3829RESEARCH ARTICLE

INTRODUCTIONPolarity is a central feature of cell function and development. Theestablishment and maintenance of cell polarity rely on restricting thedistribution of proteins to particular cortical and cytoplasmicdomains. In many cases, this is achieved through asymmetrictransport organized along a polarized microtubule (MT)cytoskeleton. Polarization of the MT network is controlled by thepositioning of the microtubule-organizing center (MTOC) and bythe interaction of the MT array with the cell cortex, particularly thecortical actin cytoskeleton. It is clear that interactions between theplasma membrane and the cytoskeleton are central to theorganization of polarized transport, but the molecular mechanismsunderlying these processes are poorly understood.

The Drosophila oocyte, in which axis-determining mRNAs arelocalized to various cortical regions, has been successfully used tostudy the specification of cell polarity and the establishment ofpolarized transport (St Johnston, 2005). During mid-oogenesis, atleast two perpendicular subsets of MTs are formed, reflecting thedorsoventral (DV) and anteroposterior (AP) axes of the oocyte(Januschke et al., 2006; MacDougall et al., 2003). The axis-determining mRNAs are localized in a MT-dependent fashion(Riechmann and Ephrussi, 2001): bicoid (bcd) mRNA is localizedto the anterior cortex of the oocyte, oskar (osk) mRNA to theposterior pole, and gurken (grk) mRNA to an anterodorsal cap nearthe oocyte nucleus. In order to identify new factors involved in thepolarized transport of mRNAs in the Drosophila oocyte, we carriedout a germline mosaic screen for mutations on chromosome arm 2R

that disrupt AP and/or DV axis formation. In the course of thisscreen, we identified a type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles (Sktl), as an essential factor for oocytepolarization.

Type I PIP5K synthesizes phosphatidylinositol 4,5 bisphosphate(PIP2) from phosphatidylinositol 4-phosphate. The membranephospholipid PIP2 is involved in the control of cell polarity andplays a role in various cellular activities (Doughman et al., 2003).PIP2 regulates the membrane localization and activity of manycellular proteins via its specific interaction with phosphoinositide-binding domains (Downes et al., 2005). Although accumulated datasuggest that PIP2 is an important regulator of actin-based cellularprocesses, in vivo analysis of type I PIP5Kα during developmenthas been lacking. In Drosophila, Sktl has been shown to be requiredfor chromatin-mediated gene regulation (Cheng and Shearn, 2004)and is important in germline development (Hassan et al., 1998), butits cytoplasmic function remains to be determined.

In this work, we demonstrate that the PIP5K Sktl controls thePIP2 level at the plasma membrane. We show that sktl mutationsdisrupt the maintenance of oocyte polarity and cause defects in actinand MT organization. We provide evidence that PIP2 synthesis bySktl is required to activate the actin-associated protein Moesin at thecortex. Moreover, our observations indicate that Sktl activity isrequired for cortical recruitment of the PAR proteins Bazooka (Baz),Par-1, Lethal (2) giant larvae [Lgl; L(2)gl – FlyBase] to the cellmembrane. This study suggests that PIP2, by regulating severalproteins, could mediate interactions between the plasma membrane,PAR proteins and the cytoskeleton that are essential for cellpolarization.

MATERIALS AND METHODSFly stocksThe sktl2.3 allele was generated in a mutagenesis screen (25 mM EMS) andwas identified by sequencing the sktl gene amplified from heterozygousmutant adult DNA extracts. The sktlM5 allele was generated in an EMSallelic screen for sktl and is lethal for all other sktl alleles. To identify thegenomic location of the locus mutated in l(2)2.3, we used the Exelixis

PIP5K-dependent production of PIP2 sustains microtubuleorganization to establish polarized transport in theDrosophila oocyteLouis Gervais1, Sandra Claret1, Jens Januschke1,2, Siegfried Roth3 and Antoine Guichet1,*

The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membraneand elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, weshow that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization ofmicrotubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controlsthe establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasmamembrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton.Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cellmembrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.

KEY WORDS: Drosophila, Microtubules, PAR proteins, PIP2, PIP5K, Polarity

Development 135, 3829-3838 (2008) doi:10.1242/dev.029009

1Institut Jacques Monod, Unité Mixte de Recherche 7592, CNRS, Universités Paris 7,2 place Jussieu, F-75251, Paris Cedex 05, France. 2Cell Division Group, ICREA andIRB, Parc Cientific de Barcelona, c/Baldiri Reixac 10-12, 08028 Barcelona, Spain.3Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstrasse 17, 50923,Köln, Germany.

*Author for correspondence (e-mail: guichet@ijm.jussieu.fr)

Accepted 28 September 2008 DEVELO

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deficiencies collection (Parks et al., 2004). A lethal complementationanalysis revealed that Df(2R)Exel6070 fails to complement l(2)2.3.Overlapping deficiencies, Df(2R)Exel7164 and Df(2R)Exel7166,complement the allele. This enabled us to locate the mutation within a 150kb fragment containing thirteen genes. l(2)2.3 is lethal over sktlΔ20. sktlΔ20,sktlΔ5 and sktlΔ15 have been described (Hassan et al., 1998). Germline cloneswere generated by the FLP/FRT technique (Chou et al., 1993). nanosGAL4VP16 (nosgal4) (Van Doren et al., 1998) and α4tubulin67c GAL4 (tubgal4)(Januschke et al., 2002) were used to express the transgenes duringoogenesis. Other fly lines: GFP-Stau (Schuldt et al., 1998), Kin-lacZ (Clarket al., 1994), Dmn-GFP (Januschke et al., 2002), UAS-Baz-GFP and UAS-BazS151A,S1085A-GFP (Benton and St Johnston, 2003b), UAS-GFP-Par-1(N1S) (Doerflinger et al., 2006), UAS-GFP-Lkb1 (Martin and St Johnston,2003) and UAS-Lgl-GFP (Tian and Deng, 2008).

