Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting

Caveolae are flask-shaped invaginations of the plasma membrane that are associated with tumor formation, pathogen entry and muscular dystrophy, through the regulation of lipids, signal transduction and endocytosis. Caveolae are generated by the fusion of caveolin-1-containing vesicles with the plasma membrane, which then participate in endocytosis via dynamin. Proteins containing membrane-sculpting F-BAR (or EFC) domains organize the membrane in clathrin-mediated endocytosis. Here, we show that the F-BAR protein PACSIN2 sculpts the plasma membrane of the caveola. The PACSIN2 F-BAR domain interacts directly with caveolin-1 by unmasking autoinhibition of PACSIN2. Furthermore, the membrane invaginations induced by the PACSIN2 F-BAR domain contained caveolin-1. Knockdown of PACSIN2 resulted in abnormal morphology of caveolin-1-associated plasma membranes, presumably as a result of decreased recruitment of dynamin-2 to caveolin-1. These results indicate that PACSIN2 mediates membrane sculpting by caveolin-1 in caveola morphology and recruits dynamin-2 for caveola fission.

PACSIN2-knockdown cells. The cellular tubulation observed in cells expressing the F-BAR domain or with PACSIN2 knockdown might be caused by the lack of recruitment of dynamin-2 to caveolae.
To further demonstrate the localization of PACSIN2 at caveolae, we examined non-transfected HeLa cells by electron microscopy. A plasma membrane sheet was prepared from HeLa cells and was labeled with anti-caveolin-1 and anti-PACSIN2 antibodies, followed by immunogold. The PACSIN2 label was found in close proximity to the caveolin-1 label (Fig. 1D,E). The PACSIN2 label was also observed at structures lacking the caveolin-1 label, which were smaller than caveolae (Fig. 1E). Thin-section electron microscopy revealed anti-PACSIN2 antibody staining in the necks of the flasklike invaginations typical of caveolae (Fig. 1F). Staining signals were also detected on the relatively flat plasma membrane and in the cytosol (Fig. 1F). No signals were identified in the absence of antibody (supplementary material Fig. S2). These data suggest that PACSIN2 functions in caveolae at the plasma membrane. (A)HeLa cells were labeled with anti-PACSIN2, anti-caveolin-1, and phalloidin to detect endogenous PACSIN2 (green), caveolin-1 (red) and actin filaments (blue), respectively. The merged image is shown. The average percentages of PACSIN2 or caveolin-1 puncta with caveolin-1 or PACSIN2, respectively, in ten cells are shown (±s.d.). (B)Each channel in A shown separately. (C)GFP-PACSIN2 (green) was coexpressed with caveolin-1-DsRed (red) in HeLa cells. The percentage of GFP puncta with DsRed in ten cells is shown (±s.d.). (D,E)A plasma membrane sheet from nontransfected HeLa cells was stained with an anti-caveolin-1 antibody with a 10-nm-gold-conjugated antibody (arrows in enlarged images in E) and an anti-PACSIN2 antibody with a 5-nm-gold-conjugated antibody (arrowheads in E). (F)Thin-section electron micrograph of non-transfected HeLa cells stained with anti-PACSIN2 antibody. Control images without anti-PACSIN2 antibody are shown in supplementary material Fig. S2.

Overexpression of the PACSIN2 F-BAR domain alters localization of caveolin-1
The canonical F-BAR-domain-containing proteins, such as FBP17 and Toca-1, induce plasma membrane invagination when overexpressed in cells. These invaginations are often observed as the mesh-like localization of the overexpressed F-BAR domain proteins. The overexpressed F-BAR domain fragment of PACSIN2 exhibited a similar mesh-like localization in HeLa cells (Fig. 2). We confirmed that this mesh-like structure corresponded to the plasma membrane invaginations, by labeling the cells with membrane-staining FM dye and the culture medium with sulforhodamine (supplementary material Fig. S3).
We then examined the localization of various membrane marker proteins to the PACSIN2 F-BAR-induced mesh-like structures, and found that caveolin-1 colocalized with PACSIN2. In cells with low expression of the PACSIN2 F-BAR domain ( Fig. 2A, left cell in large panel), the F-BAR domain and caveolin-1 colocalized along the tubules induced by F-BAR domain expression. However, caveolin-1 accumulated into several larger assemblies, which formed at the convergence of the PACSIN2-induced invaginations in cells with high F-BAR-domain expression ( Fig. 2A, right cell in large panel). This caveolin-1 accumulation was not observed in cells expressing PACSIN2 F-BAR domains with tubulation-2034 Journal of Cell Science 124 (12) defective mutations (M124T/M125T) (Shimada et al., 2010) or in those expressing FBP17 (supplementary material Fig. S4A,B). Importantly, the clathrin distribution was not altered by expression of the PACSIN2 F-BAR domain (supplementary material Fig.  S4C). PTRF was only partially localized at tubules induced by the PACSIN2 F-BAR domain (supplementary material Fig. S4D).
