pip 3,4 in clathrin mediated endocytosis

LETTERdoi:10.1038/nature12360

Spatiotemporal control of endocytosis byphosphatidylinositol-3,4-bisphosphateYork Posor1, Marielle Eichhorn-Gruenig1*, Dmytro Puchkov1*, Johannes Schoneberg2*, Alexander Ullrich2*, Andre Lampe1,Rainer Muller3, Sirus Zarbakhsh3, Federico Gulluni4, Emilio Hirsch4, Michael Krauss1, Carsten Schultz3, Jan Schmoranzer1,Frank Noe2 & Volker Haucke1,5

Phosphoinositides serve crucial roles in cell physiology, ranging fromcell signalling to membrane traffic1,2. Among the seven eukaryoticphosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasmamembrane where, among other functions, it is required for thenucleation of endocytic clathrin-coated pits36. No phosphatidyl-inositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stagesof the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P)7. How phosphatidylinositol conversion fromPI(4,5)P2-positive endocytic intermediates to PI(3)P-containingendosomes is achieved is unclear. Here we show that formationof phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class IIphosphatidylinositol-3-kinase C2a (PI(3)K C2a) spatiotemporallycontrols clathrin-mediated endocytosis. Depletion of PI(3,4)P2 orPI(3)K C2a impairs the maturation of late-stage clathrin-coatedpits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2ais required for selective enrichment of the BAR domain proteinSNX9 at late-stage endocytic intermediates. These findings providea mechanistic framework for the role of PI(3,4)P2 in endocytosisand unravel a novel discrete function of PI(3,4)P2 in a central cellphysiological process.PI(4,5)P2 generation by phosphatidylinositol phosphate-5-kinases

(phosphatidylinositol-5-kinases) is required for recruitment of earlyPI(4,5)P2-associated coat components to mediate clathrin-coated pit(CCP)nucleation in clathrin-mediated endocytosis (CME)1,5. Althoughphosphatidylinositol-5-kinases canassociatewith early coat components3,they fail to enrich at maturing CCPs8. By contrast, CCPs contain 5-phosphatases9 that degrade PI(4,5)P2 during late stages ofCME.Giventhe identification of PI(3,4)P2 4- and PI(3,4,5)P3 5-phosphatases aseffectors of endosomal Rab5 (ref. 10) we proposed that PI(3,4)P2 mightserve as an intermediate plasma membrane phosphatidylinositol spe-cies en route to PI(3)P-containing endosomes.Analysis of the cellular PI(3,4)P2 distribution using a specific anti-

PI(3,4)P2 antibody11 revealed predominant plasma membrane label-ling that overlapped with the localization of the PI(3,4)P2-sensingtandem PH-domain of TAPP112 (Supplementary Fig. 1a). In additionto larger PI(3,4)P2-positive structures11, akin to circular dorsal ruffles ofmigratory cells, anti-PI(3,4)P2 antibodies decorated diffraction-limitedpuncta that partially co-localized with plasmalemmal CCPs (Fig. 1a).To verify specificitywe analysed cells overexpressingPI(3,4)P2-specific4-phosphatase, type II inositol-3,4-bisphosphate 4-phosphatase13 fusedto a carboxy-terminal CAAX-box prenylation sequence to target it tothemembrane (INPP4BCAAX). Overexpression of INPP4BCAAXresulted indepletionof antibody-decoratedPI(3,4)P2,whereasPI(4,5)P2levels remained unchanged (Fig. 1b and Supplementary Fig. 1b, c).Selective INPP4BCAAX-mediated depletion of plasma membrane

PI(3,4)P2 but not of other phosphatidylinositols such as PI(3)P,PI(4,5)P2, or PI(3,4,5)P3 was verified by quantitative determinationof themembrane enrichment of specific phosphatidylinositol-bindingdomain-based sensors using total internal reflection (TIRF)/epifluo-rescence microscopy (Supplementary Fig. 1c). Thus, the levels anddistribution of PI(3,4)P2 are faithfully reported by anti-PI(3,4)P2antibodies or by PH-TAPP1 and overexpression of INPP4BCAAXselectively depletes plasmalemmal PI(3,4)P2.Given thepresenceofPI(3,4)P2 atCCPswe tested its functional impor-

tance for CME. Depletion of PI(3,4)P2 by INPP4BCAAX impairedtransferrin endocytosis and led to increased transferrin receptor sur-face levels, similar to depletion of PI(4,5)P2 by INPP5ECAAX, a lipidrequired for CCP nucleation (Fig. 1c). Overexpression of membrane-targeted catalytically inactive INPP4B(C842A13), thePI(3)P-phosphataseMTM1 (ref. 14), or the PI(3,4,5)P3-phosphatase PTEN (see Sup-plementary Fig. 1d, e for controls) did not affect CME of transferrin(Fig. 1c). These data reveal a hitherto unknown regulatory role forPI(3,4)P2 in CME. To dissect the underlying mechanism we analysedthe distribution and dynamics of key endocytic proteins. PI(3,4)P2depletion by INPP4B caused the accumulation of AP-2a-positiveCCPs (Fig. 1d, e) and markedly slowed CCP dynamics (Fig. 1f,Supplementary Fig. 2 and Supplementary Video 1), similar to dyna-min1/2-knockout (KO)15. No such effects were observed for catalyti-cally inactive INPP4B (C842A), MTM1 (to deplete potential plasmamembrane PI(3)P), or PTEN (to deplete PI(3,4,5)P3) (Fig. 1e andSupplementary Fig. 2). These data identify PI(3,4)P2 as a novel regu-lator of CME, possibly involved in a late stage in the pathway differentfrom PI(4,5)P2-controlled CCP initiation (Fig. 1f and SupplementaryFig. 2)4.PI(3,4)P2 canbegeneratedbywortmannin-sensitive class IPI(3)Ksand

subsequent hydrolysis of PI(3,4,5)P3 by 5-phosphatases16 downstreamof growth factor activation. Epidermal growth factor (EGF)-inducedincrease of PI(3,4,5)P3was abrogated bywortmannin inhibition of classI PI(3)K (Supplementary Fig. 1f), but had only a moderate effect onthe basal level of PI(3,4)P2 (Supplementary Fig. 1g). These data suggestthe existence of a class I PI(3)K-independent pool of PI(3,4)P2 andare consistent with CME being a constitutive process inmost cell types.A less well characterized pathway for PI(3,4)P2 production is theclass II PI(3)K-mediated phosphorylation of phosphatidylinositol-4-phosphate (PI(4)P)16. The contribution of this pathway to cellularPI(3,4)P2 synthesis is unknown. Class II PI(3)K C2a was identified asan interactor of clathrin17. PI(3)K C2a also binds to PI(4,5)P2 (ref. 18)and its activity is stimulated by clathrin17, but largely refractory to inhi-bition by wortmannin19. Quantitative proteomics showed PI(3)K C2ato be enriched in clathrin-coated vesicles (CCVs) with about 10 copiesper vesicle20. We found endogenous PI(3)K C2a to co-localize withclathrin in endocytic CCPs (Fig. 2a; in agreement with ref. 17), and

*These authors contributed equally to this work.

