM assess To .(,)Qatfcto fSc nest costeltrlflil cell follicle lateral the across intensity Sec5 of Quantification (C,F) m. a P P P a a a srqie o MPtdIns4 PM for required is D D Fatnrtows5.%o T(i.7E; (Fig. WT of 55.5% was ratio :F-actin PI4KIII ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal tiigitniywsdet oso ebae n not and membrane, of loss to due was intensity staining snee o omlPtdIns4 normal for needed is inli eaint otclFatn ftedces in decrease the If F-actin. cortical to relation in signal 123 123 PI4KIII P P 2 Ls nW,PtdIns4 WT, In GLCs. Ls eue PtdIns4 reduced GLCs, nioist muoti Tegcabr and chambers egg WT immunostain to antibodies rPtdIns(4,5) or P n d 2 0.BcueP nert scmrmsdin compromised is integrity PM Because 530). HGP loecn eotrta a also has that reporter fluorescent a PH–GFP, a a P PI4KIII D F.Br niaesadr deviations. standard indicate Bars (F). Fatnrtosmlrt hti T However, WT. in that to similar ratio :F-actin a eaiuaea recapitulate can 123 a rsi fet nteP n actin and PM the on effects drastic had P a PI4KIII tiiga h M(i.7;7.%of 76.7% 7D; (Fig. PM the at staining P Ls lhuht esrextent lesser a to although GLCs, P PtdIns(4,5) , 2 odtc PtdIns(4,5) detect To . PI4KIII P P PI4KIII 2 a n PtdIns(4,5) and P d Ruhre l,20) Low- 2000). al., et (Raucher P HGPmre h Mand PM the marked PH–GFP ewudepc Lsto GLCs expect would we , n otosPtdIns4 controls P 9;tePtdIns(4,5) the 549); a eetdaogtePM the along detected was P a tiigmgtb u to due be might staining PI4KIII n PtdIns(4,5) and a ih fetP levels PM affect might n P D 523, 123 2 PI4KIII a eetdalong detected was noealGolgi overall on a P P D P h average the , 2 123 .5.Thus, ,0.05). P a P D phenotype 123 P eesat levels ranti- or P 2 n 2 GLCs levels 2 P ,we 523, 959 was 2 :F-

Journal of Cell Science tg rltrW g hmes(i.8A,A9 (Fig. chambers egg in WT pole later posterior the or to 9 localized stage is Oskar defects. polarity exhibit EERHARTICLE RESEARCH 960 1995) al., et (Roth, 8 stage at In oocyte the 8D). to (Fig. the Gurken of successful of localization are and dorsal–anterior nucleus indicators oocyte 8B,B the polarity (Fig. of other migration Two reduced disruption. either F-actin was 8C,C (Fig. Oskar GLCs, PtdIns(4,5) of levels reduced with chambers Egg PI4KIII aldt oaiea h oslatro oiin(/ were (3/8 8D,F,G, (Fig. position WT) dorsal–anterior in 10/10 the with at compared nucleus localized, oocyte localize properly the addition, associated to In not 8F). or failed (Fig. 8E) nucleus oocyte (Fig. the chamber with egg the within concentrated ta. 08.Tu,w xmndwhether examined (Gervais we defects Thus, polarity 2008). oocyte al., have et Sktl, PIP5K the of mutation a srqie o g hme polarity chamber egg for required is 9 ttepseirpl,creaigwt h ereof degree the with correlating pole, posterior the at ) PI4KIII a D 123 Ls uknwsete o visibly not either was Gurken GLCs, PI4KIII .In ). P a 9 D 2 rmissing or ) sarsl of result a as , 123 PI4KIII Lsalso GLCs a D 123 PM. seik;qatfe nFg H.Hence, 8H). Fig. in quantified asterisks; vdneidctn htamjrrl o hsezm is enzyme this for role major a of PtdIns4 lines of that several production provide indicating We PI4KIII phosphoinositides. evidence PM that PM the suggest of affects integrity data that for our as well Furthermore, as oogenesis, itself. cortex, the during at are that organization processes actin show these we whether PI4KIII Here, unclear regulated. remained phosphoinositides, coordinately on has depend it PM the although at processes cellular Many DISCUSSION phenocopies ols n cuuaino nrclua -ci,distinctive F-actin, intracellular of accumulation and ooplasm First, a PI4KIII ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal sesnilfrcodntn ebaetafcigand trafficking membrane coordinating for essential is sktl a oaiydefects. polarity D 123 P Lsehbtnrecl ulii the in nuclei cell nurse exhibit GLCs o ovrinit PtdIns(4,5) into conversion for hmeso h eoye itd WT, listed. n genotypes the egg of in chambers found puncta of Lva diameter individual Mean spot (E) lectin 1–3). single (boxes a Lva with several associate Often, puncta 2). 