Interestingly, the overexpressed PACSIN1 F-BAR domain had a weak effect on caveolin-1 localization, whereas the overexpressed PACSIN3 F-BAR domain had a similar effect on caveolin-1 localization as the PACSIN2 F-BAR domain (Fig. 2B,C).

Binding of caveolin-1 to PACSIN2 induces membrane tubulation
The substantial colocalization of PACSIN2 with caveolin-1 upon their coexpression suggested a direct interaction between these two proteins. PACSIN2 consists of the F-BAR domain and the SH3 domain (Fig. 3A). Caveolin-1 consists of two cytoplasmic regions connected by a region embedded in the membrane (Fig. 3A). We prepared several purified fragments of caveolin-1, and analyzed their binding to purified full-length PACSIN2 in a pull-down assay. The N-terminal cytoplasmic region of caveolin-1 (residues 1-100 or 61-100) interacted with PACSIN2 ( Fig. 3B). However, the association was not observed for the C-terminal cytoplasmic region of caveolin-1 (Fig. 3B). We then mapped the binding region of PACSIN2 to caveolin-1. Similarly to the finding that the PACSIN2 F-BAR domain alone was sufficient for colocalization with caveolin-1 (Fig. 2), the F-BAR domain alone was sufficient for binding to caveolin-1 (Fig. 3C). The binding of caveolin-1 to the F-BAR domain was stronger than that to full-length PACSIN2 (Fig. 3C). The PACSIN2 C-terminal region, including the SH3 domain (residues 301-486), did not interact with caveolin-1 in a pull-down assay with purified proteins (supplementary material Fig. S5A). The association of PACSIN2 and caveolin-1 in cells was confirmed by immunoprecipitation analysis with an anticaveolin-1 antibody (supplementary material Fig. S5B).
We then examined the binding of the PACSIN1 and PACSIN3 F-BAR domains to caveolin-1. Consistent with the weak effect of the PACSIN1 F-BAR domain expression on caveolin-1 localization, the PACSIN1 F-BAR domain had weak affinity to caveolin-1. By contrast, the PACSIN3 F-BAR domain bound well to caveolin-1 (Fig. 3D).
Because the F-BAR domain binds to the membrane, we examined the binding surface of the PACSIN2 F-BAR domain for caveolin-1 with those of the F-BAR domains bearing either the R50D or M124T/M125T mutation, on the membrane-binding concave surface, and the F-BAR domain bearing the R245E mutation, on the convex surface (Fig. 3E). The binding was not altered by the R50D or M124T/M125T mutation, but it was weakened by the R245E mutation ( Fig. 3C and supplementary material Fig. S5C). Therefore, the PACSIN2 F-BAR domain appears to bind to both caveolin-1 and the membrane simultaneously, for the induction of caveolin-1-localized tubules. Interestingly, the amino acid residues conserved between PACSIN2 and PACSIN3, but not conserved in PACSIN1, were mapped on the convex surface of PACSIN2 ( Fig. 3E and supplementary material Fig. S6).
We then addressed the effect of caveolin-1 fragment binding to the F-BAR domain in the liposome association of the PACSIN2 F-BAR domain. The caveolin-1 1-100 amino acid (aa) fragment is known to form oligomers, which precipitated upon centrifugation (supplementary material Fig. S5D) (Sargiacomo et al., 1995). Therefore, the binding of the caveolin-1 fragment to liposomes could not be examined (supplementary material Fig. S5D). However, in the presence of the PACSIN2 F-BAR domain, but in the absence of liposomes, the caveolin-1 fragment did not precipitate after centrifugation, confirming the physical interaction between the PACSIN2 F-BAR domain and the caveolin-1 fragment. The presence of the caveolin-1 1-100 aa fragment did not affect the liposome binding of the PACSIN2 F-BAR domain 2035 PACSIN2 in caveolae (supplementary material Fig. S5D). The membrane tubulation induced by the F-BAR domain was not affected by the presence of the caveolin-1 fragment (data not shown).