1Leibniz Institut furMolekulare Pharmakologie (FMP)& Freie Universitat Berlin, Robert-Roessle-Strae 10, 13125Berlin, Germany. 2Freie Universitat Berlin, DFGResearchCenterMATHEON, Arnimallee 6,14195 Berlin, Germany. 3European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, 69117 Heidelberg, Germany. 4Molecular Biotechnology Center, Departments of Genetics,Biology and Biochemistry, University of Torino, Via Nizza 52, 10126 Torino, Italy. 5Charite Universitatsmedizin, NeuroCure Cluster of Excellence, Chariteplatz 1, 10117 Berlin, Germany.

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confirmed its enrichment in CCVs (Supplementary Fig. 3a). Clathrinknockdown caused dispersal of PI(3)K C2a to the cytosol, indicatingthat membrane targeting of PI(3)K C2a requires clathrin (Supplemen-tary Fig. 3b). Cells depleted of PI(3)K C2a (Supplementary Fig. 3c)showed reduced CME of transferrin and increased transferrin receptorsurface levels (2286 23% ofmock control; rescue, 1116 12% ofmock;s.e.m., n5 5 experiments), an effect rescued by re-expression of shortinterfering RNA (siRNA)-resistant PI(3)K C2a fused with enhancedgreen fluorescent protein (EGFP; Fig. 2b).CMEofEGFwas reduced to a

lesser extent (Supplementary Fig. 3d). Defective transferrinCME wasalso observed in mouse embryonic fibroblasts derived from PI(3)KC2a-KOmice, an effect rescued by re-expression of wild type, but notcatalytically inactive (Supplementary Fig. 5b) mutant PI(3)K C2a

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Figure 1 | PI(3,4)P2 regulates CME. a, Partial co-localization of PI(3,4)P2with CCPs. Confocal images of Cos7 cells stained for PI(3,4)P2 and clathrinheavy chain (CHC). Arrowheads, structures immunopositive for PI(3,4)P2 andclathrin. Scale bar, 10mm(inset: 2mm).b, Selective depletionof PI(3,4)P2 by thePI(3,4)P2-specific phosphatase INPP4B (INPP4BCAAX). Levels of PI(3,4)P2or PI(4,5)P2 were quantified by immunostaining for PI(3,4)P2 or PI(4,5)P2(mean6 s.e.m.; n5 5 experiments; *P, 0.05, t-test). c, Selective depletion ofplasma membrane PI(3,4)P2 impairs CME of transferrin. Expression ofmCherry-tagged membrane-targeted inactive INPP4B(C842A), of the PI(3)Pphosphatase MTM1, or of the PI(3,4,5)P3 phosphatase PTEN do not affectCME. INPP5E-mediated depletion of PI(4,5)P2 was used as a positive control.Bar diagrams represent ratio of internalized (10min, 37 uC) to surfacetransferrin (45min, 4 uC) (mean6 s.e.m.; n5 3 experiments, forINPP4B(C842A) n5 2; *P, 0.05, **P, 0.01, t-test). d, e, Accumulation ofAP-2a-positive CCPs in PI(3,4)P2-depleted cells. Confocal images of Cos7 cellsexpressing mCherry or mCherryINPP4BCAAX stained for endogenousAP-2a. d, Scale bar, 5mm. e, Mean intensity of endocytic AP-2a-containingCCPs (mean6 s.e.m.; n5 3 independent experiments; **P, 0.01, t-test).f, Stalled CCP dynamics in PI(3,4)P2-depleted cells analysed by TIRF imagingof EGFPclathrin. Depletion of PI(4,5)P2 by INPP5E causing loss of plasmamembrane CCPs was used as a control. Kymographs, EGFPclathrinfluorescence over 180 s in cells expressing mCherry or the indicated mCherry-tagged phosphatase. See also Supplementary Video 1.

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Figure 2 | PI(3)K C2a controls maturation of CCPs. a, Confocal images ofCos7 cells stained for endogenous PI(3)KC2a and clathrin heavy chain (CHC).Scale bar, 10mm. b, PI(3)K C2a depletion impairs CME of transferrin. Cos7cells depleted of PI(3)K C2a expressing eGFP or siRNA-resistant EGFPPI(3)K C2a wild type were assayed for CME of transferrin. Bar diagramsrepresent the ratio of internalized (10min, 37 uC) to surface transferrin(45min, 4 uC) (mean6 s.e.m.; n5 5 experiments; ***P, 0.001, t-test).c, d, PI(3)KC2a depletion impairs CCPdynamics analysed by TIRF imaging ofEGFPclathrin expressing Cos7 cells depleted of PI(3)K C2a. c, Kymographsshow increased CCP-lifetimes in cells depleted of PI(3)K C2a (seeSupplementary Videos 2 and 3). d, Lifetime distribution of CCPs binned incategories of 60 s. Data represent mean6 s.e.m. (n5 3 experiments with.1,000 CCPs per condition; *P, 0.05, **P, 0.01, t-test for scrambled vsPI(3)K C2a siRNA-treated cells). e, f, Ultrastructural analysis of CCPs incontrol or PI(3)K C2a-depleted cells. Morphological groups were shallow(stage 1), non-constricted U-shaped (stage 2), constricted V-shaped pits(stage 3), or structures containing complete clathrin coats (stage 4).e, representative images from controls (top and middle) or a PI(3)K C2a-depleted cell illustrating accumulation and clustering of U-shaped pits(bottom). Scale bar, 100 nm. f, Bar diagram detailing the relative abundance ofdifferent clathrin-coated structures in control or PI(3)K C2a-depleted cells(mean6 s.e.m.; n5 10 (mock, scrambled siRNA) or n5 11 (PI(3)K C2asiRNA) cell perimeters). g, h, Timing of recruitment of PI(3)K C2a and SNX9to CCPs analysed by TIRF microscopy. mRFP, monomeric red fluorescentprotein . g, Snapshots of endocytic proteins at single CCPs (fission at t5 0).h,Mean time course of relative fluorescence intensity atCCPs (mean6 s.e.m.; 3experiments for clathrin, dynamin 2 and PI(3)KC2a, 2 for SNX9; total numbern of CCPs: n5 58 for clathrin, n5 85 for dynamin2, n5 248 for PI(3)K C2a,n5 100 for SNX9).