TransgenesThe polyubiquitin-PH-PLCδ-GFP construct was cloned from thepEGFPN1-PH-PLCδ plasmid into a pUbi-mod poly vector (a gift fromY. Belaiche, Curie Institute, Paris, France). The cDNA of sktl from DGRC(LP03320) was amplified, sequenced and subcloned into pUASP,pUASP:GFP, pUASP:RFP and pTub (a gift from P. Roth, TemasekLife Sciences Laboratory, Singapore) vectors to prepare Drosophilatransgenes.

Immunohistochemistry and in situ hybridizationImmunolocalization and in situ hybridization were performed using standardprotocols (Tautz and Pfeifle, 1989; Wilkie and Davis, 2001). Antibodieswere used at the following dilutions: anti-β-galactosidase (Promega), 1:200;anti-Osk (Hachet and Ephrussi, 2001), 1:2000; anti-Grk (DSHB), 1:200;anti-Stau (St Johnston et al., 1991), 1:5000; anti-phospho-ERM (CellSignaling Technology), 1:100; anti-Spn-F (CG12114) (Abdu et al., 2006),1:10; anti-DPLP (Cp309) (Kawaguchi and Zheng, 2004), 1:500; and anti-Baz (Wodarz et al., 2000), 1: 2000. Western blots were probed with anti-phospho-ERM at 1:5000, anti-Moesin at 1:30,000, and anti-α-Tubulin at1:2500. F-actin was visualized after staining with Alexa Fluor 680-phalloidin (Molecular Probes). DAPI and propidium iodide were used forDNA detection. Texas Red labeled Lycopersicon esculentum (LE) lectin(Vector Laboratories) was used at 150 μg/ml and Wheat germ agglutinin(WGA) at 1/100 (Molecular Probes). Anti-Khc (AKIN02-A, Cytoskeleton)was used for MT detection. Cold-shock experiments were performed asdescribed (Januschke et al., 2006). Images were obtained with a Leica SP2AOBS microscope.

RESULTSSkittles is required for anteroposterior anddorsoventral axis formationWe carried out a germline mosaic screen for mutations onchromosome arm 2R that disrupt AP and DV axis formation in theDrosophila oocyte. To visualize the AP axis, we used, as previouslydescribed (Martin et al., 2003), the protein Staufen (Stau) tagged withgreen fluorescent protein (GFP), which moves to the posterior of theoocyte with osk mRNA (Fig. 1A) (Bolivar et al., 2001; St Johnstonand Nusslein-Volhard, 1992). To visualize DV axis formation, wescored the positioning of the nucleus relative to the anterodorsal cornerof the oocyte (Fig. 1A, asterisk). One homozygous mutant lineaffecting both nuclear positioning and Stau localization was identifiedin the screen and named l(2)2.3. In 57% of such egg chambers, Stauaccumulated in the center of the oocyte (Fig. 1B), whereas in theremainder Stau was diffusely distributed in the cytoplasm (not shown)(n=118). The nucleus was randomly positioned within the oocyte atstages 9-10 (Fig. 1B, asterisk). Eggs deposited by l(2)2.3 mutantfemales were ventralized [loss of dorsal appendages 51%, fusedappendages 43%, wild type (WT) 6%; n=116] (Fig. 1D), but alwayspresented an aeropyle at the posterior (Fig. 1D, inset). Hence, theseresults indicate that l(2)2.3 affects AP and DV axis formation withoutimpairing the early signaling step from the germline to the posterior

follicle cells (Gonzalez-Reyes et al., 1995; Roth et al., 1995). Finally,posterior follicle cell clones mutant for l(2)2.3, marked by the loss ofGFP, did not affect oocyte polarization, as revealed by the posteriorlocalization of Stau (Fig. 1E), whereas mutant clones restricted to thegermline impaired both nucleus positioning (Fig. 1F, asterisk) andStau localization (Fig. 1F). This demonstrates that the l(2)2.3phenotype is strictly germline dependent.

With the help of three overlapping deficiencies, we mappedl(2)2.3 to a 150 kb interval (see Materials and methods). We testeda series of lethal mutations in the genes of this region and found thatl(2)2.3 is an allele of sktl. Whereas sktlΔ20, which is reported to be anull allele (Hassan et al., 1998), is lethal in trans to l(2)2.3, sktlΔ5 andsktlΔ15 are female-sterile in trans to l(2)2.3. sktlΔ5/l(2)2.3 females laidventralized eggs and their oocytes had defects in the localization ofboth the nucleus and Stau (data not shown).

Molecular characterization revealed that the l(2)2.3 mutationcomprises a small deletion (276 bp) in the unique intron of sktl, asdo sktlΔ5 and sktlΔ15. l(2)2.3 results in a large reduction in sktltranscript levels in nurse cells and oocytes (Fig. 1, compare H withG), confirming that l(2)2.3 is a hypomorphic allele of sktl similar tosktlΔ5 and sktlΔ15. Finally, the lethality and oogenesis phenotypeswere rescued by ubiquitous expression of a sktl cDNA transgene,

RESEARCH ARTICLE Development 135 (23)

Fig. 1. sktl affects axis formation in the Drosophila oocyte.(A,B) Egg chambers expressing GFP-Stau. (A) Wild type (WT). (B) l(2)2.3mutant germline clone. (C,D) Dorsal view of an egg shell. (C) In WT, thetwo appendages mark the dorsal side of the egg. (D) In the l(2)2.3mutant germline clone, the egg is ventralized and lacks dorsalappendages, but the posterior aeropyle is normal (compare insets in Cand D). (E,F) Homozygous l(2)2.3 mutant clones, marked by theabsence of GFP (green). Stau protein localization (red) and nucleuspositioning are not affected when the posterior follicle cells are mutant(E), but are lost in mutant germline clones (F). (G,H) sktl mRNAexpression. The level of sktl mRNA (blue) is reduced in l(2)2.3 mutant(H) as compared with WT (G) nurse cells. Anterior is to the left andposterior is to the right. Asterisk, nucleus.