1, (boxes puncta in puncta Lva (D) PI4KII 3). (box small puncta with, lectin overlapping not close but in to, were proximity or lectin not with either associated were puncta The Lva puncta. smallest lectin or 2) 1) (box (box elongated shaped irregularly more either overlap with that puncta Lva smaller have (C) 1–3). more (boxes appear diffuse puncta lectin WT, the in although those resemble puncta associated and lectin Lva of chamber. sizes egg the the Elsewhere, of center the towards in Lectin-positive membranes (B) 3). lectin (box adjacent puncta no either or had discernible puncta barely Lva puncta small lectin 1–3); of (boxes sizes were the puncta to Lva proportional of sizes The lectin-positive puncta. to colocalize chambers partially egg WT and in visible are puncta Discrete Lva (A) lectin discernible. of where shape the puncta, trace lines Dotted 3. and right far 8 at are Images (green). lectin fluorescein– tomato and (red) anti-Lva chambers with egg stained of images Confocal (A–D) Golgi organization. regulate and PI4KII morphology and Fwd 6. Fig. n v ucawt etn(rybr) WT, bars). (gray n and lectin bars) with (black puncta Lva Lva with puncta lectin of colocalization Percentage (F) chambers). chambers); egg chambers); v uca( g chambers); egg (3 puncta Lva tnaderr * error. indicate standard Bars chambers). egg (2 puncta Lva etnad37Lapnt 3egg (3 puncta chambers); Lva 337 and lectin *** 5 5 5 P 5 uca( g chambers); egg (3 puncta 251 5 6egchambers); egg (6 554 8 etnad34Lapnt 3egg (3 puncta Lva 374 and lectin 388 , 6 .0.Saebr 10 bar: Scale 0.001. uat vra ihegre lectin engorged with overlap mutants nagmnso oe ra ,2 1, areas boxed of enlargements PI4KIII PI4KII PI4KIII P PI4KII , , a n .5 ** 0.05, , 5 n a 5 , 3 etnad228 and lectin 139 Lsaccumulate GLCs n 9 etnad356 and lectin 291 fwd 5 fwd a 3 2egg (2 132 P m PI4KIII g chambers egg m. , stePI4K the is , 0.01, fwd n P 5 PI4KIII 2 , 9 (4 399 n tthe at 5 a D 436 123 a ,

Journal of Cell Science hntpsof phenotypes Ls Fourth, GLCs. yylo asi , frE n nFG(o ) otm vrg PtdIns4 average Bottom: H). (for F,G in and E) (for C,D in bars yellow by Tegcabr.Tid muotiigrvae reduced revealed immunostaining PtdIns4 Third, of chambers. levels egg WT ooaie ihFatn(,,arwed) prxmtl 0 fteeegcabr aenrecl uliwti h ols,smlrto similar ooplasm, the within nuclei cell nurse have chambers egg these or of (C,F) 50% chambers Approximately egg WT arrowheads). (C,D,F,G) (A,B, F-actin with colocalizes EERHARTICLE RESEARCH lorsl ndcesdlvl fPtdIns(4,5) of would which levels of decreased loss 2005), in al., result et Stein also PtdIns(3,4,5)P (von the for Pten mutant phosphatase GLCs in observed also phenotypes PLC marker 7. Fig. PtdIns(4,5) reducedalongthePMin hdmn–hlodn(e) PtdIns4 (red). Rhodamine–phalloidin eod bqiosepeso fPLC of PtdIns(4,5) expression ubiquitous Second, ugs hyipneuo omnpo fPtdIns(4,5) of pool common a upon impinge they suggest fegcabr xmnd nest aisnraie oW.* WT. to normalized ratios intensity examined) chambers egg of PI4KIIIa P d 2 HGPudrcnrlof control under PH–GFP P H tiigi Tg hme bakln)cmae with compared line) (black chamber WTegg in staining (H) 2 eaiuae hspeoyei 0 fotherwise of 50% in phenotype this recapitulated , P PtdIns4 membrane plasma for required is PI4KIII PI4KIII P PI4KIII n PtdIns(4,5) and a a Lsfi oatvt n eri proteins recruit and activate to fail GLCs and a Lscmae ihW cmaearw nCwt ,Fwt ) EH o:rpeettv lto vrg nest fPtdIns4 of intensity average of plot representative Top: (E,H) G). with F D, with C in arrows (compare WT with compared GLCs P rspiaPten Drosophila PI4KIII n PtdIns(4,5) and a 1 tblnGL gen n tie ihRoaiepalii rd n or bu) PLCd (blue). ToPro and (red) Rhodamine–phalloidin with stained and (green) -tubulin-GAL4 a P Ls(,)sandwt anti-PtdIns4 with stained (D,G) GLCs 2 d nteP of PM the in HGP hc titrates which PH–GFP, P 2 r eetdo h M(ros n nrn aas vrl neste fPtdIns4 of intensities Overall canals. ring in and (arrows) PM the on detected are uat strongly mutants P 2 h similar The . n PtdIns(4,5) and P , .5 ** 0.05, PI4KIII P Fatn(E, :F-actin PI4KIII P , P 2 .0.Saebr:50 bars: Scale 0.001. a a 3 . P L rdln) vrg nest a lte sn austknfo oiin indicated positions from taken values using plotted was intensity Average line). (red GLC 2 . n 5 AB ofclscin fW g hmesepesn h PtdIns(4,5) the expressing chambers egg WT of sections Confocal (A,B) P 3 67 fegcabr xmnd n PtdIns(4,5) and examined) chambers egg of 76.7% 23; P4;CD ranti-PtdIns(4,5) or C,D) (PI4P; eil eyln Bgr ta. 07 otlsadEphrussi, and Coutelis 2007; exocyst al., the et promote (Bogard for to secretion, Sec15 recycling of component mutant regulator exocyst vesicle a the GLCs Rab6, binds which as Rab11, well in and as Sec6, found and and dRok, Sec5 activator defects subunits Moe the PM the and at Moe exhibit for Sec5 mutants subunit in exocyst seen F-actin the was retain phosphorylation or Indeed, Moe PM. recruit to and failed GLCs, GLCs in in cortex attenuated oocyte the at nw orqiePtdIns(4,5) require to known ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal m m. PI4KIII PI4KIII a a Lspeooytedsuto fcortical of disruption the phenocopy GLCs ncdw el.Additionally, cells. knockdown P 2 [PI(4,5)P P 2 Mewsdaaial reduced dramatically was pMoe . 2 ,]atbde gen and (green) antibodies F,G] ; HGPlbl h Mand PM the labels PH–GFP P P PI4KIII 2 n PtdIns(4,5) and Fatn(H, :F-actin a Ls(,arrow). (B, GLCs n 5 PI4KIII P P 3 46.9% 23; 2 2 P were E or (E) 961 a