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(Supplementary Fig. 3e). Loss of PI(3)KC 2a thus phenocopies effectsof PI(3,4)P2 depletion on CME.Nextwe analysed the dynamics of plasmalemmalCCPs inPI(3)KC2a

depleted cells by TIRF microscopy. Cells lacking PI(3)K C2a showedincreased CCP lifetimes (Fig. 2c, d and Supplementary Videos 2 and 3)and this was rescued by re-expression of siRNA-resistant EGFPPI(3)KC2a (Supplementary Fig. 4a). Although nucleation and growth of CCPswere unaltered, they frequently failed to mature to a fission-competentstate. Instead, many CCPs seemed to grow beyond the size at which theywould normally undergo fission and could be observed to split into twoor three closely neighboured CCPs (Supplementary Fig. 4b). Attenu-ated dynamics of CCPs in PI(3)K C2a-depleted cells were also seen influorescence recovery after photobleaching experiments (SupplementaryFig. 4d, e).To determine whether PI(3)K C2a regulates maturation of CCPs,

before or in conjunction with dynamin-mediated fission, we subjectedPI(3)KC2a-depleted cells to quantitativemorphometric analysis. Thisrevealed an increased number of U-shaped CCPs, a stage precedingconstriction and dynamin-mediated fission, whereas the frequenciesof early shallow CCPs, V-shaped constricted CCPs, or of free CCVswere unaltered (Fig. 2e, f). CCPs frequently appeared clustered (Sup-plementary Fig. 4f), as also seen by live imaging (Supplementary Fig.4b, c). Analysis of the dynamics of endocytic protein recruitment toCCPs showed PI(3)K C2a to follow clathrin but to precede dynamin 2(Fig. 2g, h). We conclude that PI(3)K C2a regulates CCP maturationby facilitating the transition from invaginated to V-shaped CCPs.To explore whether the function of PI(3)K C2a in CME requires its

phosphatidylinositol kinase activity we assayed catalytically inactivemutant PI(3)K C2a (Supplementary Fig. 5b). Endocytic proteins suchasAP-2a accumulate at CCPs following depletion of PI(3,4)P2 (Fig. 1d,e) or PI(3)K C2a (Fig. 3a). This defect was rescued by siRNA-resistantwild type but not catalytically inactive mutant PI(3)K C2a, althoughboth variants localized to CCPs (Supplementary Fig. 5a). Thus, PI(3)KC2a function in CME requires its PI(3)K activity.Previous studies have yielded conflicting data regarding thedominant

lipid product of PI(3)K C2a, reporting either preferential synthesis ofPI(3,4)P2 or PI(3)P21,22. Immunopurified PI(3)KC2a preferentially pro-duced PI(3,4)P2 as compared to either PI(3)P or PI(3,4,5)P3 (Figs 3b,Supplementary Fig. 5b, c), in agreement with ref. 22. If PI(3)K C2a wasto contribute to PI(3,4)P2 formation in vivo, knockdown of PI(3)K C2ashould result in reduced PI(3,4)P2 levels. Quantitative assessment ofplasmamembrane phosphatidylinositols by specific PI-binding domain-based sensors revealed a selective reduction of PI(3,4)P2 in PI(3)KC2a-knockdown cells, whereas PI(3)P, PI(4,5)P2 or PI(3,4,5)P3 remainedunchanged (Fig. 3c). Depletion of PI(3,4)P2, but not of PI(4,5)P2,was also detectable with PI-specific antibodies (Fig. 3d). Consist-ently, PI(3,4)P2 largely co-localized with the plasma membrane poolof PI(3)KC2a (Supplementary Fig. 5d). Conversely, we failed to detectPI(3,4)P2 at CCPs in PI(3)KC2a-depleted cells (Supplementary Fig. 5e).These results are consistent with the preferred production of PI(3,4)P2by PI(3)K C2a in vitro and support the hypothesis that PI(3)K C2acontributes to PI(3,4)P2 formation at CCPs in vivo.To corroborate the preferential synthesis of PI(3,4)P2 over PI(3)P by

PI(3)KC 2a in vivo we capitalized on the fact that the specificity ofPI(3)Ks is encodedwithin the phosphatidylinositol-binding activationloop23. The activation loop of PI(3,4,5)P3-producing class I PI(3)Kscontains two basic boxes that coordinate the phosphates of PI(4,5)P2.None of these basic boxes is present in PI(3)P-producing class IIIPI(3)K hVps34 (Fig. 3e). PI(3)K C2a only contains basic residues thatcoordinate the 4-phosphate group, consistent with PI(3,4)P2 synthesis.To distinguish between PI(3)K C2a-mediated formation of PI(3,4)P2or PI(3)P at CCPs we constructed a PI(3)K C2a mutant, in which the4-phosphate coordinating box was exchanged with the correspondingsequence from hVps34 (Fig. 3e). This class III-like mutant PI(3)K C2aselectively synthesized PI(3)P with wild-type PI(3)K C2a activity, butfailed to produce PI(3,4)P2 (Supplementary Fig. 5b, c). It was also unable

to rescue defective CME in PI(3)K C2a-depleted cells (Fig. 3f, g). Thus,CME requires PI(3)KC2a-mediated production of PI(3,4)P2, but not ofPI(3)P.To challenge this hypothesis by an independent approach we made

use of cell-permeable PI-derivatives24 to exogenously supply PI(3)Por PI(3,4)P2. Addition of cell-permeable PI(3,4)P2 partially rescuedendocytic protein accumulation at CCPs in PI(3)K C2a-depleted cells,whereas PI(3)P was inactive (Supplementary Fig. 5f), although it sti-mulated early endosome fusion (ref. 24 and not shown). We concludethat PI(3)KC2a is required for local PI(3,4)P2 production at endocyticCCPs.Absence of PI(3)K C2a or depletion of its lipid product PI(3,4)P2

causes a delay in CCP maturation, suggesting the presence of PI(3,4)P2effectors at CCPs. To identify such effectors we monitored CCP enrich-ment of endocytic proteins in PI(3)KC2a-depleted cells (SupplementaryFig. 6a).Of the proteins assayed the only one that failed to enrich at CCPs

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Figure 3 | PI(3,4)P2 synthesis by PI(3)K C2a at CCPs. a, Requirement forPI(3)K activity of PI(3)K C2a in CME. Mean intensity of endocytic AP-2a-containing CCPs in PI(3)K C2a-depleted Cos7 cells expressing EGFP, siRNA-resistant wild-type (WT) or kinase inactive EGFPPI(3)K C2a (mean6 s.e.m.;n5 3 experiments; *P, 0.05, **P, 0.01, ***P, 0.001, t-test). b, PI(3)K C2apreferentially synthesizes PI(3,4)P2. Enzymatic activity of immunoprecipitated63MycPI(3)K C2a. Data, mean6 s.e.m. normalized to level of PI(3)Psynthesis (n5 9 experiments; ***P, 0.001, t-test). No 3-kinase activity wasdetectable in absence of induction of PI(3)K C2a expression. c, d, Selectivereduction of PI(3,4)P2 in PI(3)K C2a-depleted cells. c, Loss of plasmamembrane association of the PI(3,4)P2-sensor 23TAPP1-PHbut not of probesfor other phosphatidylinositols determined by ratiometric TIRF/epifluorescentimaging (mean6 s.e.m.; n (experiments)5 9 (23TAPP1-PH), n5 7(23FYVE, a sensor for PI(3)P), n5 4 (PH-PLCd, a sensor for PI(4,5)P2, andPH-Btk, a sensor for PI(3,4,5)P3); **P, 0.01, ***P, 0.001, t-test). d, Levels ofPI(3,4)P2 or PI(4,5)P2 quantified by PI(3,4)P2- or PI(4,5)P2-specific antibodies(mean6 s.e.m.; n5 6 experiments; *P, 0.05, t-test). e, Alignment ofsubstrate-binding loop sequences of human phosphatidylinositol-3-kinasesand a PI(3)K C2a class III-like mutant (cl. III mut) that can only synthesizePI(3)P but not PI(3,4)P2. f, g, Requirement for PI(3)K C2a-mediated PI(3,4)P2synthesis in CME. f, Impaired CME in PI(3)K C2a-deficient cells is rescued byre-expression of wild-type (WT) but not class III-like mutant EGFPPI(3)KC2a. Bar diagrams represent the ratio of internalized (10min, 37 uC) to surfacetransferrin (45min, 4 uC) (mean6 s.e.m.; n5 3 experiments; ***P, 0.001,t-test compared to scrambled siRNA). g, Mean intensity of endocytic AP-2a-containing CCPs in PI(3)K C2a-deficient Cos7 expressingWT or class III-likemutant PI(3)K C2a (mean6 s.e.m.; n5 3 experiments; ***P, 0.001, t-test).

