Abstract
Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic1,2. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits3,4,5,6. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P)7. How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.
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Main
PI(4,5)P2 generation by phosphatidylinositol phosphate-5-kinases (phosphatidylinositol-5-kinases) is required for recruitment of early PI(4,5)P2-associated coat components to mediate clathrin-coated pit (CCP) nucleation in clathrin-mediated endocytosis (CME)1,5. Although phosphatidylinositol-5-kinases can associate with 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 of CME. Given the identification of PI(3,4)P2 4- and PI(3,4,5)P3 5-phosphatases as effectors of endosomal Rab5 (ref. 10) we proposed that PI(3,4)P2 might serve as an intermediate plasma membrane phosphatidylinositol species 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 labelling that overlapped with the localization of the PI(3,4)P2-sensing tandem PH-domain of TAPP112 (Supplementary Fig. 1a). In addition to larger PI(3,4)P2-positive structures11, akin to circular dorsal ruffles of migratory cells, anti-PI(3,4)P2 antibodies decorated diffraction-limited puncta that partially co-localized with plasmalemmal CCPs (Fig. 1a). To verify specificity we analysed cells overexpressing PI(3,4)P2-specific 4-phosphatase, type II inositol-3,4-bisphosphate 4-phosphatase13 fused to a carboxy-terminal CAAX-box prenylation sequence to target it to the membrane (INPP4B–CAAX). Overexpression of INPP4B–CAAX resulted in depletion of antibody-decorated PI(3,4)P2, whereas PI(4,5)P2 levels remained unchanged (Fig. 1b and Supplementary Fig. 1b, c). Selective INPP4B–CAAX-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 determination of the membrane enrichment of specific phosphatidylinositol-binding domain-based sensors using total internal reflection (TIRF)/epifluorescence microscopy (Supplementary Fig. 1c). Thus, the levels and distribution of PI(3,4)P2 are faithfully reported by anti-PI(3,4)P2 antibodies or by PH-TAPP1 and overexpression of INPP4B–CAAX selectively depletes plasmalemmal PI(3,4)P2.
a, Partial co-localization of PI(3,4)P2 with CCPs. Confocal images of Cos7 cells stained for PI(3,4)P2 and clathrin heavy chain (CHC). Arrowheads, structures immunopositive for PI(3,4)P2 and clathrin. Scale bar, 10 µm (inset: 2 µm). b, Selective depletion of PI(3,4)P2 by the PI(3,4)P2-specific phosphatase INPP4B (INPP4B–CAAX). Levels of PI(3,4)P2 or PI(4,5)P2 were quantified by immunostaining for PI(3,4)P2 or PI(4,5)P2 (mean ± s.e.m.; n = 5 experiments; *P < 0.05, t-test). c, Selective depletion of plasma membrane PI(3,4)P2 impairs CME of transferrin. Expression of mCherry-tagged membrane-targeted inactive INPP4B(C842A), of the PI(3)P phosphatase MTM1, or of the PI(3,4,5)P3 phosphatase PTEN do not affect CME. INPP5E-mediated depletion of PI(4,5)P2 was used as a positive control. Bar diagrams represent ratio of internalized (10 min, 37 °C) to surface transferrin (45 min, 4 °C) (mean ± s.e.m.; n = 3 experiments, for INPP4B(C842A) n = 2; *P < 0.05, **P < 0.01, t-test). d, e, Accumulation of AP-2α-positive CCPs in PI(3,4)P2-depleted cells. Confocal images of Cos7 cells expressing mCherry or mCherry–INPP4B–CAAX stained for endogenous AP-2α. d, Scale bar, 5 µm. e, Mean intensity of endocytic AP-2α-containing CCPs (mean ± s.e.m.; n = 3 independent experiments; **P < 0.01, t-test). f, Stalled CCP dynamics in PI(3,4)P2-depleted cells analysed by TIRF imaging of EGFP–clathrin. Depletion of PI(4,5)P2 by INPP5E causing loss of plasma membrane CCPs was used as a control. Kymographs, EGFP–clathrin fluorescence over 180 s in cells expressing mCherry or the indicated mCherry-tagged phosphatase. See also Supplementary Video 1.
Given the presence of PI(3,4)P2 at CCPs we tested its functional importance for CME. Depletion of PI(3,4)P2 by INPP4B–CAAX impaired transferrin endocytosis and led to increased transferrin receptor surface levels, similar to depletion of PI(4,5)P2 by INPP5E–CAAX, a lipid required for CCP nucleation (Fig. 1c). Overexpression of membrane-targeted catalytically inactive INPP4B (C842A13), the PI(3)P-phosphatase MTM1 (ref. 14), or the PI(3,4,5)P3-phosphatase PTEN (see Supplementary Fig. 1d, e for controls) did not affect CME of transferrin (Fig. 1c). These data reveal a hitherto unknown regulatory role for PI(3,4)P2 in CME. To dissect the underlying mechanism we analysed the distribution and dynamics of key endocytic proteins. PI(3,4)P2 depletion by INPP4B caused the accumulation of AP-2α-positive CCPs (Fig. 1d, e) and markedly slowed CCP dynamics (Fig. 1f, Supplementary Fig. 2 and Supplementary Video 1), similar to dynamin1/2-knockout (KO)15. No such effects were observed for catalytically inactive INPP4B (C842A), MTM1 (to deplete potential plasma membrane PI(3)P), or PTEN (to deplete PI(3,4,5)P3) (Fig. 1e and Supplementary Fig. 2). These data identify PI(3,4)P2 as a novel regulator of CME, possibly involved in a late stage in the pathway different from PI(4,5)P2-controlled CCP initiation (Fig. 1f and Supplementary Fig. 2)4.