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confirming that these defects are caused by mutations in this gene.Germline clones, mutant for sktlΔ20 and sktlM5 (see Materials andmethods), arrested oogenesis at stage 4 (see Fig. S1A in thesupplementary material).

Early oogenesis can be separated into different steps:determination, polarization along the AP axis and, subsequently, themaintenance of this polarity (for a review, see Huynh and StJohnston, 2004). The early arrest found in null alleles of sktl is notdue to oocyte determination defects. Indeed, Orb, a suitable markerfor oocyte determination, was restricted as normal to one cell in thecyst (see Fig. S1I in the supplementary material). Furthermore, Orb,and the centrosomes as revealed by γ-Tubulin, were correctlytranslocated to the posterior of the oocyte suggesting that early APpolarization takes place normally in the mutant egg chambers (seeFig. S1G,I in the supplementary material). However, starting fromstage 2, posterior components such as Orb started to lose theirposterior restriction (see Fig. S1I, white arrowhead, in thesupplementary material) and were eventually found mislocalizedthroughout the cyst. Thus, Sktl is not required for the determinationof the oocyte during its polarization in region 3 of the germarium.However, Sktl appears to be required for the maintenance of oocytepolarity. We therefore conducted our analysis with the hypomorphicsktl alleles from which we were able to obtain vitellogenic oocytes,enabling us to study the function of Sktl during the later stages ofoocyte polarization (mid-oogenesis).

We investigated whether Sktl is required for polarized transport, i.e.for asymmetric mRNA localization. To score for mRNA localizationdefects along the DV axis, we analyzed grk mRNA. In sktl2.3 germlineclones, up until stage 8, grk mRNA accumulated above the nucleus inthe anterodorsal corner of the oocyte (Fig. 2C), as in the WT (Fig. 2A).However, during stage 9, grk mRNA became mislocalized into thecytoplasm, where it accumulated around the mispositioned nucleus(Fig. 2D) (85%, n=64). Likewise, when analyzing mRNA localizationalong the AP axis, we found that bcd mRNA was localized correctlyto the anterior margin up until stage 8 (Fig. 2G), but becamemislocalized in the oocyte during stage 9, forming a ring around themispositioned nucleus (Fig. 2H) (88%, n=43). In the WT, osk mRNAtransport toward the posterior pole of the oocyte is achieved duringstage 9, and Osk is subsequently translated at the posterior (Markussenet al., 1995; Rongo et al., 1995). During stage 8, when bcd and grkhave reached their final location (Fig. 2A,E), osk mRNA accumulatestemporarily in the center of the WT oocyte (Fig. 2I), before it reachesthe posterior pole (Fig. 2J). In sktl mutant oocytes, osk mRNAlocalized normally to the center of the cytoplasm during stage 8 (Fig.2K). However, during the subsequent stages, it failed to reach theposterior and remained in the middle of the oocyte (Fig. 2L) (98%,n=57). Moreover, Osk protein was never translated in these mutants(see Fig. S2B in the supplementary material). These results indicatethat Sktl is required for mRNA transport along the AP and DV axes ofthe oocyte.

3831RESEARCH ARTICLEPIP5K sustains microtubule organization and polarized transport

Fig. 2. mRNA localization and nucleus positioning defects in the sktl mutant oocyte. Wild-type (A,B,E,F,I,J) and sktl2.3 mutant (C,D,G,H,K,L)Drosophila germline clones. (A-D) grk mRNA distribution. In stage 8 sktl2.3 mutants, grk mRNA (blue) localizes as in WT (compare C with A).However, in sktl2.3 stage 10 oocytes, grk mRNA is mislocalized with the nucleus (compare D with B). (E-H) bcd mRNA distribution. In stage 8 sktl2.3

mutant oocytes, bcd mRNA (blue) localizes as in WT (compare G with E). In stage 10 sktl2.3 oocytes, bcd mRNA is mislocalized in the form of acortical ring close to the nucleus (compare H with F). (I-L) osk mRNA localization. In stage 8 sktl2.3 oocytes, osk mRNA (brown) localizes as in WT(compare K with I). However, during stage 10, osk mRNA does not reach the posterior, unlike in WT (compare L with J), and is mislocalized in thecytoplasm. (M,N) Stage 10 sktl2.3 mutant egg chamber expressing GFP-Stau (green) and stained for DNA (red). (M) The oocyte nucleus is notanchored to the anterior cortex and is mislocalized in the cytoplasm. (N) Optical cross-section (at the level of the opposing arrowheads in M)showing that the anteriorly detached nucleus remains attached to the lateral cortex of the oocyte. Asterisk, nucleus.