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in PI(3)K C2a-depleted cells was the PX-BAR domain protein sortingnexin 9 (SNX9) (Supplementary Fig. 6a).We thus analysed the ability ofSNX9 to associate with phosphatidylinositol liposomes. EndogenousSNX9 (Fig. 4a) or its PX-BARmodule (Supplementary Fig. 6b) preferen-tially bound to phosphatidylinositol-3-phosphates including PI(3,4)P2,PI(3)P and PI(3,4,5)P3, but also associated with PI(4,5)P2 in vitro. Asbinding experiments with purified proteins might poorly reflect the situ-ation in vivo we directly compared phosphatidylinositol association ofSNX9with that of other endocytic proteins in brain extracts. Only SNX9preferred association with PI(3,4)P2 over PI(4,5)P2, whereas AP180,epsin 1 and AP-2a showed preferential PI(4,5)P2 binding (Fig. 4b, c).Thus, SNX9 is a putative PI(3,4)P2 effector in CME. To test this, weanalysed the localization of SNX9 at CCPs in cells depleted of PI(3)KC2a or PI(3,4)P2. Loss of dynamins results in accumulation of SNX9assemblies on elongated necks of arrested CCPs15. We confirmed theenrichment of endogenous SNX9 at AP-2a-coated endocytic intermedi-ates in cells depletedof dynamin2 (Fig. 4d, e).Co-silencingofPI(3)KC2awith dynamin2 prevented SNX9 accumulation at arrestedCCPs (Fig. 4d,e and Supplementary Fig. 7a), whereas other endocytic proteins accumu-lated irrespective of the presence of PI(3)KC2a (Supplementary Fig. 7b).Similar effects were caused by INPP4BCAAX-mediated depletion ofPI(3,4)P2 (Fig. 4f, g and Supplementary Fig. 7d). Knockdown of SNX9or PI(3)K C2a also interfered with the formation or stability of ARP2/3-positive tubular membrane invaginations in dynamin 2-depleted cells(Supplementary Fig. 7c). Thus, PI(3)K C2a-mediated PI(3,4)P2 produc-tion is required for SNX9 recruitment during late stages of CME.Previous work has shown that depletion of SNX9 interferes with

CME in HeLa cells and we confirmed this (Supplementary Fig. 8a). Inother cell lines (that is, Cos7) SNX9 is functionally redundant with itsparalogue SNX1825 (ref. 25). In agreement, depletion of SNX9 andSNX18 in Cos7 cells (Supplementary Fig. 8b) inhibited transferrin-CME (Fig. 4h) and interfered with CCP dynamics evidenced by AP-2a accumulation (SupplementaryFig. 8c), similar to the effects seenupondepletion of PI(3,4)P2 or PI(3)K C2a (compare Fig. 1e with Fig. 2c, d).Defective CME or AP-2a accumulation were rescued by siRNA-resistantwild-type EGFPSNX9 but not mutants of SNX9, in which key residuesrequired for binding to phosphatidylinositol-3-phosphates (Supplemen-tary Fig. 6c, ref. 26) had been mutated (Fig. 4h).Thus, PI(3)K C2a via its lipid product PI(3,4)P2 facilitates enrich-

ment of PI(3,4)P2-binding effector proteins, most notably SNX9before dynamin-mediated fission. Total internal reflection fluorescence(TIRF) microscopy analysis indeed revealed that accumulation ofmCherrySNX9 was delayed by about 20 s with respect to EGFPPI(3)K C2a, but preceded arrival of dynamin 2 (Fig. 2g, h). These dataagreewith a spatiotemporal computationalmodel that suggests amech-anism by which PI(3,4)P2 production at CCPs triggers selective SNX9recruitment (for details see Schoneberg et al. in preparation, preprint athttp://arxiv.org/find/physics/1/au:1Noe_F/0/1/0/all/0/1).The present work identifies a novel function for PI(3,4)P2, a lipid

previously implicated in the late sustained phase of growth factorsignalling1,2, in constitutive CME. We show PI(3)K C2a-mediatedPI(3,4)P2 synthesis to be required for CCP maturation and for recruit-ment of the PX-BAR domain protein SNX9 to CCPs at a late stageprecedingdynamin-mediated fission.Our analysis of the timing of endo-cytic protein arrival at CCPs indicates a hitherto unknown functionalinterplay betweenPI(4,5)P2 andPI(3,4)P2 in controllingdistinct stages ofCME in mammalian cells. We further suggest that the combined activ-ities of PI(4,5)P2-phosphatases9 and of PI(3)K C2a catalyse phosphati-dylinositol conversion from PI(4,5)P2 to PI(3,4)P2. Phosphatidylinositolconversion regulates CCPmaturation and constriction andmay therebyprepare endocytic vesicles for fusionwith PI(3)P-containing endosomes.Similar conversion mechanisms involving Rab proteins and phosphati-dylinositols regulate further endosomal progression27. The identificationof PI(3)K C2a as a major PI(3,4)P2-synthesizing enzyme will pave theway for the further study of this exciting lipid in cell physiological pro-cesses other thanCMEand indisease including cancer13 anddiabetes21,22.

METHODS SUMMARYTotal internal reflection fluorescence (TIRF) microscopy. TIRF imaging wasperformed using a Zeiss Axiovert200M microscope equipped with an incubationchamber (37 uC and 5% CO2), a 3100 TIRF objective and a dual-colour TIRFsetup (Visitron Systems) using Slidebook imaging software (3i Inc.). For analysisof CCP dynamics, time-lapse series of 3min with a frame rate of 0.5Hz wererecorded.