PI(3,4)P2 can be generated by wortmannin-sensitive class I PI(3)Ks and subsequent hydrolysis of PI(3,4,5)P3 by 5-phosphatases16 downstream of growth factor activation. Epidermal growth factor (EGF)-induced increase of PI(3,4,5)P3 was abrogated by wortmannin inhibition of class I PI(3)K (Supplementary Fig. 1f), but had only a moderate effect on the basal level of PI(3,4)P2 (Supplementary Fig. 1g). These data suggest the existence of a class I PI(3)K-independent pool of PI(3,4)P2 and are consistent with CME being a constitutive process in most cell types. A less well characterized pathway for PI(3,4)P2 production is the class II PI(3)K-mediated phosphorylation of phosphatidylinositol-4-phosphate (PI(4)P)16. The contribution of this pathway to cellular PI(3,4)P2 synthesis is unknown. Class II PI(3)K C2α was identified as an interactor of clathrin17. PI(3)K C2α also binds to PI(4,5)P2 (ref. 18) and its activity is stimulated by clathrin17, but largely refractory to inhibition by wortmannin19. Quantitative proteomics showed PI(3)K C2α to be enriched in clathrin-coated vesicles (CCVs) with about 10 copies per vesicle20. We found endogenous PI(3)K C2α to co-localize with clathrin in endocytic CCPs (Fig. 2a; in agreement with ref. 17), and confirmed its enrichment in CCVs (Supplementary Fig. 3a). Clathrin knockdown caused dispersal of PI(3)K C2α to the cytosol, indicating that membrane targeting of PI(3)K C2α requires clathrin (Supplementary Fig. 3b). Cells depleted of PI(3)K C2α (Supplementary Fig. 3c) showed reduced CME of transferrin and increased transferrin receptor surface levels (228 ± 23% of mock control; rescue, 111 ± 12% of mock; s.e.m., n = 5 experiments), an effect rescued by re-expression of short interfering RNA (siRNA)-resistant PI(3)K C2α fused with enhanced green fluorescent protein (EGFP; Fig. 2b). CME of EGF was reduced to a lesser extent (Supplementary Fig. 3d). Defective transferrin–CME was also observed in mouse embryonic fibroblasts derived from PI(3)K C2α-KO mice, an effect rescued by re-expression of wild type, but not catalytically inactive (Supplementary Fig. 5b) mutant PI(3)K C2α (Supplementary Fig. 3e). Loss of PI(3)KC 2α thus phenocopies effects of PI(3,4)P2 depletion on CME.
a, Confocal images of Cos7 cells stained for endogenous PI(3)K C2α and clathrin heavy chain (CHC). Scale bar, 10 µm. b, PI(3)K C2α depletion impairs CME of transferrin. Cos7 cells depleted of PI(3)K C2α expressing eGFP or siRNA-resistant EGFP–PI(3)K C2α wild type were assayed for CME of transferrin. Bar diagrams represent the ratio of internalized (10 min, 37 °C) to surface transferrin (45 min, 4 °C) (mean ± s.e.m.; n = 5 experiments; ***P < 0.001, t-test). c, d, PI(3)K C2α depletion impairs CCP dynamics analysed by TIRF imaging of EGFP–clathrin expressing Cos7 cells depleted of PI(3)K C2α. c, Kymographs show increased CCP-lifetimes in cells depleted of PI(3)K C2α (see Supplementary Videos 2 and 3). d, Lifetime distribution of CCPs binned in categories of 60 s. Data represent mean ± s.e.m. (n = 3 experiments with >1,000 CCPs per condition; *P < 0.05, **P < 0.01, t-test for scrambled vs PI(3)K C2α siRNA-treated cells). e, f, Ultrastructural analysis of CCPs in control or PI(3)K C2α-depleted cells. Morphological groups were shallow (stage 1), non-constricted U-shaped (stage 2), constricted Ω-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 C2α-depleted cell illustrating accumulation and clustering of U-shaped pits (bottom). Scale bar, 100 nm. f, Bar diagram detailing the relative abundance of different clathrin-coated structures in control or PI(3)K C2α-depleted cells (mean ± s.e.m.; n = 10 (mock, scrambled siRNA) or n = 11 (PI(3)K C2α siRNA) cell perimeters). g, h, Timing of recruitment of PI(3)K C2α and SNX9 to CCPs analysed by TIRF microscopy. mRFP, monomeric red fluorescent protein. g, Snapshots of endocytic proteins at single CCPs (fission at t = 0). h, Mean time course of relative fluorescence intensity at CCPs (mean ± s.e.m.; 3 experiments for clathrin, dynamin 2 and PI(3)K C2α, 2 for SNX9; total number n of CCPs: n = 58 for clathrin, n = 85 for dynamin2, n = 248 for PI(3)K C2α, n = 100 for SNX9).
Next we analysed the dynamics of plasmalemmal CCPs in PI(3)K C2α depleted cells by TIRF microscopy. Cells lacking PI(3)K C2α showed increased CCP lifetimes (Fig. 2c, d and Supplementary Videos 2 and 3) and this was rescued by re-expression of siRNA-resistant EGFP–PI(3)K C2α (Supplementary Fig. 4a). Although nucleation and growth of CCPs were unaltered, they frequently failed to mature to a fission-competent state. Instead, many CCPs seemed to grow beyond the size at which they would normally undergo fission and could be observed to split into two or three closely neighboured CCPs (Supplementary Fig. 4b). Attenuated dynamics of CCPs in PI(3)K C2α-depleted cells were also seen in fluorescence recovery after photobleaching experiments (Supplementary Fig. 4d, e).