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Sktl controls nuclear anchoring and microtubulenetwork organizationIn the oocyte, the anterodorsal positioning of the nucleus involvesat least two distinct steps. First, during the transition from stage 6 to7, the oocyte nucleus migrates from a posterior to an anterodorsalposition in the cytoplasm. Then, the nucleus is anchored at theanterodorsal cortex. This attachment is a MT-based processinvolving at least two distinct mechanisms for anchoring to theanterior and the lateral cortex, respectively (Guichet et al., 2001;Januschke et al., 2002; Koch and Spitzer, 1983). In sktl2.3 mutantoocytes, there was no defect in oocyte nucleus migration duringstage 7 (data not shown), and at stage 8 the nucleus was alwayscorrectly localized (Fig. 2C,K, asterisks). However, in 80% of stage9 (n=54) and 96% of stage 10 (n=48) oocytes, the nucleus wasmislocalized (Fig. 2M). Optical cross-sections of sktl2.3 mutantoocytes revealed that the mislocated nucleus was still tightlyassociated with the lateral cortex (Fig. 2N). These results indicatethat Sktl is required for the anterodorsal maintenance of the nucleusand, more specifically, for its anterior anchorage.

Previous work has indicated that the nucleus appears to have aninfluence on MT organization (Guichet et al., 2001; Januschke et al.,2006). We investigated whether the inactivation of Sktl affects MTorganization. In sktl2.3 germline clones, we observed that up to stage8, the MT network is organized as in controls (Fig. 3A,B). However,from stage 9 onwards, the anterior-to-posterior MT organizationseemed to be lost in the sktl2.3 germline clones. Indeed, the MT arraywas organized around the mislocalized nucleus (Fig. 3D), in contrastto the MT organization in the WT (Fig. 3C). We next analyzed thedistribution of various markers for MT polarity. MT plus ends can bemarked by a fusion of the Kinesin heavy chain with β-galactosidase(Khc-β-gal) (Clark et al., 1994). In WT oocytes, Khc-β-gal localizesto the posterior cortex during stage 9, revealing that the MT plus endsare pointing towards the posterior (Fig. 3E). In sktl2.3 germline clones,Khc-β-gal was never detected at the posterior and, in 23% of the eggchambers (n=79), it accumulated in anterior regions of the cytoplasm(Fig. 3F). Likewise, with a different marker for MT plus ends,Dynamitin-GFP (Dmn-GFP), (Duncan and Warrior, 2002; Januschkeet al., 2002), we found that the posterior localization of Dmn-GFP

RESEARCH ARTICLE Development 135 (23)

Fig. 3. Microtubule organization in sktl mutantoocyte. WT (A,C,E,G,I,K,N,O) and sktl2.3 mutant(B,D,J,L,M) Drosophila germline clones andsktl2.3/sktlΔ5 mutant egg chambers (F,H,P,Q).(A-D) Microtubule (MT) network detected with Khcantibody. In stage 8 sktl2.3 oocytes, MT networkorganization is not affected (compare B with A).However, in stage 10 sktl2.3 oocytes, MTorganization is disrupted (compare D with C). MTbundles appear to project from the mislocalizednucleus towards the center of the cytoplasm(arrowheads in D). (E,F) Khc-β-gal (red) and DNA(green) staining. In sktl2.3/sktlΔ5 oocytes, theposterior pool of Khc-β-gal is lost and it accumulatesin the anterior part of the oocyte (F). The inset in Fshows Khc-β-gal staining alone. (G,H) Dmn-GFP. Insktl2.3/sktlΔ5 oocytes, the posterior pool of Dmn-GFPis lost and an accumulation is found in the mostanterior part of the cytoplasm (H). However, cortical-and nuclear-associated localization are unaffected,even when the nucleus is mislocalized. (I,J) In bothmutant and WT, the centrosome component DPLP(red) is detected around the oocyte nucleus and as adot close to it (arrowhead). The dots surroundingthe oocyte correspond to the centrosomes of thefollicular cells. (K-M) Projections of confocal sectionsfrom oocytes immunostained with Spn-F antibodyrevealing the minus ends of the MTs. Spn-F is foundmislocalized in a characteristic cortical ring close tothe mislocalized nucleus in stage 10 sktl2.3 oocytes(compare M with K), but not in stage 8 (L). (N-Q) MTdetection by anti-Khc. WT (N) and sktl2.3/sktlΔ5 (P)oocytes showing complete depolymerization of MTsafter 30 minutes of cold shock. (O,Q) WT andmutant oocytes after a 10-minute recovery at 25°C.MTs regrow from the misplaced nucleus (arrowheadsin Q). Asterisk, nucleus.

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seen in WT was always lost in sktl2.3 mutant oocytes, where itaccumulated at the anterior in 33% of the cases studied (n=35) (Fig.3, G versus H). This is in agreement with the results obtained withKhc-β-gal (Fig. 3F). Hence, it appears that MT plus ends can beectopically located close to the anterior of the oocyte, indicating thatsktl mutation prevents the correct orientation of the MTs.

Interestingly, Dmn is also a suitable marker for revealing the MTminus ends associated with the nucleus of the oocyte (Duncan andWarrior, 2002; Januschke et al., 2002) (Fig. 3G, red arrowhead). Insktl2.3 germline clones, Dmn-GFP was found around themislocalized nucleus (Fig. 3H, red arrowhead). In addition, MTminus ends in the WT are also distributed along the anterior marginof the oocyte (Clark et al., 1997; Theurkauf et al., 1993). These MTminus end extremities are especially highlighted by Spindle-F (Spn-F) (Fig. 3K) (Abdu et al., 2006) and can also be revealed by thelocalization of bcd mRNA (Fig. 2E,F) (Cha et al., 2001). In sktl2.3

mutant oocytes, Spn-F and bcd mRNA were distributed along theanterior margin up to stage 8 (Fig. 3L; Fig. 2G). However, fromstage 9 onwards, Spn-F formed a cortical ring at the position of themisplaced nucleus (Fig. 3M). This localization is very reminiscentof the localization of bcd mRNA in sktl2.3 mutant oocytes (Fig. 2H).Taken together, these observations suggest that MT minus ends arepresent as a cortical ring around the mispositioned nucleus.