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Figure 4 | SNX9 is a PI(3,4)P2 effector at CCPs. a, Binding of endogenousSNX9 from Hek293 cells to liposomes containing 5mol% of the indicatedphosphatidylinositol in flotation assays. Input, 10mg protein for bound (top) or30mg (bottom) for unbound fractions (representative of 3 experiments).b, c, Association of SNX9 affinity-isolated from rat brain extracts withPI(3,4)P2- beads. Endocytic proteins AP180, AP-2a or epsin 1 preferentiallyassociate with PI(4,5)P2-beads. Clathrin, negative control. b, Densitometricquantification of data in a (mean6 s.e.m.; n5 3 experiments; **P, 0.01,***P, 0.001, t-test). d, e, SNX9 accumulation at endocytic intermediatesrequires PI(3)K C2a. Confocal images of Cos7 cells depleted of PI(3)K C2a,dynamin2, or both, stained for AP-2a and SNX9. d, Scale bar, 10mm.e, Quantitative analysis of SNX9 levels at endocytic intermediates as shown ind (mean6 s.e.m.; n5 3 experiments; *P, 0.05, t-test). f, g, PI(3,4)P2 isrequired for accumulation of SNX9 at stalled CCPs. f, Confocal images ofendocytic protein accumulation in dynamin 2-deprived Cos7 cells depleted ofPI(3,4)P2 by mCherryINPP4BCAAX. Depletion of PI(3,4)P2 preventsaccumulation of SNX9 but not of AP-2a at endocytic intermediates. Scale bar,10mm. g, Quantification of SNX9 levels at stalled CCPs as shown inf (mean6 s.e.m.; n5 3 experiments; *P, 0.05, t-test). h, Impaired CME oftransferrin in Cos7 cells depleted of SNX9 and its close paralogue SNX18 isrescued by re-expression of wild-type (WT) EGFPSNX9 but not of PI-bindingdeficient PX-domain mutants RYK (SNX9(R286A, Y287A, K288); ref. 26) orK267N, R327N (see Supplementary Fig. 6c). Bar diagrams represent the ratio ofinternalized (10min, 37 uC) to surface transferrin (45min, 4 uC)(mean6 s.e.m.; n5 5 experiments, except n5 4 (EGFPSNX9(RYK) andEGFPSNX9(K267N, R327N) and n5 2 (SNX18); **P, 0.01, ***P, 0.001,t-test vs scrambled siRNA).

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Electron microscopy. Glutaraldehyde-fixed Cos7 cells treated with siRNAs werescraped, pelleted, and subsequently processed for electron microscopy and mor-phometric analysis.Lipid kinase assays.Kinase activity was assessed by a radioactivity-based assay (inkinase buffer: 5mMHEPES/KOH pH7.2, 25mMKCl, 2.5mMMgOAc, 150mMKGlu, 10mM CaCl2, 0.2% CHAPS) using recombinant 63mycPI(3)K C2aimmunoprecipitated from overexpressing HEK293 cells. 200mM phosphoinosi-tides, 200mM ATP and 8mCi of [c-32P]ATP were combined with 1 recombinant63mycPI(3)KC2a and incubated at 37 uC for 10min. Reactionswere stopped byaddition of 500ml coldmethanol:H2O:32%HCl (10:10:1), followed by lipid extrac-tion and thin-layer-chromatography (TLC) analysis.

Full Methods and any associated references are available in the online version ofthe paper.

Received 23 August 2012; accepted 6 June 2013.

Published online 3 July 2013.

1. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membranedynamics. Nature 443, 651657 (2006).

2. Wymann,M. P. & Schneiter, R. Lipid signalling in disease.Nature Rev. Mol. Cell Biol.9, 162176 (2008).

3. Krauss, M., Kukhtina, V., Pechstein, A. & Haucke, V. Stimulation ofphosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2m-cargo complexes. Proc. Natl Acad. Sci. USA 103,1193411939 (2006).

4. Loerke, D. et al. Cargo and dynamin regulate clathrin-coated pit maturation. PLoSBiol. 7, e57 (2009).

5. McMahon,H. T.&Boucrot, E.Molecularmechanismandphysiological functions ofclathrin-mediated endocytosis. Nature Rev. Mol. Cell Biol. 12, 517533 (2011).

6. Zoncu, R. et al. Loss of endocytic clathrin-coated pits upon acute depletion ofphosphatidylinositol 4,5-bisphosphate.Proc. Natl Acad. Sci. USA104,37933798(2007).

7. Gruenberg, J. Lipids in endocyticmembrane transport and sorting. Curr. Opin. CellBiol. 15, 382388 (2003).

8. Antonescu, C. N., Aguet, F., o., Danuser, G. & Schmid, S. L. Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size.Mol. Biol. Cell 22, 25882600 (2011).

9. Chang-Ileto, B. et al. Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulatedbymembrane curvature and facilitates membrane fission. Dev. Cell 20, 206218(2011).

10. Shin, H.-W. et al. An enzymatic cascade of Rab5 effectors regulatesphosphoinositide turnover in the endocytic pathway. J. Cell Biol. 170, 607618(2005).

11. Bae, Y. H. et al.Profilin1 regulates PI(3,4)P2 and lamellipodin accumulation at theleading edge thus influencing motility of MDA-MB-231 cells. Proc. Natl Acad. Sci.USA 107, 2154721552 (2010).

12. Dowler, S. et al. Identification of pleckstrin-homology-domain-containing proteinswith novel phosphoinositide-binding specificities. Biochem. J. 351, 1931 (2000).

13. Gewinner, C. et al. Evidence that inositol polyphosphate 4-phosphatase type II is atumor suppressor that inhibits PI3K signaling. Cancer Cell 16, 115125 (2009).

14. Fili, N., Calleja, V., Woscholski, R., Parker, P. J. & Larijani, B. Compartmental signalmodulation: endosomal phosphatidylinositol 3-phosphate controls endosomemorphology and selective cargo sorting. Proc. Natl Acad. Sci. USA 103,1547315478 (2006).

15. Ferguson, S. M. et al. Coordinated actions of actin and BAR proteins upstream ofdynamin at endocytic clathrin-coated pits. Dev. Cell 17, 811822 (2009).

16. Rameh, L. E.&Cantley, L. C. The role ofphosphoinositide3-kinase lipidproducts incell function. J. Biol. Chem. 274, 83478350 (1999).

17. Gaidarov, I., Smith, M. E., Domin, J. & Keen, J. H. The class II phosphoinositide3-kinase C2a is activated by clathrin and regulates clathrin-mediatedmembranetrafficking.Mol. Cell 7, 443449 (2001).

18. Stahelin, R. V. et al. Structural and membrane binding analysis of the Phoxhomology domain of phosphoinositide 3-kinase-C2a. J. Biol. Chem. 281,3939639406 (2006).

19. Domin, J. et al. Cloning of a human phosphoinositide 3-kinase with a C2 domainthat displays reduced sensitivity to the inhibitor wortmannin. Biochem. J. 326,139147 (1997).

20. Borner, G. H. H. et al.Multivariate proteomic profiling identifies novel accessoryproteins of coated vesicles. J. Cell Biol. 197, 141160 (2012).

21. Falasca, M. et al. The role of phosphoinositide 3-kinase C2a in insulin signaling.J. Biol. Chem. 282, 2822628236 (2007).

22. Leibiger, B. et al. Insulin-feedback via PI3KC2a activated PKBa/Akt1 is requiredfor glucose-stimulated insulin secretion. FASEB J. 24, 18241837 (2010).