Journal of Cell Science kl n ehp loPPK9,t rdc olo PtdIns4 of pool PtdIns(4,5) a produce PM to feeds PIP5K59B, that also perhaps and Sktl, in that suggest spoal u oteosre peuaino w PIP5Ks, two of upregulation observed the This to 2012). PIPKI due al., et probably (Nakatsu labeling is metabolic by assessed when fRoGPs aiymmesdrn oeei causes similar F-actin, oogenesis cortical of during loss members and canals to family ring versions of GTPase dominant-negative aggregation of Rho expression that of and (Murray migration 2012), mesoderm al., during et activation Rho in functions PI4KIII that another observations with from redundant partially least at Drosophila is observations). Sktl unpublished that (our suggests follicles This WT among thrive to fail hra lbllvl fPtdIns(4,5) of levels global whereas neetnl,in Interestingly, PtdIns(4,5) inln n stability. and signaling h I5 kl o xml,po oaiaini lodefective also is localization pMoe example, in For Sktl. PIP5K the EERHARTICLE RESEARCH 962 Verdier PtdIns(4,5) 2002; al., to Thus, et 2005). Langevin Polesello al., precursor 2007; et 2005; Wu al., al., 2006; et al., et et Murthy Januschke 2005; 2002; al., al., et et Jankovics 2007; PtdIns(4,5) PtdIns(4,5) Grase l,2008), al., et of (Gervais GLCs marked Although PI4KIII ossetwt this, with Consistent osof Loss PI4KIII sktl b a yoopi Ls(evi ta. 08.However, 2008). al., et (Gervais GLCs hypomorphic n PIPK1 and oeei eet r o dnia otoeof those to identical not are defects oogenesis a P P P PI4KIII D I5 uigognss upr o hsie stems idea this for Support oogenesis. during PIP5K 2 2 2 123 n hs htddwr essrnl affected. strongly less were reduced did showed that GLCs those of and percentage , smaller a ; eotr r rmtclyrdcda h PM, the at reduced dramatically are reporters Drosophila Ls(upyadMnel 96.Hne we Hence, 1996). Montell, and (Murphy GLCs a PI4KIII c ec,i spsil htcompensatory that possible is it Hence, . a rae feto PtdIns4 on effect greater a has PI4KIII PI4KIII P P a 2 2 oeei PI4KIII oogenesis xrspoon fet nPM on effects profound exerts , . sktl ncotMF,PtdIns4 MEFs, knockout a a a D ulallshv enreported been have alleles null D 123 n IK9 ar u similar out carry PI5K59B and 123 P PI4KIII 2 Lsmd nti manner this in made GLCs Lsrsml Lsfor GLCs resemble GLCs r nymdsl affected modestly only are a ysnhszn the synthesizing by , a csusra of upstream acts P hnon than P sktl and P . fetof effect ietyrsosbefrsm fteosre phenotypes. observed PtdIns4 the PtdIns(4,5) either that that of fact indicates some the for However, responsible directly peuainof upregulation oaiain(evi ta. 08 o ti ta. 05.A 2005). al., et Stein von in on observed 2008; effect suggesting al., pole, similar et posterior (Gervais the localization from missing PI4KIII or phenotypes reduced are the either at Gurken anchor of or with migrate shared mislocalization to nucleus and oocyte dorsal-anterior the of Failure GLCs. ietrgltr rcro,o oh ean noe question. open an remains both, or precursor, a regulator, direct PtdIns4 PtdIns4 of PI4KIII of on ob ein nangtv hreta ol epoie by provided be could that charge PtdIns4 negative a either on reliant be to found in oefntospeiul trbtdt MPtdIns(4,5) PM to attributed previously PtdIns4 functions PM some for functions independent ohPtdIns4 both ta. 09 aaae l,21) PI4KIII 2011), (Krauss al., on F-actin et and Tanaka based 2009; Oskar al., between Indeed, et links organization. reported Oskar previously the F-actin of PI4KIII extent Moesin-dependent of suggesting the effect disruption, that F-actin the of that noted degree the We with correlated 2002). defect al., et Polesello eea set fcl oaiywr irpe in disrupted were polarity cell of aspects Several u eut ev pntepsiiiyo PtdIns(4,5) of possibility the open leave results Our PI4KIII P a like , PI4KIII ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal otiue oP ucinin function PM to contributes P a a P hdmn–hlodn(e)adTPo(le.