Since the nucleus may function as a MT nucleation center in theoocyte (Januschke et al., 2006), we investigated whether themisplaced nucleus is still associated, as in WT, with centrosomalcomponents such as DPLP (Cp309 – FlyBase) (Januschke et al.,2006). In both WT (Fig. 3I) and sktl mutant (Fig. 3J) oocytes, DPLPwas localized around the nucleus, and as a bright dot in the vicinity ofthe nucleus possibly corresponding to the centrosome. In order to testthe MT-nucleating capacity of the mispositioned nucleus, we used acold-induced MT disassembly assay (Januschke et al., 2006). As in

the WT, during the initial period of recovery at 25°C after completedepolymerization through cold-shock treatment (Fig. 3P), MTpolymerization only took place in the immediate vicinity of themispositioned oocyte nucleus (Fig. 3Q). Therefore, as in WT oocytes,MT nucleation in the sktl mutant is asymmetric and mainly restrictedto the area surrounding the nucleus. However, we cannot exclude thepossibility that we might have missed some MT nucleation activity atthe cortex as a result of the experimental set-up. These results confirmthe previously described role of the nucleus as the main active MTOCin the oocyte (Januschke et al., 2006). To conclude, in sktl mutants, theMT array is reorganized around the delocalized nucleus with a reverseposterior-to-anterior orientation and this is very likely to beresponsible for mRNA mislocalization.

The PIP5K Sktl is cortically localized and controlsthe level of PIP2 at the plasma membranesktl encodes a putative ortholog of a type I PIP5K (Knirr et al., 1997)that catalyzes the phosphorylation of phosphatidylinositol 4-phosphate to generate the phosphatidylinositol 4,5 bisphosphate(PIP2), a major component of the plasma membrane. We examinedthe localization of a GFP-tagged Sktl protein by expressing a UASp-GFP-sktl transgene under the control of the germline-specific drivernanosGal4 (nosgal4). This combination rescues the phenotypes ofsktl2.3/sktl2.3 and sktlΔ5/sktl2.3 mutant oocytes, indicating that theGFP-sktl transgene is functional. From the early stages of oogenesisuntil the development of mature egg chambers, Sktl-GFPaccumulated at the cortex just below the plasma membrane of theoocyte and nurse cells, where it colocalized with the actincytoskeleton (Fig. 4A-D). A fraction of Sktl-GFP was detected ascytoplasmic particles (Fig. 4A). The localization of Sktl at theplasma membrane is consistent with its involvement in PIP2synthesis (Oude Weernink et al., 2004).

3833RESEARCH ARTICLEPIP5K sustains microtubule organization and polarized transport

Fig. 4. Sktl controls the PIP2 level at the oocyte cortex.(A-D) nosgal4>GFP-Sktl egg chambers. GFP-Sktl (A, green in C,D)colocalizes with actin (B, red in C,D) along nurse cell and oocytecortex (merge in C,D). (E-H) PH-GFP distribution. PH-GFP (E, greenin G,H) localizes at the actin (F, red in G,H) cortex of follicular cells,nurse cells and oocyte (merge in G,H). (I-K) Colocalization betweenPH-GFP (I, green in K) and nosgal4>RFP-Sktl (J, red in K) along theoocyte cortex and in the cytoplasm. (L-N) Wild-type Drosophila eggchamber expressing PH-GFP (L, green in N), co-stained with theplasma membrane marker LE lectin (M, red in N). PH-GFP localizesall along the plasma membrane of the oocyte (merge in N). Insetsin L and N are magnifications of anterior margin and lateral cortex,respectively. (O-Q) sktl2.3 mutant germline clones expressing PH-GFP (O, green in Q) co-stained with LE lectin (P, red in Q). Theaccumulation of PIP2 marker is greatly reduced all along the oocyteplasma membrane. Insets in O and Q are magnifications of anteriormargin and lateral cortex, respectively. Arrows and arrowheads(I,J,K,L,N,O,Q) indicate the plasma membrane of the oocyte andfollicle cells, respectively. Asterisk, nucleus.

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In order to monitor the distribution and the level of PIP2, wegenerated a PLCδPH-GFP transgene containing GFP fused to thePIP2-specific pleckstrin-homology domain of phospholipase Cδ(Balla et al., 1998). During oogenesis, the PIP2 reporter wasspecifically distributed along the plasma membrane in both thegermline and follicle cells (Fig. 4E,I, arrow and arrowhead).Moreover, PIP2 reporter colocalized with the glycosamyl-modifiedproteins present in the oocyte plasma membrane (Fig. 4L-N) andwith the cortical actin cytoskeleton (Fig. 4E-H). Sktl (Fig. 4J) andPIP2 (Fig. 4I) colocalized along the oocyte cortex (Fig. 4K, arrowin the inset).

To address the potential PIP5K activity of Sktl at the oocyteplasma membrane, we analyzed the distribution of the PIP2 reporterin sktl mutant germline clones (Fig. 4O-Q). We observed a severedecrease in the level of PIP2 reporter all along the oocyte cortex(Fig. 4, compare arrows in the insets, O with L and Q with N). Ourresults strongly implicate Sktl in the control of PIP2 levels at theplasma membrane, suggesting that Sktl is required for PIP2synthesis in the Drosophila oocyte.