To determine whether PI(3)K C2α regulates maturation of CCPs, before or in conjunction with dynamin-mediated fission, we subjected PI(3)K C2α-depleted cells to quantitative morphometric analysis. This revealed an increased number of U-shaped CCPs, a stage preceding constriction and dynamin-mediated fission, whereas the frequencies of early shallow CCPs, Ω-shaped constricted CCPs, or of free CCVs were unaltered (Fig. 2e, f). CCPs frequently appeared clustered (Supplementary Fig. 4f), as also seen by live imaging (Supplementary Fig. 4b, c). Analysis of the dynamics of endocytic protein recruitment to CCPs showed PI(3)K C2α to follow clathrin but to precede dynamin 2 (Fig. 2g, h). We conclude that PI(3)K C2α regulates CCP maturation by facilitating the transition from invaginated to Ω-shaped CCPs.
To explore whether the function of PI(3)K C2α in CME requires its phosphatidylinositol kinase activity we assayed catalytically inactive mutant PI(3)K C2α (Supplementary Fig. 5b). Endocytic proteins such as AP-2α accumulate at CCPs following depletion of PI(3,4)P2 (Fig. 1d, e) or PI(3)K C2α (Fig. 3a). This defect was rescued by siRNA-resistant wild type but not catalytically inactive mutant PI(3)K C2α, although both variants localized to CCPs (Supplementary Fig. 5a). Thus, PI(3)K C2α function in CME requires its PI(3)K activity.
a, Requirement for PI(3)K activity of PI(3)K C2α in CME. Mean intensity of endocytic AP-2α-containing CCPs in PI(3)K C2α-depleted Cos7 cells expressing EGFP, siRNA-resistant wild-type (WT) or kinase inactive EGFP–PI(3)K C2α (mean ± s.e.m.; n = 3 experiments; *P < 0.05, **P < 0.01, ***P < 0.001, t-test). b, PI(3)K C2α preferentially synthesizes PI(3,4)P2. Enzymatic activity of immunoprecipitated 6×Myc–PI(3)K C2α. Data, mean ± s.e.m. normalized to level of PI(3)P synthesis (n = 9 experiments; ***P < 0.001, t-test). No 3-kinase activity was detectable in absence of induction of PI(3)K C2α expression. c, d, Selective reduction of PI(3,4)P2 in PI(3)K C2α-depleted cells. c, Loss of plasma membrane association of the PI(3,4)P2-sensor 2×TAPP1-PH but not of probes for other phosphatidylinositols determined by ratiometric TIRF/epifluorescent imaging (mean ± s.e.m.; n (experiments) = 9 (2×TAPP1-PH), n = 7 (2×FYVE, a sensor for PI(3)P), n = 4 (PH-PLCδ, a sensor for PI(4,5)P2, and PH-Btk, a sensor for PI(3,4,5)P3); **P < 0.01, ***P < 0.001, t-test). d, Levels of PI(3,4)P2 or PI(4,5)P2 quantified by PI(3,4)P2- or PI(4,5)P2-specific antibodies (mean ± s.e.m.; n = 6 experiments; *P < 0.05, t-test). e, Alignment of substrate-binding loop sequences of human phosphatidylinositol-3-kinases and a PI(3)K C2α class III-like mutant (cl. III mut) that can only synthesize PI(3)P but not PI(3,4)P2. f, g, Requirement for PI(3)K C2α-mediated PI(3,4)P2 synthesis in CME. f, Impaired CME in PI(3)K C2α-deficient cells is rescued by re-expression of wild-type (WT) but not class III-like mutant EGFP–PI(3)K C2α. Bar diagrams represent the ratio of internalized (10 min, 37 °C) to surface transferrin (45 min, 4 °C) (mean ± s.e.m.; n = 3 experiments; ***P < 0.001, t-test compared to scrambled siRNA). g, Mean intensity of endocytic AP-2α-containing CCPs in PI(3)K C2α-deficient Cos7 expressing WT or class III-like mutant PI(3)K C2α (mean ± s.e.m.; n = 3 experiments; ***P < 0.001, t-test).
Previous studies have yielded conflicting data regarding the dominant lipid product of PI(3)K C2α, reporting either preferential synthesis of PI(3,4)P2 or PI(3)P21,22. Immunopurified PI(3)K C2α preferentially produced 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 C2α was to contribute to PI(3,4)P2 formation in vivo, knockdown of PI(3)K C2α should result in reduced PI(3,4)P2 levels. Quantitative assessment of plasma membrane phosphatidylinositols by specific PI-binding domain-based sensors revealed a selective reduction of PI(3,4)P2 in PI(3)K C2α-knockdown cells, whereas PI(3)P, PI(4,5)P2 or PI(3,4,5)P3 remained unchanged (Fig. 3c). Depletion of PI(3,4)P2, but not of PI(4,5)P2, was also detectable with PI-specific antibodies (Fig. 3d). Consistently, PI(3,4)P2 largely co-localized with the plasma membrane pool of PI(3)K C2α (Supplementary Fig. 5d). Conversely, we failed to detect PI(3,4)P2 at CCPs in PI(3)K C2α-depleted cells (Supplementary Fig. 5e). These results are consistent with the preferred production of PI(3,4)P2 by PI(3)K C2α in vitro and support the hypothesis that PI(3)K C2α contributes 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 2α in vivo we capitalized on the fact that the specificity of PI(3)Ks is encoded within the phosphatidylinositol-binding activation loop23. The activation loop of PI(3,4,5)P3-producing class I PI(3)Ks contains 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 III PI(3)K hVps34 (Fig. 3e). PI(3)K C2α only contains basic residues that coordinate the 4-phosphate group, consistent with PI(3,4)P2 synthesis. To distinguish between PI(3)K C2α-mediated formation of PI(3,4)P2 or PI(3)P at CCPs we constructed a PI(3)K C2α mutant, in which the 4-phosphate coordinating box was exchanged with the corresponding sequence from hVps34 (Fig. 3e). This class III-like mutant PI(3)K C2α selectively synthesized PI(3)P with wild-type PI(3)K C2α activity, but failed to produce PI(3,4)P2 (Supplementary Fig. 5b, c). It was also unable to rescue defective CME in PI(3)K C2α-depleted cells (Fig. 3f, g). Thus, CME requires PI(3)K C2α-mediated production of PI(3,4)P2, but not of PI(3)P.