23. Pirola, L. et al. Activation loop sequences confer substrate specificity tophosphoinositide 3-kinase a (PI3Ka). Functions of lipid kinase-deficient PI3Ka insignaling. J. Biol. Chem. 276, 2154421554 (2001).

24. Subramanian, D. et al. Activation of membrane-permeant caged PtdIns(3)Pinduces endosomal fusion in cells. Nature Chem. Biol. 6, 324326 (2010).

25. Park, J. et al. SNX18 shares a redundant role with SNX9 andmodulates endocytictrafficking at the plasma membrane. J. Cell Sci. 123, 17421750 (2010).

26. Yarar, D., Surka, M. C., Leonard, M. C. & Schmid, S. L. SNX9 activities are regulatedby multiple phosphoinositides through both PX and BAR domains. Traffic 9,133146 (2008).

27. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism ofprogression from early to late endosomes. Cell 122, 735749 (2005).

Supplementary Information is available in the online version of the paper.

AcknowledgementsWe thank E. Ungewickell, P. Di Fiore, P. De Camilli, H. McMahon,E. Wancker, T. Sudhof and S. Carlsson for antibodies, L. Cantley, T. Takenawa,M. Wymann, T. Ross, O. Daumke and W. Yang for plasmids, and O. Daumke, B. Eickoltand F. Wieland for critical comments. Supported by grants from the DeutscheForschungsgemeinschaft (SFB 740/C8; SFB 740/D7; SFB 958/A04; SFB 958/A07;SFB 958/Z02).

Author Contributions Y.P., M.E.-G., D.P., M.K. performed experiments; R.M., S.Z., C.S.provided reagents; A.L. and J.S. aided with microscopy; Y.P., M.E.-G., J.S., F.N. and V.H.designed research; F.G. and E.H. contributed reagents; J.S., A.U. and. F.N. conductedsimulations. Y.P., F.N. and V.H. wrote the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondenceand requests for materials should be addressed to V.H. ([email protected]).

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METHODSAntibodies. An overview of all antibodies used in this study is given in Sup-plementary Table 1.siRNAs. All siRNA sequences were used as 21-mers or 23-mers including 39-dTdT overhangs. The sequences of the PI(3)K C2a-targeting siRNAs used in thisstudy are as follows: siRNA 1 59-ggatctttttaaacctatt-39; siRNA 2 59-gcacaaacccaggctattt-39. The dynamin 2 siRNA sequence used is: 59-gcaactgaccaaccacatc-39.For silencing of SNX9 expression in HeLa cells, a pool of 4 siRNAs was obtainedfrom Dharmacon (Thermo Scientific). The SNX9 siRNA sequence used for Cos7cells lies within the 39-UTR of the mRNA and is: 59-ggacagaacgggccttgaa-39. Forsilencing of SNX18 expression the siRNA sequence used is: 59-caccgacgagaaagccuggaa-39. The scrambled control siRNA used in all experiments corresponds tothe scrambled m2 adaptin sequence 59-gtaactgtcggctcgtggt-39.Lipid reagents. Phosphatidylinositols for lipid binding assays were obtained fromAvanti Polar Lipids, phosphatidylcholine (PC), phosphatidylserine (PS), and cho-lesterol were from Sigma-Aldrich, L-a-phosphaditylethanolamine (PE) was fromJena Bioscience and rhodaminePE was from Avanti Polar Lipids.Plasmids. The sequence encoding full-length human INPP4B was amplified fromcDNA provided by L. Cantley and inserted in frame between the EcoRV and NotIsites of a pcDNA3.1(1)-based HA-expression vector (sequence between NdeI andEcoRV exchangedwith that frompcHA2)modified to encode the carboxy-terminalCAAX-box prenylation sequence from K-ras (KSKTKCVIM-Stop28) directly fol-lowing the Not I site. The INPP4BCAAX encoding sequence was subcloned intoan mCherry-expression vector for live cell imaging. Expression plasmids encodingINPP4BCAAX(C842A), full-length human MTM1CAAX14, full-length humanPTENCAAX and residues 214614 of human INPP5ECAAX29 were designedidentically.TheRFP23PHTAPP1 constructwas a gift fromT.Takenawa and theGFPPH domain of Brutons tyrosine kinase was provided by M. Wymann. Foranalysis of recruitment of proteins to CCPs in live cells, a fusion of mRFP to ratclathrin light chain inserted between KpnI and ApaI of pcDNA5/FRT/TO and amouse dynamin 2mCherry construct provided by O. Daumke were used in con-junction with a pEGFPC3PI(3)K C2a construct encoding human full-lengthPI(3)K C2a assembled from HeLa cDNA (verified by sequencing). A kinase-inactive mutant of PI(3)K C2a was obtained by mutating the ATP-binding site(K1138A,D1157A) and the catalytic loop (D1250A)30. Constructs ofwild-type andkinase-inactive PI(3)KC2a resistant to siRNA1were generated by creating 4 silentmutations: 59-agatctattcaaaccgatt-39. The PI(3)P-restricted class III mutant ofPI(3)K C2a resulted from the mutation of 1283KRDR1286 to 1283KPLP1286.For visualization of proteins at CCPs, a 33HAHip1R construct was provided byT. Ross and a clone encoding epsin 1GFP was purchased from OriGene Tech-nologies. All constructs encoding full-length SNX9 or domains thereof werederived from human SNX9 cDNA provided by W. Yang. For GSTPXBAR,cDNA encoding amino acids 204 to 595 of human SNX9 (ref. 31) was cloned inbetween the EcoRI and NotI sites of pGex-4T-1.Cell lines. All cell lines used (Cos7, HEK293, HeLa) were obtained from ATCCandnot used beyond passage 30 from original derivation byATCC. Cell lines wereroutinely tested for mycoplasma contamination.siRNA and plasmid transfections. HeLa or Cos7 cells seeded on day 0 weretransfectedwith siRNAs usingOligofectamine (Invitrogen) according to themanu-facturers instructions on day 1, expanded on day 2, transfected a second time onday 3, seeded for the experiment onday 4, andused for the experiment on day 5. Forexpression of recombinant proteins in knockdown cells, plasmids were transfectedusing lipofectamine 2000 (Invitrogen) according to themanufacturers instructionson day 4. In the case of INPP4BCAAX constructs, cells were sequentially trans-fected first with siRNAs and then with plasmid both on day 3 to allow for a totalexpression time of 40h.Upon plasmid transfection of untreated cells, cells were generally allowed to

express protein overnight and analysed the next day except for INPP4BCAAXconstructs where expression for two days was found to give better results.Transferrin uptake and surface labelling. HeLa cells treated with siRNAs ortransfected with mCherryINPP4BCAAX were seeded on Matrigel (BD bios-ciences)-coated coverslips. On the day of the experiment, cells were serum-starvedfor 1.5 h and used for either transferrin uptake or transferrin receptor surfacelabelling. For transferrin uptake, cells were incubated with 25mgml21 transferrinAlexa568 or transferrinAlexa647 (Molecular Probes, Invitrogen) for 10min at37 uC. After two washes with ice-cold PBS cells were acid washed at pH5.3 (0.2Msodiumacetate, 200mMsodiumchloride) on ice for 2min to remove surface-boundtransferrin, washed 2 times with ice-cold PBS and fixed with 4% paraformaldehyde(PFA) for 45min at room temperature. For surface labelling, cells were incubatedwith 25mgml21 transferrinAlexa568 at 4 uC for 45min to block endocytosis andlabel transferrin receptors on the cell surface. Cellswerewashed 3 timeswith ice-coldPBS on ice for onemin and fixed with 4% PFA for 45min at room temperature.