(A (blue). ToPro and (green), (red) antibodies Rhodamine–phalloidin anti-Oskar with stained chambers fnra bu a)adanra rdbr ula oiinin position nuclear bar) (red versus abnormal WT and bar) Frequency (blue (H) normal nucleus. of (G) oocyte mislocalized nucleus. with oocyte chamber the egg or to (E) relative dispersed (F) either mislocalized is Gurken nucleus, 10 In anchored stage asterisk). in (outline, oocyte is the chambers of which egg corner nucleus, anterior oocyte dorsal the the in above anchored directly WT, In found is (D) Gurken (blue). ToPro (green), and anti-Gurken (red) non-specific. with Rhodamine–phalloidin is stained cells chambers follicle Egg the (D–F) in of Rhodamine–phalloidin staining (compare Oskar chamber B,C). the egg throughout the disruption of F-actin rest of degree the with correlated chambers egg later or (B 9 stage (A in WT oocyte in the of concentrated posterior is the protein at Oskar areas. boxed of views magnified in defects Polarity 8. Fig. n fteohrP4scudspl ml amount small a supply could PI4Ks other the of one , D sktl moesin 2 htsre sapeusrt PtdIns(4,5) to precursor a as serves that 123 P 9 P sseiial eurd eas PI4KIII Because required. specifically is ,C sktl 9 uat Grase l,20) sa rti was protein Oskar 2008). al., et (Gervais mutants rPtdIns(4,5) or n PtdIns(4,5) and a ros.Vraiiyo sa oaiainin localization Oskar of Variability arrows). , Ls ti osbeta ako PtdIns4 of lack that possible is it GLCs, Drosophila nPtdIns(4,5) on 9 oskar uategcabr Jnoise l,2002; al., et (Jankovics chambers egg mutant ro) u sete eue rasn in absent or reduced either is but arrow), , and PI4KIII a Pten sktl RAadpoenwsfrequently was protein and mRNA a nOkri eitdi atthrough part in mediated is Oskar on oye.Saebr:50 bars: Scale oocytes. P I5sacut o h weaker the for accounts PIP5Ks P a loaffect also may , Lsso iia phenotypes similar show GLCs 2 ln sntsfiin rthat or sufficient not is alone P P Hmode l,21) Thus, 2012). al., et (Hammond 2 2 h ehns ywhich by mechanism the , absence the in Alternatively, . PI4KIII Drosophila P ti oeotythat noteworthy is It . a PI4KIII GLCs. a a tmlt a stimulate may a AC Egg (A–C) m oye ihan with oocytes oskar hte sa as whether , (A–G). m PI4KIII P 9 a ,B 2 PI4KIII . PI4KIII regulates 9 P ,C PI4KIII mRNA 2 9 )2 a were P a D 6 P a 123 - a 2 is -

Journal of Cell Science nvriy e ae,C)and CT) Haven, New University, pcfcpos ned eei neatosidct that indicate interactions of genetic inhibition control Patched Indeed, cell, that relieves the factors Hedgehog within pools. identifying lipid of same specific importance the al., of the in et pools emphasizing results (Maffucci different probably enzymes cells of the production cervical of and regulation Differential ovarian 2005). human cultured migration lysophosphatidic-acid-dependent of for necessary is latter rmEa cetr(ezanIsiue eoo,Israel). Rehovot, Institute, (Weizmann Schejter Eyal from IKioom a vle samcaimfrspatial for mechanism the a in as processes PI4KIII of with phosphoinositide-dependent evolved cell, diversity of functional has phosphoinositide that coordination of isoforms appears roles it PI4K the multifaceted metazoans, at the in effectors a molecular signaling Given to and Hence, signals cortex. lipids 2011) 2010). control relaying cell to thereby al., al., is PM, enzyme et et the this (Yan at of (Garrenton property flies conserved yeast and in crucial in signaling signaling Hippo MAPK 2009), al., et PI4KIII 2010). PI4KIII that suggesting PtdIns3 produce both fet.Freape h ls IP3sPI3K-C2 PI3Ks II cellular class and the in physiological example, different For theme vastly cellular effects. have recurrent produce can non-overlapping to lipid a same act a the nominally underscore that performs enzymes results biology: it phosphoinositide our that Indeed, shows function. and flies, oocyte control to oocyte the with chambers and egg In PFCs migration. in nucleus events PI4KIII the molecular that of These suggest in 2011). seen localization observations those al., to et turn similar and (Yan are in defects Staufen migration which determinant polarity signaling, nucleus posterior Hippo oocyte control to regulates (PFCs) cells follicle polarization. cell actin and phosphoinositides, trafficking coordinates membrane organization, that loop feedback positive ARTICLE RESEARCH le eerie nsadr onelmlse gra 25 at agar molasses cornmeal standard on raised were crosses Flies genetic and stocks Fly METHODS AND MATERIALS rlkadHwr isiz(nvriyo oot,Trno ON, Toronto, Toronto, of (University Lipshitz Howard Canada). and Erclik ui SrdigadRbn 92.PeeetG38 a obtained was GE3785 Korea). P-element (Taejon, 1982). GenExel Rubin, from and (Spradling Rubin Ahunr 90.Vsbemresadblne hoooe are Germline chromosomes 1992). Zimm, balancer and of (Lindsley and Zimm transformation and markers Lindsley by Visible described 1990). (Ashburner, nbt h oyeadtePC o otne communication continued for determinants. PFCs polarity posterior the of and maintenance oocyte and the Tapon, that and both suggesting adjacent Polesello 2008), in 2007; al., observed al., et normal et Yu is (Meignin signaling, 2007; PFCs localization Hippo WT the