To confirm the caveolin-1 binding to PACSIN2 on the tubulated membrane, we expressed the caveolin-1 fragment with PACSIN2 in cells. The caveolin-1 fragment was found on the PACSIN2induced tubular structures, indicating the interaction of PACSIN2 with the caveolin-1 fragment on the tubulated membrane Pull-down assay of purified full-length PACSIN2 (1mM) with glutathione S-transferase (GST)-caveolin-1 fragments (2mM) containing aa 1-100, aa 61-100, or aa 137-179. GST fusion proteins were immobilized on the beads, and the bound PACSIN2 was visualized by western blotting. (C)Pull-down assay of purified caveolin-1 (aa 1-100) and PACSIN2 F-BAR domain. Concentration-dependent binding of full-length PACSIN2 or wild-type (WT), R50E, or R245E PACSIN2 F-BAR domain with GST-caveolin-1 (aa 1-100) (2mM) was analyzed. Bound proteins were visualized by western blotting. After washing, a 10% portion of the reaction mixture was blotted with 5% of the initial reaction mixture of 5mM PACSIN2 protein. (D)Pull-down assay of purified F-BAR domains of PACSIN1, PACSIN2 and PACSIN3 (1mM) with glutathione S-transferase (GST)-caveolin-1 fragments (1mM) containing aa 1-100, aa 61-100 or aa 137-179. GST fusion proteins were immobilized on the beads, and the bound PACSINs were visualized by Coomassie Brilliant Blue staining. (E)Structure of pacsin2 F-BAR domain Mutated amino acid residues on the structure of the F-BAR domain of PACSIN2 are colored cyan. The amino acid residues that are not conserved in PACSIN1 are colored magenta. Left, bottom view (concave side); right, side view. Because PACSIN2 F-BAR domain forms a dimer, the same amino acid residues are present symmetrically.(F) Competitive binding of the GST-SH3 domain (aa 387-486) of PACSIN2 (2mM) with the PACSIN2 F-BAR domain (1mM) in the presence or absence of 10mM caveolin-1 fragment (aa 1-100) and/or dynamin-1 PxxP peptide (200mM). GST-SH3 protein was immobilized on the beads, and bound PACSIN2 F-BAR domain protein was visualized by western blotting. (G)Folch liposome tubulation by full-length PACSIN2 (1mM), in the absence or presence of the caveolin-1 aa 1-100 fragment (10mM) and/or dynamin-1 PxxP peptide (200mM). Liposomes incubated with caveolin-1 fragment alone (15mM) and liposomes without protein incubation are also shown. (H)Percentage ± s.e.m. of liposomes with >200 nm diameters that have tubulation in G. Liposomes with smaller diameters (<200 nm) lacked tubulation. Note that the diameter of tubules induced by PACSIN2 is ~50-100 nm (Shimada et al., 2010). Therefore, the liposomes with smaller diameters (<200 nm) could not have tubulation. The significance to the liposomes incubated with PACSIN2 alone was calculated using the Student's t-test. *P<0.05.
(supplementary material Fig. S7). Consistently, the cellular tubulation induced by the expression of the PACSIN2 F-BAR domain was decreased by the knockdown of caveolin-1, suggesting that caveolin-1 enhances the membrane localization of PACSIN2 F-BAR domain in cells (supplementary material Fig. S8).

Caveolin-1 inhibits the intramolecular interaction of PACSIN2
We next addressed the autoinhibition of PACSIN2 through the intramolecular interaction between its SH3 and F-BAR domains, by pull-down assay. We found that this interaction was decreased remarkably by incubation with the caveolin-1 fragment (aa 1-100) (Fig. 3F). The SH3 domain of PACSIN2 is also known to bind to dynamin, and the binding to dynamin is also suggested to affect the intramolecular interaction. The intramolecular interaction was further decreased by incubation with the dynamin-derived PxxP peptide and the caveolin-1 fragment (Fig. 3F).
We also examined the tubulation induced by full-length PACSIN2 in vitro. We found that the tubulation was markedly strengthened in the presence of the caveolin-1 fragment (Fig.  3G,H). The tubulation occurred more efficiently in the presence of both the caveolin-1 fragment and PxxP peptide (Fig. 3G,H). The caveolin-1 fragment alone, or the PxxP peptide alone, had no effect on liposome morphology (Fig. 3G,H). Therefore, the membrane interaction might be stimulated by the release of the SH3 domain from the F-BAR domain, thus allowing membrane tubulation in the presence of caveolin-1.