To challenge this hypothesis by an independent approach we made use of cell-permeable PI-derivatives24 to exogenously supply PI(3)P or PI(3,4)P2. Addition of cell-permeable PI(3,4)P2 partially rescued endocytic protein accumulation at CCPs in PI(3)K C2α-depleted cells, whereas PI(3)P was inactive (Supplementary Fig. 5f), although it stimulated early endosome fusion (ref. 24 and not shown). We conclude that PI(3)K C2α is required for local PI(3,4)P2 production at endocytic CCPs.
Absence of PI(3)K C2α or depletion of its lipid product PI(3,4)P2 causes a delay in CCP maturation, suggesting the presence of PI(3,4)P2 effectors at CCPs. To identify such effectors we monitored CCP enrichment of endocytic proteins in PI(3)K C2α-depleted cells (Supplementary Fig. 6a). Of the proteins assayed the only one that failed to enrich at CCPs in PI(3)K C2α-depleted cells was the PX-BAR domain protein sorting nexin 9 (SNX9) (Supplementary Fig. 6a). We thus analysed the ability of SNX9 to associate with phosphatidylinositol liposomes. Endogenous SNX9 (Fig. 4a) or its PX-BAR module (Supplementary Fig. 6b) preferentially 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. As binding experiments with purified proteins might poorly reflect the situation in vivo we directly compared phosphatidylinositol association of SNX9 with that of other endocytic proteins in brain extracts. Only SNX9 preferred association with PI(3,4)P2 over PI(4,5)P2, whereas AP180, epsin 1 and AP-2α 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, we analysed the localization of SNX9 at CCPs in cells depleted of PI(3)K C2α or PI(3,4)P2. Loss of dynamins results in accumulation of SNX9 assemblies on elongated necks of arrested CCPs15. We confirmed the enrichment of endogenous SNX9 at AP-2α-coated endocytic intermediates in cells depleted of dynamin 2 (Fig. 4d, e). Co-silencing of PI(3)K C2α with dynamin2 prevented SNX9 accumulation at arrested CCPs (Fig. 4d, e and Supplementary Fig. 7a), whereas other endocytic proteins accumulated irrespective of the presence of PI(3)K C2α (Supplementary Fig. 7b). Similar effects were caused by INPP4B–CAAX-mediated depletion of PI(3,4)P2 (Fig. 4f, g and Supplementary Fig. 7d). Knockdown of SNX9 or PI(3)K C2α 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 C2α-mediated PI(3,4)P2 production is required for SNX9 recruitment during late stages of CME.
a, Binding of endogenous SNX9 from Hek293 cells to liposomes containing 5 mol% of the indicated phosphatidylinositol in flotation assays. Input, 10 µg protein for bound (top) or 30 µg (bottom) for unbound fractions (representative of 3 experiments). b, c, Association of SNX9 affinity-isolated from rat brain extracts with PI(3,4)P2- beads. Endocytic proteins AP180, AP-2α or epsin 1 preferentially associate with PI(4,5)P2-beads. Clathrin, negative control. b, Densitometric quantification of data in a (mean ± s.e.m.; n = 3 experiments; **P < 0.01, ***P < 0.001, t-test). d, e, SNX9 accumulation at endocytic intermediates requires PI(3)K C2α. Confocal images of Cos7 cells depleted of PI(3)K C2α, dynamin2, or both, stained for AP-2α and SNX9. d, Scale bar, 10 µm. e, Quantitative analysis of SNX9 levels at endocytic intermediates as shown in d (mean ± s.e.m.; n = 3 experiments; *P < 0.05, t-test). f, g, PI(3,4)P2 is required for accumulation of SNX9 at stalled CCPs. f, Confocal images of endocytic protein accumulation in dynamin 2-deprived Cos7 cells depleted of PI(3,4)P2 by mCherry–INPP4B–CAAX. Depletion of PI(3,4)P2 prevents accumulation of SNX9 but not of AP-2α at endocytic intermediates. Scale bar, 10 µm. g, Quantification of SNX9 levels at stalled CCPs as shown in f (mean ± s.e.m.; n = 3 experiments; *P < 0.05, t-test). h, Impaired CME of transferrin in Cos7 cells depleted of SNX9 and its close paralogue SNX18 is rescued by re-expression of wild-type (WT) EGFP–SNX9 but not of PI-binding deficient PX-domain mutants RYK (SNX9(R286A, Y287A, K288); ref. 26) or K267N, R327N (see Supplementary Fig. 6c). Bar diagrams represent the ratio of internalized (10 min, 37 °C) to surface transferrin (45 min, 4 °C) (mean ± s.e.m.; n = 5 experiments, except n = 4 (EGFP–SNX9(RYK) and EGFP–SNX9(K267N, R327N) and n = 2 (SNX18); **P < 0.01, ***P < 0.001, t-test vs scrambled siRNA).