for Oskar to mutant only and are Staufen half Alternatively, and posterior the WT of process. are in posterior or PFCs defective the dorsal-anterior be nucleus the complete could to initiating oocyte nucleus to signal the of unknown fails tethering the but receiving migration, of capable a In that is suggesting 2011). oocyte, the al., the in of et nucleus middle at unknown (Yan oocyte the repolarization tightly the send oocyte contrast, to positioned initiates fail that consistently PFCs signal mutant is that indicating nucleus posterior, oocyte the PI4KIII u td dniisPI4KIII identifies study Our w a ;P{w a rvosysont erqie nposterior in required be to shown previously was + a , w UASp::PLC lopooe G inln nzbaih(Ma zebrafish in signaling FGF promotes also a 1118 ciga e euao ttePM. the at regulator key a as acting P mro a are u si paln and Spradling in as out carried was embryos n r oepesd oee,ol the only however, co-expressed; are and a ciiyi euae Yvr tal., et (Yavari regulated is activity d PH-GFP PI4KIII w;;D a PI4KIII w ;P{w steesnilP IKin PI4K PM essential the as PI4KIII a – Sb/TM2 2–3 a rmLn oly(Yale Cooley Lynn from was } rbbyrgltsdistinct regulates probably a + PI4KIII PI4KIII , Ls hnhl fthe of half When GLCs. a tubP-GAL4 PI4KIII a Lsi on nthe in found is GLCs Drosophila Ls oee,our However, GLCs. , a a Ubx a uatgermline mutant a erequired be may a n PI3K-C2 and uatPFCs, mutant a rmTed from was }/ TM3,Sb PI4KIII fwd was and ˚ a C b , epae eepeae yPRapiiaino eoi N from DNA genomic of amplification PCR by w prepared were templates using pCaSpeR4 into subcloned 3 the containing ta. 02.Sok rmteBloomington the from were: IN) Stocks (Bloomington, 2012). al., et PI4KII n DAfamnswr ue tauiu internal with unique pBluescript a at into fused were fragments cDNA and ACGCGGCTACAAT-3 TCCGCACGCGTACCTAC-3 Kpn oiiaino E759rmvda diinl18k rmte3 the from kb 1.8 additional an of removed GE3785-9 between of region mobilization of intergenic exon of the first groups the removed into and deletion pooled initial flies, candidate An 750 10. from extracted DNA genomic using imprecisely ermn 92,the P{FRT(w 1992), Perrimon, Busrp with pBluescript 3 5 primers using PI4KIII FM7i ognrt eeinin deletion a generate To the of Generation uin h 5 The fusion. 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AAGAATGCCATATTTCGC-3 AGACGATG-3 TTCTCGCTAAGTA-3 control, GFP the of ability the assess odtrietelta eidof period lethal the determine To tesde sL ave n eepeue obe to presumed were and larvae, L1 as died All develop. others only to but allowed and hatched, plates juice embryos agar onto collected were embryos N otiigte5 the containing DNA Drosophila 3 the to joined xn6sat(5 start 6 exon (5 AATCCAACCT-3 were: sequences sequence Gene-specific promoter lgncetdsicue tpsrn)a5 a strand) (top included Oligonucleotides AATTAACCCTCACTAAAGGGAGA-3 9 1118 PI4KIII l(1)3Ah l(1)3Ah Mi B w y FM7i, ovo w ognrt Lsb h ehdo huadPrio Co and (Chou Perrimon and Chou of method the by GLCs generate To P{FRT(w sgg l(1)3Bb ete homozygous Neither PI4KIII I– n 5 and Ymlswr rse to crossed were males /Y PI4KIII 1 flies. Mlu eae eecosdto crossed were females /FM7a/Dp(1;2;Y)w a a uat eedsrbdpeiul Bile l,20;Burgess 2000; al., et (Brill previously described were mutants D D } GFP hs 21 4 21 eercvrdi qa ubr ofml siblings. female to numbers equal in recovered were /+ a a with ligated was fragment I 123 a P{FRT(w ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal /FM7a/Dp(1;2;Y)w ) , slik } hs /FM7a/Dp(1;2;Y)w iraryCnr,Msisua nai,Cnd) Genomic Canada). Ontario, Mississauga, Centre, Microarray 9 ( is a 14A-B -ATCTGCTGCACACCCTGGTA-3 9 PI4KIII ) fwd ae eercvrd indicating recovered, were males } af ossigo eoi N encoding DNA genomic of consisting half, 9 9 zw2 ,P{ 9 14A-B sN a mlfe rmtepGPN plasmid. pEGFP-N2 the from amplified was dsRNA sN a mlfe rmETL345 sanegative a As LD34405. EST from amplified was dsRNA ecetaseewsgnrtda genomic–cDNA a as generated was transgene rescue -CGATCAGTACCTCTCCTTTC-3 9 af otiigcN ETcoeS115 Canadian SD12145; clone (EST cDNA containing half, n 59 and -GCTCTAGAGCTTCGATATTTTCCGCTTTTTAGC- , Xba D 9 9 c21 T rmS115 mlfe sn 5 using amplified SD12145, from UTR Act-GFP n 5 and PI4KII – rnpss.Dltoswr dniidb C of PCR by identified were Deletions transposase. 2–3 hs n aacdover balanced and PI4KIII sgg a nall of allele an , Xba and I ) D } -CGGCAGACTATTGGGATTGT-3 9 9 123 ); 14A-B/C(1)DX/ PI4KIII 9 u etG38 nat(E759.Subsequent (GE3785-9). intact GE3785 left but , ecigrgo a mlfe from amplified was region rescuing noplecitwith pBluescript into , 9 and I -ACGAACAGTTCCAGCAGCTT-3 + PI4KIII ). 