Sktl regulates PAR polarity along the AP axisHow might Sktl regulate the positioning of the nucleus and thelocalization of mRNAs in the oocyte? The mutually antagonisticinteractions between the PAR proteins Baz and aPKC at theanterior, and between Par-1 and Lgl at the posterior, are required formRNA localization and MT organization in the oocyte during mid-oogenesis (Benton and St Johnston, 2003a; Martin and St Johnston,

2003; Shulman et al., 2000; Tian and Deng, 2008; Tomancak et al.,2000). Furthermore, some elements of these PAR complexes, suchas Lgl and Par-1, are also involved in the anterodorsal positioningof the nucleus (Doerflinger et al., 2006; Tian and Deng, 2008).Interestingly, it has recently been shown that in MDCK cells, Par-3 recruitment to the plasma membrane requires PIP2 (Wu et al.,2007). Thus, we examined whether Sktl activity is required for Bazlocalization. In the oocyte, Baz-GFP localizes strongly at theanterior and lateral cortex, but is excluded from the posterior (Fig.5A) (Benton and St Johnston, 2003a; Benton and St Johnston,2003b). In sktl2.3/sktlΔ5 mutant oocytes, Baz-GFP was less enrichedalong the anterolateral cortex, but became distributed in thecytoplasm (Fig. 5B) (52%, n=63). Furthermore, the requirement forSktl to control Baz localization is not restricted to the oocytebecause in follicle cell clones mutant for sktlM5, the apicalrestriction of Baz was lost and the protein became ectopicallydistributed in the cytoplasm (Fig. 5C,D). Thus, Sktl activity isrequired to recruit Baz at the membrane in the oocyte and epithelialfollicle cells. Because the deficit of PIP2 might have a global effecton the oocyte cortex, we tested whether Lkb1 (the Drosophila PAR-4 homolog), which is known to be bound to the plasma membranethrough a prenylation motif (Martin and St Johnston, 2003), wasstill localized in the absence of Sktl. We found that in sktl2.3

germline clones, GFP-Lkb1 localized to the oocyte cortex as in WT(Fig. 5E,F). This indicates that the delocalization of Baz observedin sktl mutants is due to the decrease in PIP2 level, rather than to acomplete disorganization of the cortex.

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Fig. 5. Sktl is required to properly organize the cortical domains of the oocyte. (A) Baz-GFP localizes strongly at the anterolateral cortex ofthe WT Drosophila oocyte. (B) In sktl2.3/sktlΔ5 oocytes, Baz-GFP has lost most of its cortical localization and is found as puncta in the cytoplasm.(C,D) skltlM5 follicle cell clone (FCC; dashed line) marked by the absence of GFP (green) and stained for Baz (red in C, white in D). (E,F) Corticallocalization of Lkb1 (E) is maintained in sktl2.3/sktlΔ5 oocytes (F). (G) Baz-GFPS151A,S1085A overexpression induces oocyte polarity defect phenotypes,with nucleus mispositioning and ventralized eggs (inset in G). (H) Lgl-GFP is localized at the posterior of the oocyte. (I) In sktl2.3/sktlΔ5 oocytes, Lgl-GFP is no longer enriched at the posterior and is instead detected mainly in the cytoplasm and along the cortex. (J) GFP-Par-1 (N1S) is enriched atthe posterior at stage 9 (arrowhead). (K) In sktl2.3/sktlΔ5 oocytes, GFP-Par-1 (N1S) is no longer enriched at the posterior and instead has started toaccumulate all along the cortex and in the cytoplasm. Asterisk, nucleus.

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Previous analysis revealed that the restricted localization of Bazis essential for oocyte polarity. Indeed, overexpression of a mutatedform of Baz that cannot be phosphorylated by Par-1(BazS151A,S1085A-GFP), induced a delocalization of Baz all along thecortex and some accumulation in the cytoplasm associated withpenetrant oocyte polarity phenotypes (Benton and St Johnston,2003b). Interestingly, we found that the overexpression of thismutated form of Baz affected the positioning of the nucleus, as insktl mutants (Fig. 5G). Thus, we questioned whether Sktl and Bazmight act together to polarize the oocyte. Using a sktl2.3/sktlΔ15

hypomorphic combination, in which the delocalization of the oocytenucleus occurs in only 31% of cases examined (Table 1), we foundthat the nucleus was mislocalized in 63% when the gene dosage ofbaz is lowered (Table 1). Thus, sktl and baz interact genetically.Taken together, these results suggest that the targeting of Baz to thecortex, as mediated by Sktl, could be required for the positioning ofthe nucleus and for MT organization.

Cross-regulatory interactions between the aPKC-Baz complexand Lgl and Par-1 are crucial in order to establish complementarycortical domains in polarized cells. It has been shown that aPKCphosphorylation of Lgl restricts Lgl activity and Par-1 enrichmentto the posterior of the oocyte (Tian and Deng, 2008) (Fig. 5H,J).Thus, we analyzed the localization of the fusion proteins Lgl-GFPand GFP-Par-1 (N1S isoform) in sktl2.3/sktlΔ5 oocytes. Interestingly,Lgl and Par-1 were no longer restricted to the posterior but becamedistributed uniformly along the cortex and started to accumulate inthe cytoplasm (Fig. 5I,K) (Lgl mislocalization, 100%, n=30; Par-1mislocalization, 100%, n=8). These results indicate that Sktl isrequired for the maintenance of complementary compartments at theoocyte cortex along the AP axis, and by this means might regulateoocyte polarity during mid-oogenesis.