Previous work has shown that depletion of SNX9 interferes with CME in HeLa cells and we confirmed this (Supplementary Fig. 8a). In other cell lines (that is, Cos7) SNX9 is functionally redundant with its paralogue SNX1825 (ref. 25). In agreement, depletion of SNX9 and SNX18 in Cos7 cells (Supplementary Fig. 8b) inhibited transferrin-CME (Fig. 4h) and interfered with CCP dynamics evidenced by AP-2α accumulation (Supplementary Fig. 8c), similar to the effects seen upon depletion of PI(3,4)P2 or PI(3)K C2α (compare Fig. 1e with Fig. 2c, d). Defective CME or AP-2α accumulation were rescued by siRNA-resistant wild-type EGFP–SNX9 but not mutants of SNX9, in which key residues required for binding to phosphatidylinositol-3-phosphates (Supplementary Fig. 6c, ref. 26) had been mutated (Fig. 4h).
Thus, PI(3)K C2α via its lipid product PI(3,4)P2 facilitates enrichment of PI(3,4)P2-binding effector proteins, most notably SNX9 before dynamin-mediated fission. Total internal reflection fluorescence (TIRF) microscopy analysis indeed revealed that accumulation of mCherry–SNX9 was delayed by about 20 s with respect to EGFP–PI(3)K C2α, but preceded arrival of dynamin 2 (Fig. 2g, h). These data agree with a spatiotemporal computational model that suggests a mechanism by which PI(3,4)P2 production at CCPs triggers selective SNX9 recruitment (for details see Schöneberg et al. in preparation, preprint at http://arxiv.org/find/physics/1/au:+Noe_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 factor signalling1,2, in constitutive CME. We show PI(3)K C2α-mediated PI(3,4)P2 synthesis to be required for CCP maturation and for recruitment of the PX-BAR domain protein SNX9 to CCPs at a late stage preceding dynamin-mediated fission. Our analysis of the timing of endocytic protein arrival at CCPs indicates a hitherto unknown functional interplay between PI(4,5)P2 and PI(3,4)P2 in controlling distinct stages of CME in mammalian cells. We further suggest that the combined activities of PI(4,5)P2-phosphatases9 and of PI(3)K C2α catalyse phosphatidylinositol conversion from PI(4,5)P2 to PI(3,4)P2. Phosphatidylinositol conversion regulates CCP maturation and constriction and may thereby prepare endocytic vesicles for fusion with PI(3)P-containing endosomes. Similar conversion mechanisms involving Rab proteins and phosphatidylinositols regulate further endosomal progression27. The identification of PI(3)K C2α as a major PI(3,4)P2-synthesizing enzyme will pave the way for the further study of this exciting lipid in cell physiological processes other than CME and in disease including cancer13 and diabetes21,22.
Methods Summary
Total internal reflection fluorescence (TIRF) microscopy
TIRF imaging was performed using a Zeiss Axiovert200M microscope equipped with an incubation chamber (37 °C and 5% CO2), a ×100 TIRF objective and a dual-colour TIRF setup (Visitron Systems) using Slidebook imaging software (3i Inc.). For analysis of CCP dynamics, time-lapse series of 3 min with a frame rate of 0.5 Hz were recorded.
Electron microscopy
Glutaraldehyde-fixed Cos7 cells treated with siRNAs were scraped, pelleted, and subsequently processed for electron microscopy and morphometric analysis.
Lipid kinase assays
Kinase activity was assessed by a radioactivity-based assay (in kinase buffer: 5 mM HEPES/KOH pH 7.2, 25 mM KCl, 2.5 mM MgOAc, 150 mM KGlu, 10 µM CaCl2, 0.2% CHAPS) using recombinant 6×myc–PI(3)K C2α immunoprecipitated from overexpressing HEK293 cells. 200 µM phosphoinositides, 200 µM ATP and 8 µCi of [γ-32P]ATP were combined with 1 recombinant 6×myc–PI(3)K C2α and incubated at 37 °C for 10 min. Reactions were stopped by addition of 500 µl cold methanol:H2O:32% HCl (10:10:1), followed by lipid extraction and thin-layer-chromatography (TLC) analysis.
Online Methods
Antibodies
An overview of all antibodies used in this study is given in Supplementary Table 1.
siRNAs
All siRNA sequences were used as 21-mers or 23-mers including 3′-dTdT overhangs. The sequences of the PI(3)K C2α-targeting siRNAs used in this study are as follows: siRNA 1 5′-ggatctttttaaacctatt-3′; siRNA 2 5′-gcacaaacccaggctattt-3′. The dynamin 2 siRNA sequence used is: 5′-gcaactgaccaaccacatc-3′. For silencing of SNX9 expression in HeLa cells, a pool of 4 siRNAs was obtained from Dharmacon (Thermo Scientific). The SNX9 siRNA sequence used for Cos7 cells lies within the 3′-UTR of the mRNA and is: 5′-ggacagaacgggccttgaa-3′. For silencing of SNX18 expression the siRNA sequence used is: 5′-caccgacgagaaagccuggaa-3′. The scrambled control siRNA used in all experiments corresponds to the scrambled µ2 adaptin sequence 5′-gtaactgtcggctcgtggt-3′.