Transferrin labelling was analysed using a Zeiss Axiovert200Mmicroscope andSlidebook imaging software (3i Inc.). Internalized transferrin per cell was quan-tified and normalized to the amount of surface-bound transferrin determined inthe same experiment as a measure for the efficiency of internalization.Immunocytochemistry. Staining of proteins in cultured cells seeded on glasscoverslips was performed as described32. For lipid antibody stainings, Cos7 cellswere fixed in 2% PFA at room temperature for 20min and permeabilized withsaponin (30min at room temperature in 0.5% saponin, 1% bovine serum albumin(BSA) in PBS). Cells were labelled with lipid-specific antibodies (see Supplemen-tary Table 1) diluted in 1%BSA in PBS for 2 h at room temperature. After washingthree times for 5min with PBS, cells were incubated with appropriate fluorescentsecondary antibodies for 1 h andwashed three times 10minwith PBS. Protein andlipid immunocytochemistry stainings were routinely analysed and quantifiedusing a spinning disk confocal microscope (Ultraview ERS, Perkin Elmer) andVolocity imaging software (Improvision, Perkin Elmer).Total internal reflection fluorescence (TIRF) microscopy. TIRF imaging wasperformed using a Zeiss Axiovert200M microscope equipped with an incubationchamber (37 uC and 5% CO2), a 3100 TIRF objective and a dual-colour TIRFsetup from Visitron Systems using Slidebook imaging software (3i Inc.). For ana-lysis of CCP dynamics, time-lapse series of 3min with a frame rate of 0.5Hz wererecorded. CCP lifetimes were assessed by arbitrarily selecting 50 or 25 CCPs percell in the centre frame of the time-lapse series and determining the frame ofappearance and disappearance. In case CCPs already existed in the first frameor persisted until the last frame, these frames were counted. For the analysis ofrecruitment time courses of proteins to CCPs, only CCPs were used that bothappeared and disappeared within the time lapse series. From these, fluorescenceintensities over time were quantified and aligned on the time axis by the last frameof GFPPI(3)K C2a presence (t5 0, fission). Fluorescence intensities for all timepoints in relation to t5 0 were averaged over all CCPs in the analysis and renor-malized to the resulting peak value. For analysis of GFPPHBtk membrane asso-ciation, TIRF and epifluorescence images of the same cell were acquired and theTIRF fluorescence intensity was normalized to the epifluorescence signal in orderto achieve intrinsic correction for expression level variations between cells.Fluorescence recovery after photobleaching (FRAP). FRAP experiments wereperformed using a spinning disk confocal microscope equipped with an incuba-tion chamber (37 uC) and a photokinesis unit (Ultraview ERS, Perkin Elmer). Oneto three regions of interest in the peripheral, flat part of an EGFPCLC expressingcell were selected. A time-lapse series at 0.5Hzwas recorded with 10 frames beforeand 60 frames after bleaching. For quantification, the sum EGFPCLC fluor-escence intensity at CCPs was quantified over time and normalized to the meansum intensity during the pre-bleaching period.PIP/AM experiments. Cell-permeable acetoxy methylester (AM)-protectedphosphatidylinositol derivatives were synthesized as described33. For treatmentof cells, PI(3)P/AM or PI(3,4)P2/AM dissolved in dry DMSO were mixed with anequal volume of 10% pluronic F127 in DMSO (Sigma-Aldrich) to enhance solu-bility in aqueous buffers and diluted inDMEM to a final concentration of 200mM.Cells on coverslips were treated with DMSO1 pluronic (control) or PIP /AMs for10min at 37 uC and then processed for immunocytochemistry as described above.Electron microscopy. Ultrastructural analysis was performed as described34.Glutaraldehyde-fixed Cos7 cells treated with siRNAs were scraped, pelleted, andsubsequently processed for electron microscopy and morphometric analysis aspreviously described15. Briefly, after epoxy resin embedding and sectioning,micro-graphs were taken along the cell perimeter at320,000magnification. Images werecombined to reconstruct the cell perimeter and numbers of clathrin-coated inter-mediates were determined.Purification of clathrin-coated vesicles. CCVs were purified essentially asdescribed35. Briefly, calf brain was homogenized and the cytosolic andmicrosomalfraction was obtained by sequential centrifugation at 17,000g and 30,000g. Lightmembranes were pelleted at 150,000g, resuspended and mixed with an equalvolume of 12.5% Ficoll, 12.5% sucrose solution to adjust the density of the solutionto that of CCVs. Contaminating, heavier membranes were removed by centrifu-gation at 90,000g and the CCV-containing supernatant was diluted in order toallow sedimentation of CCVs at 150,000g. For stripping of coat proteins, purifiedCCVswere incubated over night at room temperaturewith 0.8MTris-HCl pH7.4to disrupt proteinmembrane interactions. Vesicles including integral membraneproteins were sedimented at 250,000g.In vitro kinase activity assays. Kinase activity of recombinant PI(3)K C2a wasassessed using a radioactivity-based assay essentially as described36. In brief, one10-cm dish of HEK293 cells transiently overexpressing 63myc-PI(3)K C2a waslysed in immunoprecipitation (IP) buffer (20mM HEPES, 100mM KCl, 2mMMgCl2, 1% CHAPS, 1mM PMSF, 0.3% protease inhibitor cocktail (Sigma)), andcentrifuged for 10min at 20,500g at 4 uC. The resulting supernatant was centri-fuged at 65,000 r.p.m. in a TLA-110 rotor (Beckman Coulter). PI(3)K C2a was