9 PI4KIII a -TTAATACGACTCACTATAGGGAGA-3 ovo w Kpn FM7i } PI4KIII and deletion PI4KIII M3C1D,yf y JMR3/C(1)DX, + 9 MiAct-GFP/ FM7i + Xma 9 a PI4KIII .cN ucoe rmS115a a as SD12145 from subcloned cDNA I. Xba n 5 and PI4KIII n 59 and rngn orsu viability, rescue to transgene Ylra ece h dl tg.The stage. adult the reached larvae /Y a sktl D a zw2 PI4KIII .TegnmccN osrc was construct genomic–cDNA The I. a xn6ed(5 end 6 exon n h blunt-cutters the and I P{ a D eeinwsrcmie with recombined was deletion E75(i.1)wsexcised was 1A) (Fig. GE3785 , 9 123 and , -CCCCCCGGGGGGCGTTAGA- FRT(w obesrne N (dsRNA) RNA double-stranded a -GGAGCACATCAGACACAGG- Y FM7i D a ; 9 123 r(otmsrn)a5 a strand) (bottom or ) P{hsFLP xn5(5 5 exon a 9 eae o hemizygous nor females ;; l(1)3Bb D ˚ 3pooe eune(5 sequence promoter T3 Lswr rdcdby produced were GLCs . hs Mlu 123 ae aha 8 2and 72 48, at bath water C ae.NnGP(male) Non-GFP males. 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Journal of Cell Science isses albd A.Eulaonso h 3adT transcription 95 T7 to and heated T3 mixed, were the products of amounts Equal CA). Carlsbad, Biosystems, T3 and T7 MegaScript using TT-3 o h oto ape 8 sample, control the For GAGGAGCAGGAACCAC-3 GAT-3 Lf ehoois n u naVi elTm C ytm(Life System ACTATGTGCTCAA-3 were: PCR amplification Real-Time for 7 5 used Kit ViiA primers Mix a Transcription Master Gene-specific on Select Technologies). run Reverse SYBR and with cDNA up Technologies) set (Life Capacity were reactions High qPCR TRIzol (Invitrogen). the using extracted was using cDNA RNA instructions. total prepared manufacturer’s and to PBS according Technologies) with (Life once rinsed were cells 0 MKH mM 100 niLa( itfo onSso,Uiest fTxsa utn 1:2000), Austin; at rabbit Texas 1987), of University al., Sisson, al., (Murthy et John et (Patel from (Queenan gift 1:200) 1:50) (a 1:300) (DSHB; (Cell anti-Lva (DSHB; 7G10 (DSHB; anti-FasIII 1D12 anti-phospho-Moe 22A2 mouse anti-Gurken 1999), mouse rabbit anti-Sec5 2003), al., mouse 1:700), et 1:700), University; Signaling; McGill Lasko, 2009). al., performed et (Hammond was previously described phosphates as phosphatidylinositol of Immunolocalization nehnlbfr diin(nirgnCr. albd A.ToPro in mounted CA). were samples Carlsbad, (0.1 and (Invitrogen) Corp., 1:1000 at (Invitrogen PPD used was addition dye before DNA ethanol in MgCl eiidb R-C splmnaymtra i.S1). Fig. material of (supplementary qRT-PCR knockdown by Double verified anneal. to temperature room nlclzto faGliPtdIns4 Golgi a of localization on TGTCGATACCCTT-3 GCAGGA-3 5 nlssfrcmaio fpo eesin treatments. dsRNA levels triplicate pMoe of PI4KII comparison for analysis 3 ARTICLE RESEARCH 964 distilled of solution (3:2:1 fixative B (Ma buffer H in procedures fixed standard were Ovaries using 2004). performed was Immunolocalization Immunocytochemistry 4 knockdown, double medium ml 1 PI4KIII in 8 of plates total 24-well a in containing 1 plated and were L-glutamine (Invitrogen) nM streptomycin 16.5 with supplemented Sigma) 420; experiments dsRNA in Drosophila knockdown mRNA of Quantification re-probed then anti- and 2004). MA) rabbit Danvers, al., (no. with Inc., anti-phospho-Ezrin/Radixin/Moesin Technology et Signaling rabbit (Hipfner Cell with 3141; were previously probed immunoblotting were described and Blots lysis as cell essentially treatments, performed dsRNA culture, cell treatment S2 dsRNA and culture Cell 5 AGTAGGGAGTGCCAATGAAGGTGT-3 CTG-3 AACA-3 GCGAGACCAT-3 xrse stertoo hshrltdt oa o inl,adwere and signals, were Moe in total levels observed to ratio phosphorylation the phosphorylated Moe to of normalized ratio 1.42q. the ImageJ as of expressed function using quantified ‘Gel’ were immunoblots the in Signals NC)]. Durham, University, (5 9 9 9 9 2 ); 9 -AAGAAGCGCACCAAGCACTTCATC-3 -TGACGCCGATCATCTCTTGTCCAT-3 -AGTCCCTCCAGGCAAACC-3 h olwn nioiswr sd abtat-sa agf rmPaul from gift (a anti-Oskar rabbit used: were antibodies following The :6 aaomleyebfe )fr1 iue.Bfe osssof consists B Buffer minutes. 