Lipid reagents
Phosphatidylinositols for lipid binding assays were obtained from Avanti Polar Lipids, phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol were from Sigma-Aldrich, l-α-phosphaditylethanolamine (PE) was from Jena Bioscience and rhodamine–PE was from Avanti Polar Lipids.
Plasmids
The sequence encoding full-length human INPP4B was amplified from cDNA provided by L. Cantley and inserted in frame between the EcoRV and NotI sites of a pcDNA3.1(+)-based HA-expression vector (sequence between NdeI and EcoRV exchanged with that from pcHA2) modified to encode the carboxy-terminal CAAX-box prenylation sequence from K-ras (KSKTKCVIM-Stop28) directly following the Not I site. The INPP4B–CAAX encoding sequence was subcloned into an mCherry-expression vector for live cell imaging. Expression plasmids encoding INPP4B–CAAX(C842A), full-length human MTM1–CAAX14, full-length human PTEN–CAAX and residues 214–614 of human INPP5E–CAAX29 were designed identically. The RFP–2×PH–TAPP1 construct was a gift from T. Takenawa and the GFP–PH domain of Bruton’s tyrosine kinase was provided by M. Wymann. For analysis of recruitment of proteins to CCPs in live cells, a fusion of mRFP to rat clathrin light chain inserted between KpnI and ApaI of pcDNA5/FRT/TO and a mouse dynamin 2–mCherry construct provided by O. Daumke were used in conjunction with a pEGFP–C3–PI(3)K C2α construct encoding human full-length PI(3)K C2α assembled from HeLa cDNA (verified by sequencing). A kinase-inactive mutant of PI(3)K C2α was obtained by mutating the ATP-binding site (K1138A, D1157A) and the catalytic loop (D1250A)30. Constructs of wild-type and kinase-inactive PI(3)K C2α resistant to siRNA 1 were generated by creating 4 silent mutations: 5′-agatctattcaaaccgatt-3′. The PI(3)P-restricted class III mutant of PI(3)K C2α resulted from the mutation of 1283KRDR1286 to 1283KPLP1286. For visualization of proteins at CCPs, a 3×HA–Hip1R construct was provided by T. Ross and a clone encoding epsin 1–GFP was purchased from OriGene Technologies. All constructs encoding full-length SNX9 or domains thereof were derived from human SNX9 cDNA provided by W. Yang. For GST–PX–BAR, cDNA encoding amino acids 204 to 595 of human SNX9 (ref. 31) was cloned in between the EcoRI and NotI sites of pGex-4T-1.
Cell lines
All cell lines used (Cos7, HEK293, HeLa) were obtained from ATCC and not used beyond passage 30 from original derivation by ATCC. Cell lines were routinely tested for mycoplasma contamination.
siRNA and plasmid transfections
HeLa or Cos7 cells seeded on day 0 were transfected with siRNAs using Oligofectamine (Invitrogen) according to the manufacturer’s instructions on day 1, expanded on day 2, transfected a second time on day 3, seeded for the experiment on day 4, and used for the experiment on day 5. For expression of recombinant proteins in knockdown cells, plasmids were transfected using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions on day 4. In the case of INPP4B–CAAX constructs, cells were sequentially transfected first with siRNAs and then with plasmid both on day 3 to allow for a total expression time of 40 h.
Upon plasmid transfection of untreated cells, cells were generally allowed to express protein overnight and analysed the next day except for INPP4B–CAAX constructs where expression for two days was found to give better results.
Transferrin uptake and surface labelling
HeLa cells treated with siRNAs or transfected with mCherry–INPP4B–CAAX were seeded on Matrigel (BD biosciences)-coated coverslips. On the day of the experiment, cells were serum-starved for 1.5 h and used for either transferrin uptake or transferrin receptor surface labelling. For transferrin uptake, cells were incubated with 25 µg ml−1 transferrin–Alexa568 or transferrin–Alexa647 (Molecular Probes, Invitrogen) for 10 min at 37 °C. After two washes with ice-cold PBS cells were acid washed at pH 5.3 (0.2 M sodium acetate, 200 mM sodium chloride) on ice for 2 min to remove surface-bound transferrin, washed 2 times with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) for 45 min at room temperature. For surface labelling, cells were incubated with 25 µg ml−1 transferrin–Alexa568 at 4 °C for 45 min to block endocytosis and label transferrin receptors on the cell surface. Cells were washed 3 times with ice-cold PBS on ice for one min and fixed with 4% PFA for 45 min at room temperature.
Transferrin labelling was analysed using a Zeiss Axiovert200M microscope and Slidebook imaging software (3i Inc.). Internalized transferrin per cell was quantified and normalized to the amount of surface-bound transferrin determined in the same experiment as a measure for the efficiency of internalization.
Immunocytochemistry
Staining of proteins in cultured cells seeded on glass coverslips was performed as described32. For lipid antibody stainings, Cos7 cells were fixed in 2% PFA at room temperature for 20 min and permeabilized with saponin (30 min at room temperature in 0.5% saponin, 1% bovine serum albumin (BSA) in PBS). Cells were labelled with lipid-specific antibodies (see Supplementary Table 1) diluted in 1% BSA in PBS for 2 h at room temperature. After washing three times for 5 min with PBS, cells were incubated with appropriate fluorescent secondary antibodies for 1 h and washed three times 10 min with PBS. Protein and lipid immunocytochemistry stainings were routinely analysed and quantified using a spinning disk confocal microscope (Ultraview ERS, Perkin Elmer) and Volocity imaging software (Improvision, Perkin Elmer).