Sktl controls the cortical organization of theF-actin cytoskeleton and the activation of MoesinInterestingly, in a hypomorphic combination of sktl2.3 and sktlΔ15

alleles, the delocalization of the oocyte nucleus occurred in only 31%of the oocytes, as compared with 90% in sktl2.3/sktl2.3 (Table 2), andthe mislocalization of bcd and grk mRNAs was similarly decreased.However, osk mRNA was still found diffusely distributed in thecytoplasm of 62% of the oocytes (n=47) (Table 2). This suggests thatSktl might also control osk mRNA by a mechanism independent ofnucleus positioning. Previous work has shown that the anchorage ofosk mRNA to the posterior oocyte cortex is actin dependent (StJohnston, 2005). We addressed the relationship between Sktl and actinorganization. In WT oocytes, the microfilaments form a continuouslayer along the entire cortex and the orientation of the actin bundles is

parallel to the plasma membrane (Robinson and Cooley, 1997) (Fig.6C-E). Sktl colocalizes with actin (Fig. 4C,D). In the sktl2.3 mutant,the organization of actin filaments appeared to be normal during earlyoogenesis (data not shown), but from stage 8 onwards we detected twodistinct types of actin defect. In 36% of oocytes (n=118), the corticalactin network was disrupted at the border between the anterior marginand the lateral cortex (Fig. 6H-J). In 15%, the actin microfilaments inthe oocyte were loosely bound to the lateral cortex, including to theposterior pole, and they delaminated into the cytoplasm (Fig. 6M-O).It is important to emphasize that the continuity of the plasmamembrane was unaffected in the sktl mutant, as revealed by LE lectinlabeling (Fig. 6G,L). Thus, impairing Sktl function leads todisorganization of the microfilament scaffold along the oocyte cortexand to detachment of cortical actin. Mutations in several actin-relatedgenes disrupt mRNA localization by inducing premature MT-basedcytoplasmic streaming (Emmons et al., 1995; Manseau et al., 1996;Theurkauf, 1994). However, in sktl mutant oocytes, we did notobserve the early movement (stages 7-9) of yolk particles that ischaracteristic of premature cytoplasmic streaming (data not shown).

Several proteins known to modulate the actin cytoskeleton areregulated by PIP2 (Niggli, 2005). Among these, Moesin, which linksthe actin cytoskeleton to the plasma membrane, is required for oskmRNA localization without affecting cytoplasmic streaming(Jankovics et al., 2002; Polesello et al., 2002). Previous ex-vivostudies have shown that Moesin activation is promoted by PIP2binding to its FERM domain (Barret et al., 2000; Niggli et al., 1995).We evaluated whether Sktl function is required for Moesinactivation by examining the level of Moesin phosphorylation in sktlmutants using an antibody specific to phosphorylated ERM proteins(Polesello et al., 2002). In the WT, we detected the phosphorylatedform of Moesin (P-Moe) all along the cortex of the oocyte and nursecells (Fig. 7A). In sktl2.3 germline clones, P-Moe distribution at thecortex was lost (Fig. 7B). Accordingly, an immunoblot analysisshowed that P-Moe was dramatically reduced in sktl2.3/sktlΔ5 eggchambers (Fig. 7C), whereas the level of total Moesin remainedunchanged (Fig. 7D). Taken together, these results indicate that Sktlis required for the activation of Moesin in the oocyte and couldexplain the defects in actin organization in the sktl mutant.

DISCUSSIONBinding interactions between the plasma membrane and thecytoskeleton are important for the establishment of cell polarity.PIP2 is essential for the maintenance of these interactions byinfluencing the activity of several proteins that regulate thearchitecture of the cytoskeleton. This study provides in vivoevidence of a function for PIP5KIα in cell polarization.

3835RESEARCH ARTICLEPIP5K sustains microtubule organization and polarized transport

Table 1. Genetic interactions between sktl and baz allelesWT sktl2.3/sktl2.3 sktl2.3/sktlΔ15 baz4/+; sktl2.3/sktlΔ15

Mislocalized nucleus (%) 0 87 31 63*n† 48 102 74 87

*P=0.001, comparison with sktl2.3/sktlΔ15, χ2 test.†Number of oocytes scored at stage 9-10.

Table 2. Mislocalization of nucleus and mRNAs in sktl mutant oocytesGenotype Mislocalized nucleus % (n) Mislocalized bcd mRNA % (n) Mislocalized grk mRNA % (n) Mislocalized osk mRNA % (n)

WT 0 (48) 3 (37) 4 (43) 3 (75)sktl2.3/sktl2.3 87 (102) 85 (64) 88 (43) 98 (57)sktl2.3/sktlΔ15 31 (97) 20 (43) 18 (47) 62 (47)

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Our results indicate that the type I PIP5K, Sktl, is essential forPIP2 synthesis in the Drosophila oocyte. PIP2 directly controls thelocalization and activity of many proteins via its interaction withphosphoinositide-binding domains. Among them, the activation ofERM proteins such as Moesin, results in the unmasking of theirfunctional binding sites. Our study indicates that in vivo, PIP2provided by a PIP5K is required for Moesin phosphorylation,supporting results obtained previously with cellular systems (Fievetet al., 2004; Lacalle et al., 2007). Since PIP2 is also required for PIP3synthesis, it is possible that Sktl also affects PIP3 production. It willbe interesting to investigate the requirements of PIP3Ks duringmiddle oogenesis and to compare them with those of Sktl.