15 for B) paraformaldehyde:buffer O:16% -GGTTGGTGGCCATGAACAA-3 PI4KII 9 2 hdmn–hlodnwsue t4Um,didadresuspended and dried U/ml, 4 at used was Rhodamine–phalloidin . n 59 and 9 9 ); a ncdw xeiet a efre ihrslsfrom results with performed was experiments knockdown 9 n 5 and sN ls4 plus dsRNA slik n 5 and 6 (5 2clsaatdt rwhi eu-remdu (EX-CELL medium serum-free in growth to adapted cells S2 9 2 9 -TGTTCTGCTGGTAGTGGTCG-3 ; PO -TTCGTGGAGGGTTACAAGG-3 (5 slik B,9%gyeo,1mg/ml 1 glycerol, 90% PBS, 9 9 -CGTAGACCTTGAAGCGGAAG-3 -TTAAGCCAAAGGACGAGGAACCCT-3 9 4 -CTCCAGTCACCACGGCTATTG-3 Drosophila /K ,5 9 ); 2 9 HPO -CCTGCATCGCAACAAAGTCATCCA-3 sktl m m 9 9 gof h feto Nio o hshrlto and phosphorylation Moe on RNAi of effect The . sN.Frsnl ncdws 4 knockdowns, single For dsRNA. g ; 4 m m PI4KII xn1sat(5 start 1 exon H68 5 MKl 5 MNC,2 mM 20 NaCl, mM 150 KCl, mM 450 6.8, pH fcnrl( control of g gof PI4KII 9 nvitro in o agf rmDne ihr (Duke Kiehart Daniel from gift [a Moe ); GFP lacZ ,5 P ˚ o 0mntsadcoe lwyto slowly cooled and minutes 10 for C 9 GFP akrwsvrfe ntshown). (not verified was marker 9 ls4 plus n 5 and 9 -TCTTCAGCTTGGCCTTCTGTC- sN eeue.Fv aslater, days Five used. were dsRNA rncito is(min Applied (Ambion, kits transcription (5 n 5 and 9 dRAtetdcls Statistical cells. -dsRNA-treated -GACGTAAACGGCCACAAG- lacZ 9 9 m 9 ; -AGCTGCAGAATAAACA- 9 -GCAGCAGGAACTGAAC- 9 gof -ACGCTGACGATTCATA- 9 rpl32 n 5 and sN eeue.For used. were dsRNA ) PI4KIII n 5 and 9 9 .dRAwsprepared was dsRNA ). n 59 and fwd p 9 9 9 fwd frnormalization), (for -phenylenediamine). -TTCTCGGTGCG- -ACGCACTCTGT- ); sN eeused. were dsRNA 9 a sktl -AAGGGAAAA- n 5 and versus and exon1middle 6 9 m ; PI4KII 9 gof penicillin– -CGACG- PI4KIII 9 fwd n 5 and slik ´ fwd was the and or a 9 ´ - , , , r140[anti-PtdIns(4,5) (anti-PtdIns4 1:100 at (Echelon 1:400 used were antibodies UT) or City, Lake IgM Salt Inc., phosphate Biosciences anti-phosphatidylinositol mouse g rIMscnayatbde ojgtdt lx lo 8 r568 1:1000. or at 488 used Fluor were Alexa CA) to Carlsbad, Invitrogen, conjugated Probes, antibodies 150 (Molecular secondary at IgM or used IgG was CA) Burlingame, esculentum Lycopersicon NV olwdb ue’ ariecmaio otts.Sec5, post-test. comparison pairwise Tukey’s for by PtdIns4 two-way distance and one-way followed cross-sectional using performed the greatest ANOVA was using analysis the scored Statistical across spot. were each chamber tool egg measurement representative were a line of plane Images one within 2003). Microscopy (Advanced Rollins, USA). software MA, Woburn, acquisition and Techniques, AmtV542 (Bazinet using obtained previously chamber, described egg the to relative morphology. oocyte cell the follicle of and shape most and the size and was of vitellogenesis, position WT basis nucleus in the oocyte on scored The staged hallmark(s). were developmental Spradling GLCs Photoshop prominent to Mutant Adobe according performed 1993). using was chambers manner (Spradling, levels, egg scanning identical WT for of an adjusted Staging inverted in CS5. were contrast Ti NY). comparison and (Melville, for Eclipse brightness software used AR images Nikon necessary, Elements Baden- a When NIS phosphate using (Oberkochen, with microscope phosphatidylinositol acquired confocal inverted software of were 4.8 100 staining Images a Axiovision Axiovert on Germany). Axiocam using or Wu¨rttemberg, Zeiss Zeiss a camera software with a microscope LSM510 CCD fluorescence upright using either 2 microscope Axioplan Zeiss on confocal scanning acquired laser were Images analysis and Imaging upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.129031/-/DC1 online available material Supplementary material Supplementary Cancer The and J.A.B.]; J.A.B.]. [to [to to Fund Inc. #106426 Society, Connaught and Toronto Research #81187 of MOP Canadian University numbers the the J.T.]; [grant D.R.H.]; [to Research Restracomp Health SickKids of by Institutes supported was work This Funding the co-wrote and project the the edited designed manuscript. and and experiments conceived dsRNA J.A.B. the manuscript; analyzed and the designed experiments; for D.R.H. dsRNA screen performed the K.O. designed manuscript; dsRNA J.B. the except co-wrote experiments and all experiments analyzed and performed designed, J.T. contributions Author interests. competing no declare authors The interests Competing for Heng Meng Yew microscopy. and electron Holmyard transmission with Doug with assistance Rollins, assistance for Janet Facility) and Michael Imaging imaging; reagents; (SickKids confocal and Paroutis flies Paul providing and generously Woodside for Bloomington GenExel the and Sisson, Bank, Center John Cooley, Hybridoma Lipshitz, Lynn Studies Howard manuscript; Erclik, Developmental Ted the the Kiehart, on Dan comments Lasko, Paul valuable Dorothea Schejter, helpful assistance; for Eyal for technical Lasko Schupbach for Paul Trudi Ma and and Jonathan Godt labs and Hipfner Bel and Del Lauren Brill discussions; the of members thank We Acknowledgements neste omlzdt WT. to normalized intensities hmeswr uniiduigIae .3.Saitclaayi of analysis and Statistical WT between 1.43u. PI4KIII ratios ImageJ intensity using phosphate:F-actin phosphatidylinositol quantified were chambers v n etnpnt iewsmaue sn ooiy4 Puncta 4. Volocity using measured was size puncta lectin and as Lva microscopy electron transmission for prepared were Ovaries P a PtdIns(4,5) , a efre ihtepie Student’s paired the with performed was ora fCl cec 21)17 5–6 doi:10.1242/jcs.129031 954–966 127, (2014) Science Cell of Journal PI4KIII P 2 n -ci nest eeso ersnaieegg representative of levels intensity F-actin and tmt)lci Vco aoaoisInc., Laboratories (Vector lectin (tomato) a P PI4KIII 2 Lsjde ob tg rltrby later or 8 stage be to judged GLCs .Fursen rTexas-Red-labeled or Fluorescein- ]. a m uatadeie h manuscript; the edited and mutant /l nirbi n anti-mouse and Anti-rabbit g/ml. ts sn average using t-test Drosophila Stock P )