Total internal reflection fluorescence (TIRF) microscopy
TIRF imaging was performed using a Zeiss Axiovert200M microscope equipped with an incubation chamber (37 °C and 5% CO2), a ×100 TIRF objective and a dual-colour TIRF setup from Visitron Systems using Slidebook imaging software (3i Inc.). For analysis of CCP dynamics, time-lapse series of 3 min with a frame rate of 0.5 Hz were recorded. CCP lifetimes were assessed by arbitrarily selecting 50 or 25 CCPs per cell in the centre frame of the time-lapse series and determining the frame of appearance and disappearance. In case CCPs already existed in the first frame or persisted until the last frame, these frames were counted. For the analysis of recruitment time courses of proteins to CCPs, only CCPs were used that both appeared and disappeared within the time lapse series. From these, fluorescence intensities over time were quantified and aligned on the time axis by the last frame of GFP–PI(3)K C2α presence (t = 0, fission). Fluorescence intensities for all time points in relation to t = 0 were averaged over all CCPs in the analysis and renormalized to the resulting peak value. For analysis of GFP–PHBtk membrane association, TIRF and epifluorescence images of the same cell were acquired and the TIRF fluorescence intensity was normalized to the epifluorescence signal in order to achieve intrinsic correction for expression level variations between cells.
Fluorescence recovery after photobleaching (FRAP)
FRAP experiments were performed using a spinning disk confocal microscope equipped with an incubation chamber (37 °C) and a photokinesis unit (Ultraview ERS, Perkin Elmer). One to three regions of interest in the peripheral, flat part of an EGFP–CLC expressing cell were selected. A time-lapse series at 0.5 Hz was recorded with 10 frames before and 60 frames after bleaching. For quantification, the sum EGFP–CLC fluorescence intensity at CCPs was quantified over time and normalized to the mean sum intensity during the pre-bleaching period.
PIP/AM experiments
Cell-permeable acetoxy methylester (AM)-protected phosphatidylinositol derivatives were synthesized as described33. For treatment of cells, PI(3)P/AM or PI(3,4)P2/AM dissolved in dry DMSO were mixed with an equal volume of 10% pluronic F127 in DMSO (Sigma-Aldrich) to enhance solubility in aqueous buffers and diluted in DMEM to a final concentration of 200 µM. Cells on coverslips were treated with DMSO + pluronic (control) or PIP /AMs for 10 min at 37 °C 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, and subsequently processed for electron microscopy and morphometric analysis as previously described15. Briefly, after epoxy resin embedding and sectioning, micrographs were taken along the cell perimeter at ×20,000 magnification. Images were combined to reconstruct the cell perimeter and numbers of clathrin-coated intermediates were determined.
Purification of clathrin-coated vesicles
CCVs were purified essentially as described35. Briefly, calf brain was homogenized and the cytosolic and microsomal fraction was obtained by sequential centrifugation at 17,000g and 30,000g. Light membranes were pelleted at 150,000g, resuspended and mixed with an equal volume of 12.5% Ficoll, 12.5% sucrose solution to adjust the density of the solution to that of CCVs. Contaminating, heavier membranes were removed by centrifugation at 90,000g and the CCV-containing supernatant was diluted in order to allow sedimentation of CCVs at 150,000g. For stripping of coat proteins, purified CCVs were incubated over night at room temperature with 0.8 M Tris-HCl pH 7.4 to disrupt protein–membrane interactions. Vesicles including integral membrane proteins were sedimented at 250,000g.
In vitro kinase activity assays
Kinase activity of recombinant PI(3)K C2α was assessed using a radioactivity-based assay essentially as described36. In brief, one 10-cm dish of HEK293 cells transiently overexpressing 6×myc-PI(3)K C2α was lysed in immunoprecipitation (IP) buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, 1% CHAPS, 1 mM PMSF, 0.3% protease inhibitor cocktail (Sigma)), and centrifuged for 10 min at 20,500g at 4 °C. The resulting supernatant was centrifuged at 65,000 r.p.m. in a TLA-110 rotor (Beckman Coulter). PI(3)K C2α was immunoprecipitated from the extract using ∼15 µg c-myc antibody and 1.5 mg of protein as IP input. The IP was washed twice with IP buffer and once in kinase buffer (5 mM HEPES/KOH pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate (Mg(CH3COO)2), 150 mM KGlu, 10 µM CaCl2, 0.2% CHAPS). Phosphatidylinositols were dissolved in kinase buffer (note that presence of 0.2% CHAPS was required for full solubility of PI(4)P), incubated on ice for 30 min, sonicated for 1 min using a small tip sonicator (Bandelin Sonoplus) 1 s on 1 s off at 70% intensity. 200 µM phosphoinositides, 200 µM ATP and 8 µCi of [γ-32P]ATP were combined with 1/8 of one IP sample and incubated at 37 °C for 10 min. The reactions were stopped by the addition of 500 µl cold methanol:H2O:32%HCl (10:10:1) and lipid extraction and thin-layer-chromatography (TLC) analysis were performed as described36.
Liposome flotation assay
Liposome preparation. A total of 600–800 µg of lipids were dissolved in a mixture of CHCl3:methanol:1 N HCl (2:1:0.01) to the desired concentration, combined in a glass vial and dried under pressurized N2 followed by vacuum for 30 min. Liposomes were rehydrated in 300 µl HEPES buffer (50 mM HEPES pH 7.4, 100 mM KCl (140 mM KCl for experiments with GFP–SNX9 WT or K267N, R327N; 200 mM KCl for experiments with GST–SNX9 PX–BAR)) for 1 h at room temperature under frequent vortexing. After the addition of 1.7 ml H2O the liposomes were centrifuged in a TLA110 rotor at 20,000 r.p.m. at 4 °C for one hour. The resulting pellet was resuspended in HEPES buffer to a final lipid concentration of 3 mg ml−1. Liposome mixtures were extruded 14 times through an 800-nm polycarbonate membrane (Whatman) using a manually operated extruder (LiposoFast, Avestin, Inc.). The final concentration of lipid species in mol% were: 50% PC, 20% cholesterol, 19% PE, 1% rhodamine–PE, 5% PS, and 5% phosphatidylinositols.