RESEARCH LETTER


immunoprecipitated from the extract using,15mg c-myc antibody and 1.5mg ofprotein as IP input. The IP was washed twice with IP buffer and once in kinasebuffer (5mM HEPES/KOH pH7.2, 25mM KCl, 2.5mM magnesium acetate(Mg(CH3COO)2), 150mM KGlu, 10mM CaCl2, 0.2% CHAPS). Phosphati-dylinositols were dissolved in kinase buffer (note that presence of 0.2% CHAPSwas required for full solubility of PI(4)P), incubated on ice for 30min, sonicatedfor 1min using a small tip sonicator (Bandelin Sonoplus) 1 s on 1 s off at 70%intensity. 200mMphosphoinositides, 200mMATP and 8mCi of [c-32P]ATP werecombined with 1/8 of one IP sample and incubated at 37 uC for 10min. Thereactions were stopped by the addition of 500ml cold methanol:H2O:32%HCl(10:10:1) and lipid extraction and thin-layer-chromatography (TLC) analysis wereperformed as described36.Liposome flotation assay. Liposome preparation. A total of 600800mg of lipidswere dissolved in a mixture of CHCl3:methanol:1N HCl (2:1:0.01) to the desiredconcentration, combined in a glass vial and dried under pressurized N2 followedby vacuum for 30min. Liposomes were rehydrated in 300ml HEPES buffer(50mM HEPES pH7.4, 100mM KCl (140mM KCl for experiments with GFPSNX9WT or K267N, R327N; 200mMKCl for experiments with GSTSNX9 PXBAR)) for 1 h at room temperature under frequent vortexing. After the addition of1.7ml H2O the liposomes were centrifuged in a TLA110 rotor at 20,000 r.p.m. at4 uC for one hour. The resulting pellet was resuspended in HEPES buffer to a finallipid concentration of 3mgml21. Liposome mixtures were extruded 14 timesthrough an 800-nmpolycarbonatemembrane (Whatman) using amanually oper-ated extruder (LiposoFast, Avestin, Inc.). The final concentration of lipid species inmol% were: 50%PC, 20% cholesterol, 19%PE, 1% rhodaminePE, 5% PS, and 5%phosphatidylinositols.Flotation assay. 450mg liposomes in HEPES buffer were combined with either

2mg of purified GSTSNX9 PX-BAR protein or with 30mg of HEK293 cell extractcontaining overexpressed HASNX9 PX-BAR or GFPSNX9 full-length proteinand incubated for 15min at 4 uC on a rotating wheel. The mixture was thenadjusted to 30% sucrose by adding 75% sucrose in HEPES buffer and transferredto a TLS-55 centrifuge tube. This was overlaid with 200ml of 25% sucrose inHEPES buffer followed by 50ml of HEPES buffer. Liposomes were floated bycentrifuging one hour at 55,000 r.p.m. (,240,000g) in a TLS 55 swing out rotor(Beckman Coulter). The fractions were collected using a blunt-ended needleattached to a calibrated syringe by removing the bottom layer first (,250ml totalvolume), followedby themiddle layer (200ml) and in the end the top layer contain-ing the liposomes and any bound protein. Top and bottom fractions were sepa-rated on 8% acrylamide gels and stained with Coomassie for GSTSNX9 PX-BARand immunoblotted for HASNX9 PX-BAR or GFPSNX9 full length.PIP bead-based affinity purification. Agarose PIP Beads (Echelon Biosciences)containing 10 nanomoles of bound PI(4,5)P2 or PI(3,4)P2 were used to pull down

proteins fromrat brain extract. Rat brain extractwas prepared from2.5 g frozen ratbrain homogenized in homogenization buffer (4mM HEPES pH7.4, 320mMsucrose, 1mM PMSF, 0.3% protease inhibitor cocktail) using 13 strokes of aglassTeflon-homogenizer at 900 r.p.m. The homogenate was centrifuged at900g for 10min at 4 uC. To the supernatant PIP pull-down buffer (20mMHEPES pH7.4, 50mM NaCl, 0.25% NP-40) was added to 13 concentrationand incubated on ice for 30min followed by centrifugation at 43,500g at 4 uC.The supernatant was centrifuged again at 265,000g for 15min at 4 uC to removeaggregated proteins. 68mgof proteinwere added to 100ml of 1:1 washed agarose-bead slurry and incubated at 4 uC for 1.5 h on a rotatingwheel. Beadswere pelleted,washed 3 times with PIP pull-down buffer and bound proteins were eluted twotimes in 30ml of 13Laemmli sample buffer. 30ml of the pooled eluate were thenloaded onto an 8% acrylamide gel for SDSPAGE followed by immunoblotting.Statistical methods. For analyses comprising multiple independent experiments(n), sample size within each experiment was chosen to provide statistically signifi-cant estimates for each sample, corresponding to 20 to 40 images per sample formicroscopy-based quantifications. In all experiments, cells were arbitrarily chosenbased on the signal in a separate channel independent from the signal to bequantified. All statistical tests performed were two-tailed, unpaired t-tests asjudged appropriate for the respective experiments.

28. Malecz, N. et al. Synaptojanin 2, a novel Rac1 effector that regulates clathrin-mediated endocytosis. Curr. Biol. 10, 13831386 (2000).

29. Varnai, P., Thyagarajan, B., Rohacs, T. & Balla, T. Rapidly inducible changes inphosphatidylinositol 4,5-bisphosphate levels influence multiple regulatoryfunctions of the lipid in intact living cells. J. Cell Biol. 175, 377382 (2006).

30. Gaidarov, I., Zhao, Y. & Keen, J. H. Individual phosphoinositide 3-kinase C2adomain activities independently regulate clathrin function. J. Biol. Chem. 280,4076640772 (2005).

31. Pylypenko, O., Lundmark, R., Rasmuson, E., Carlsson, S. R. & Rak, A. ThePX-BARmembrane-remodeling unit of sorting nexin 9. EMBO J. 26, 47884800(2007).

32. Maritzen, T. et al. Gadkin negatively regulates cell spreading and motility viasequestration of the actin-nucleating ARP2/3 complex. Proc. Natl Acad. Sci. USA109, 1038210387 (2012).

33. Laketa, V. et al.Membrane-permeant phosphoinositide derivatives asmodulators of growth factor signaling and neurite outgrowth. Chem. Biol. 16,11901196 (2009).

34. von Kleist, L. et al. Role of the clathrin terminal domain in regulating coated pitdynamics revealed by small molecule inhibition. Cell 146, 471484 (2011).

35. Campbell, C., Squicciarini, J., Shia, M., Pilch, P. F. & Fine, R. E. Identification of aprotein kinase as an intrinsic component of rat liver coated vesicles.Biochemistry23, 44204426 (1984).

36. Wieffer,M.,Haucke, V.&Krauss,M.Regulationofphosphoinositide-metabolizingenzymes by clathrin coat proteins.Methods Cell Biol. 108, 209225 (2012).

LETTER RESEARCH


TitleAuthorsAbstractMethods SummaryTotal internal reflection fluorescence (TIRF) microscopyElectron microscopyLipid kinase assays

ReferencesMethodsAntibodiessiRNAsLipid reagentsPlasmidsCell linessiRNA and plasmid transfectionsTransferrin uptake and surface labellingImmunocytochemistryTotal internal reflection fluorescence (TIRF) microscopyFluorescence recovery after photobleaching (FRAP)PIP/AM experimentsElectron microscopyPurification of clathrin-coated vesiclesIn vitro kinase activity assaysLiposome flotation assayPIP bead-based affinity purificationStatistical methods

Methods ReferencesFigure 1 PI(3,4)P2 regulates CME.Figure 2 PI(3)K C2a controls maturation of CCPs.Figure 3 PI(3,4)P2 synthesis by PI(3)K C2a at CCPs.Figure 4 SNX9 is a PI(3,4)P2 effector at CCPs.

pip 3,4 in clathrin mediated endocytosis

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