Journal of Cell Science akvc,F,Sna . uacoih .adEd´y,M. Erde´lyi, and T. Luka´csovich, R., Sinka, F., Jankovics, ife,D . elr .adChn .M. S. Cohen, and N. Keller, R., D. W. Guo, Hipfner, and J. Zhang, X., Zhang, M. F., G. Xi, Carman, B., and He, D. S. Emr, J., D. Markley, A., Audhya, S., G. Han, auck,J,Ncls . opgo,J,Frsehr . od .and B. Goud, E., Formstecher, J., Compagnon, E., Nicolas, J., Januschke, amn,G . ice,M . nesn .E,Hlih . oei . Balla, A., Koteci, J., Holdich, E., K. Anderson, J., M. Fischer, R., G. Hammond, amn,G . civ,G n rie .F. R. Irvine, and G. Schiavo, R., G. Hammond, A. Guichet, and S. Roth, J., Januschke, S., Claret, L., Gervais, arno,L . tfn .J,MMra,M . m,S .adThorner, and D. S. Emr, A., M. McMurray, J., C. Stefan, S., L. Garrenton, cad A. Echard, adrv .adKe,J H. J. Keen, and I. Gaidarov, ain . e,H . oln,J,Ngci . lnesi,J T., J. Blankenship, T., Noguchi, J., Rollins, C., H. Wei, L., Fabian, olr . tukof . ihu,J n oe,R S. R. Cohen, and A. J. M. Michaud, Matteis, E., De Struckhoff, G., and Dollar, C. Wilson, M., Vicinanza, G., D’Angelo, ivt .T,Gura,A,Ry . e asr,L,Mnet . ovr,D. Louvard, P., Mangeat, L., Maestro, Del C., Roy, A., Gautreau, T., B. Fievet, otls .B n prsi A. Ephrussi, and B. J. Coutelis, J., N. Rollins, Perrimon, G., and B. Polevoy, T. Chou, B., Barylko, I., C. Ma, M., A. L. Wilde, Bel, and Del J., R. T.Burgess, Wong, M. Fuller, A., and J. M. Brill, Scharer-Schuksz, R., G. Hime, A., J. Brill, oad . a,L,X,J n oe,R S. R. Cohen, and J. Xu, L., Lan, Tepass, and N., J. Bogard, Sisson, M., Pellikka, O., Papoulas, P., Laprise, S., E. Beronja, J. Rollins, and C. Bazinet, al,A,Tyeoa . soek,A,V´ni .adBla T. Balla, and P. Va´rnai, A., Tsiomenko, G., Tuymetova, A., Balla, T. Balla, D. and A. S. Balla, Emr, and M. Foti, A., Audhya, D. S. Emr, and A. Audhya, M. Ashburner, References ARTICLE RESEARCH rslnsatnadcl ebaei rspiaoctsadi eurdfor required is and oocytes Drosophila in membrane anchoring. cell OSKAR and actin crosslinks euae osnatvt opooeeihla nert uigtsu growth. tissue plasma during the integrity epithelial to promote Dev. exocyst to Genes activity the Moesin of regulates targeting the mediates membrane. and 4- phospholipids phosphatidylinositol encodes gene LSB6 activity. kinase cerevisiae Saccharomyces The Drosophila. A. Guichet, ii eemnnso ebaeidentity. membrane of determinants F. lipid R. Irvine, and T. ehiusrva utpe itntclua ol fPdn4 and PtdIns4P of pools cellular distinct multiple, PtdIns(4,5)P(2). reveal establish techniques to oocyte. organization Drosophila microtubule the sustains in transport PIP2 polarized of production dependent hshtdlnstl45bshsht itiuini eurdfrMAPK for required is signaling. distribution phosphatidylinositol-4,5-bisphosphate J. 764. yokltn(Hoboken) Cytoskeleton o agtn opam ebaecahi-otdpits. clathrin-coated membrane plasma to targeting for 659. elmod,K,Plvy . evi,L,Giht . ulr .T tal. et T. M. Drosophila. Fuller, in and A., polarity localization cell Guichet, exocyst spermatid L., directs 4,5-bisphosphate Gervais, Phosphatidylinositol G., (2010). Polevoy, K., Bellamkonda, irtbl raiain n sa RAlclzto n translation. and localization mRNA oskar and trafficking, Development membrane between organization, link novel a microtubule oocyte: Drosophila the of polarization function. complex Golgi 270. in Phosphoinositides 1419-1430. eunilyi h ciainmcaimo ezrin. of mechanism activation the in M. sequentially Arpin, and n eemnn oaiaindrn rspiaoogenesis. Drosophila during localization determinant and Drosophila. Drosophila. in in chimeras germline proteins female produce granule secretory of trafficking Development A. regulates J. Brill, 4-kinase and Kra¨mer, H. P., J. Albanesi, bridge intercellular cytokinesis. and organization cytokinesis. actin germline during regulates formation kinase phospholipid oncin ewe emiese el n ih el nteDrosophila the in cells niche and cells stem ovary. germline between connections cells. photoreceptor U. spermiogenesis. 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