We showed that Sktl is necessary for the positioning of the oocytenucleus, for MT organization and for the localization of grk, bcd andosk mRNAs. The nature of the MT regrowth after cold-induced MTdisassembly in sktl mutants is consistent with previous observationssuggesting that the nucleus functions as the main MT nucleation centerin the oocyte, influencing the organization of the MT network and,thereby, mRNA asymmetric transport (Guichet et al., 2001; Januschkeet al., 2006). Since MT minus ends are found as a cortical ring aroundthe nucleus, we propose a model whereby, in sktl mutants, MTsnucleated from the nucleus might be translocated to, and anchored at,the surrounding cortex, as previously proposed for anchoring at theanterior margin in the WT (Guichet et al., 2001; Januschke et al., 2008).

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Fig. 6. Sktl is required for actin organization. Wild-type (A-E) and sktl2.3 mutant (F-O) Drosophila germline clones co-stained for plasmamembrane (B,G,L, green in merge A,F,K) and F-actin (C-E,H-J,M-O, red in merge A,F,K). Interruption of the actin cortex along the anterior margin isfound in 36% of mutant oocytes (arrowheads in I,J). Ectopic F-actin structures are observed in 15% of mutant oocytes. D,I,N are magnificationsfrom C,H,M, respectively. E,J,O are optical cross-sections of C,H,M, respectively, at the level of the red arrowheads. Asterisk, nucleus.

Fig. 7. Sktl activity is required for Moesin phosphorylation. (A,B) Immunostaining using an antibody specific for the phosphorylated form ofMoesin (P-Moe). P-Moe staining is found at the cortex in WT Drosophila oocytes (red in A) and is abolished in sktl2.3 mutant germline clonesmarked by the loss of GFP (B). (C,D) Western blot of ovary extracts (1, 2 and 4 mean that the equivalent of 1, 2 and 4 ovaries were loaded) fromWT and sktl2.3/sktlΔ5 probed with anti-P-Moe (C), anti-Moesin (D) and anti-Tubulin (α-tub) as a loading control (C,D). (E,F) Immunostaining using anantibody against Moesin. Moesin is localized at the cortex in both WT and sktl2.3/sktlΔ5 oocytes (E,F, arrowheads). Asterisk, nucleus. D

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How could Sktl control the anchoring of the nucleus? Recentresults in the C. elegans embryo indicate that PPK-1, a PIP5K,controls spindle movements by regulating the heterotrimeric Gproteins GPR-1/2 and LIN-5, which are similar to Pins (Raps –FlyBase) and Mud (Mushroom body defect), respectively, inDrosophila (Panbianco et al., 2008). Pins requirement has not beenreported in the oocyte. However, Mud is distributed around thenucleus (Yu et al., 2006), like the Dynein-Dynactin complex withwhich it has been reported to control spindle attachment in othersystems (Gonczy, 2008). However, although the positioning of thenucleus is Dynein dependent (Januschke et al., 2002), it does notnecessarily require Mud (Yu et al., 2006). It would however beinteresting to investigate whether Pins and Mud act redundantly withother factors to control the positioning of the nucleus.

Our results also indicate that Sktl regulates PAR polarity proteinsalong the AP axis. In the absence of Sktl function, Baz, Lgl and Par-1 are mislocalized in the oocyte. In MDCK cells, PIP2 has beenshown to bind to Par-3 and to participate in its recruitment at theplasma membrane. In the absence of Sktl, Baz localization isaffected in both the oocyte and follicle cells. One hypothesis is thatin the absence of a sufficient level of PIP2, Baz is not recruited at thecortex of the oocyte, compromising the equilibrium betweenanterior and posterior PAR complexes and inducing themislocalization of Lgl and Par-1. It is also possible that in theabsence of Sktl, the defective actin cytoskeleton in the oocytecompromises the localization of Lgl, as occurs in neuroblasts(Betschinger et al., 2005), and of Par-1 (N1S) to the posterior cortex(Doerflinger et al., 2006).

PAR proteins are well-known regulators of MT organization andMT-based transport in polarized cells (Munro, 2006). Furthermore,ectopic expression along the entire cortex of Par-1 (N1S)(Doerflinger et al., 2006), Lgl (Tian and Deng, 2008) or Baz (thisstudy), as well inactivation of lgl (Tian and Deng, 2008), affect thepositioning of the nucleus, as in absence of Sktl. This furthersuggests that the correct positioning of the PAR proteins along theAP axis is crucial for the anterodorsal anchorage of the nucleus. It ishowever possible that the defects in the localization of mRNAsobserved in sktl mutant oocytes are not only caused by themispositioning of the nucleus, but also involve a more direct effectof the defective distribution of PAR proteins on MT organization.

It is interesting to note that in the C. elegans embryo, PARproteins such as PAR-2 and PAR-3 regulate the activity and thedistribution of the PIP5K, PPK-1, but that PAR-2 and PAR-3 are notregulated by PPK-1 (Panbianco et al., 2008), whereas in theDrosophila oocyte Sktl controls the distribution of the PAR proteins.Hence, phosphoinositide regulators and PAR proteins are closelyassociated in the control of polarity establishment in differentorganisms; however, they can act at different levels relative to eachother.

We thank S. Delga and R. Naaman for their work on Skittles; C. Moch for thedesign of pUbi vectors; F. Payre, D. St Johnston, H. Bellen and W. Deng forreagents; J. Compagnon for suggestions; and J. R. Huynh, A. Gautreau, J.Rothman, I. Becam and A. Kropfinger for critical comments on the manuscript.Drosophila embryo injections were carried out by BestGene. L.G. wassupported by a fellowship from ‘Ligue contre le cancer’. This work wassupported by grants from the CNRS, Association pour la recherche sur lecancer (ARC; subvention number 4446, 3297), ACI ‘Jeune chercheur’programme of the Ministere de la Recherche, ANR Blanche (grant Cymempol,Blan06-3-139786).

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/135/23/3829/DC1

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