Flotation assay. 450 µg liposomes in HEPES buffer were combined with either 2 µg of purified GST–SNX9 PX-BAR protein or with 30 µg of HEK293 cell extract containing overexpressed HA–SNX9 PX-BAR or GFP–SNX9 full-length protein and incubated for 15 min at 4 °C on a rotating wheel. The mixture was then adjusted to 30% sucrose by adding 75% sucrose in HEPES buffer and transferred to a TLS-55 centrifuge tube. This was overlaid with 200 µl of 25% sucrose in HEPES buffer followed by 50 µl of HEPES buffer. Liposomes were floated by centrifuging 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 needle attached to a calibrated syringe by removing the bottom layer first (∼250 µl total volume), followed by the middle layer (200 µl) and in the end the top layer containing the liposomes and any bound protein. Top and bottom fractions were separated on 8% acrylamide gels and stained with Coomassie for GST–SNX9 PX-BAR and immunoblotted for HA–SNX9 PX-BAR or GFP–SNX9 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 from rat brain extract. Rat brain extract was prepared from 2.5 g frozen rat brain homogenized in homogenization buffer (4 mM HEPES pH 7.4, 320 mM sucrose, 1 mM PMSF, 0.3% protease inhibitor cocktail) using 13 strokes of a glass–Teflon-homogenizer at 900 r.p.m. The homogenate was centrifuged at 900g for 10 min at 4 °C. To the supernatant PIP pull-down buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 0.25% NP-40) was added to 1 × concentration and incubated on ice for 30 min followed by centrifugation at 43,500g at 4 °C. The supernatant was centrifuged again at 265,000g for 15 min at 4 °C to remove aggregated proteins. 6–8 mg of protein were added to 100 µl of 1:1 washed agarose-bead slurry and incubated at 4 °C for 1.5 h on a rotating wheel. Beads were pelleted, washed 3 times with PIP pull-down buffer and bound proteins were eluted two times in 30 µl of 1 × Laemmli sample buffer. 30 µl of the pooled eluate were then loaded onto an 8% acrylamide gel for SDS–PAGE followed by immunoblotting.
Statistical methods
For analyses comprising multiple independent experiments (n), sample size within each experiment was chosen to provide statistically significant estimates for each sample, corresponding to 20 to 40 images per sample for microscopy-based quantifications. In all experiments, cells were arbitrarily chosen based on the signal in a separate channel independent from the signal to be quantified. All statistical tests performed were two-tailed, unpaired t-tests as judged appropriate for the respective experiments.
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Acknowledgements
We thank E. Ungewickell, P. Di Fiore, P. De Camilli, H. McMahon, E. Wancker, T. Südhof and S. Carlsson for antibodies, L. Cantley, T. Takenawa, M. Wymann, T. Ross, O. Daumke and W. Yang for plasmids, and O. Daumke, B. Eickolt and F. Wieland for critical comments. Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 740/C8; SFB 740/D7; SFB 958/A04; SFB 958/A07; SFB 958/Z02).
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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. conducted simulations. Y.P., F.N. and V.H. wrote the manuscript.
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This file contains Supplementary Figures 1-8 and Supplementary Table 1. (PDF 10942 kb)
Depletion of PI(3,4)P2 attenuates CCP dynamics
The peripheral regions of two neighbouring eGFP-clathrin light chain expressing cells imaged by TIRF microscopy shown for 3 min. The cell on the right suffers from PI(3,4)P2 depletion due to co-expression of mCherry-INPP4B-CAAX . For clarity, a dotted line has been drawn along the border between the two cells. Note the strikingly attenuated CCPs dynamics in the PI(3,4)P2-depleted cell. (MOV 7431 kb)
Attenuated CCP dynamics upon depletion of PI3K C2α
Videos 2 and 3 show representative areas from eGFP-clathrin light chain expressing cells treated with scrambled or PI3K C2α siRNAs, respectively, imaged by TIRF microscopy for 3 min. CCPs in control cells (video 2; scrambled siRNA) display a dynamic succession of appearance, growth, and disappearance (internalization). By contrast, CCPs in PI3K C2α-depleted cells (video 3; PI3K C2α-siRNA) are long-lived and stable over time, indicative of defective CCP maturation. (MOV 4251 kb)
Attenuated CCP dynamics upon depletion of PI3K C2α
Videos 2 and 3 show representative areas from eGFP-clathrin light chain expressing cells treated with scrambled or PI3K C2α siRNAs, respectively, imaged by TIRF microscopy for 3 min. CCPs in control cells (video 2; scrambled siRNA) display a dynamic succession of appearance, growth, and disappearance (internalization). By contrast, CCPs in PI3K C2α-depleted cells (video 3; PI3K C2α-siRNA) are long-lived and stable over time, indicative of defective CCP maturation. (MOV 3910 kb)
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Posor, Y., Eichhorn-Gruenig, M., Puchkov, D. et al. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, 233–237 (2013). https://doi.org/10.1038/nature12360
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DOI: https://doi.org/10.1038/nature12360
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