2036 Journal of Cell Science 124 (12) We then examined whether this mechanism functioned in cells. We expressed dynamin-2 proline-rich peptide or the caveolin-1 fragment with PACSIN2 in cells, and the enhancement of PACSIN2-induced tubulation was observed, supporting the disruption of the intramolecular interaction by these two fragments (supplementary material Fig. S7). The overexpression of full-length caveolin-1 with PACSIN2 also enhanced PACSIN2-induced tubulation (supplementary material Fig. S7).
We also co-expressed the wild-type or dominant-negative mutant of dynamin-2 (K44A) with the wild-type PACSIN2 in HeLa cells, and quantified the tubulation formation. PACSIN2-expressing cells with the dynamin-2 K44A mutant exhibited more tubulation than the cells expressing PACSIN2 alone (supplementary material Fig.  S9). The PACSIN2-expressing cells with wild-type dynamin-2 displayed less tubulation than the cells expressing PACSIN2 alone (supplementary material Fig. S9). The co-expression of another SH3 binding protein, N-WASP, minimally affected the tubulation (supplementary material Fig. S9). These results suggested that the tubules induced by PACSIN2 could be activated by K44A dynamin-2 or dynamin-2 proline-rich peptide, but were antagonized by wild-type dynamin-2, presumably as a result of its membrane scission activity.

Knockdown of PACSIN2 alters the morphology of caveolin-1-associated membranes
The results mentioned above indicated that PACSIN2 mediates membrane tubulation upon binding to caveolin-1, thus sculpting the membranes of caveolae. Therefore, we performed RNA interference (RNAi) of PACSIN2 and analyzed the morphology of the caveolin-1-associated membranes by electron microscopy. HeLa cells treated with small interfering RNA (siRNA) to knock down PACSIN2 contained significantly less PACSIN2 (Fig. 4A). The amounts of caveola-related proteins, including dynamin-2, caveolin-1 and PTRF/cavin-1, were not affected by RNAi of PACSIN2 (Fig.  4A). We then analyzed the morphology of caveolae in cells treated with PACSIN2 siRNA. These cells were labeled with an anticaevolin-1 antibody and then with a colloidal gold-labeled secondary antibody. Portions of the plasma membrane with caveolin-1 in PACSIN2 siRNA cells showed non-flask-shaped invaginations (Fig. 4B,C). To confirm this morphological defect, the plasma membrane associated with caveolin-1 was traced (Fig.  4D,E). Because PACSIN2 was localized at the neck of caveolae (Fig. 1F), the neck width of the caveolin-associated invaginations was measured (Fig. 4F). The average diameter at the neck was 62±2 nm in control siRNA cells, whereas it was 79±3 nm in PACSIN2 siRNA cells (values are mean ± s.e.m.; P<0.05 by Student's t-test), clearly indicating the function of PACSIN2 at the caveolae neck. The depths of the caveolin-associated invaginations 2037 PACSIN2 in caveolae were also measured (Fig. 4G). The depths were 106±6 nm in control cells and 189±9 nm in PACSIN2 siRNA cells. Although the change in the depth of caveolae was not very large, for observations by light microscopy, the increase in the depth might suggest the failure of proteins, such as dynamin, to interact with caveolae.
PACSIN2 recruits dynamin-2 to caveolin-1 spots for caveolae endocytosis Finally, we addressed the possible defects in dynamin recruitment to caveolae in PACSIN2 siRNA cells. The caveolae associated with dynamin-2 are believed to execute endocytosis, and thus the quantification of dynamin-2 localization at caveolin-1 spots would be difficult. Therefore, we examined the localization of the dynamin-2 K44A mutant at caveolin-1 spots, in control and PACSIN2 siRNA cells. Many caveolin-1 spots associated with dynamin K44A were observed in the control siRNA cells, whereas very few were detected in the PACSIN2 siRNA cells (Fig. 5A,B).
To confirm the defect in dynamin-2 recruitment upon RNAi of PACSIN2, the cells were briefly treated with an inhibitor of dynamin, dynasore, to prevent the scission of caveolae into vesicles. The cells were then stained with anti-caveolin-1 and anti-dynamin- 2 antibodies. The number of caveolin-1 spots colocalized with dynamin-2 significantly decreased upon PACSIN2 RNAi (Fig.  5C-E). The defect in dynamin-2 recruitment to caveolae in PACSIN2 siRNA cells was then examined by electron microscopy. The plasma membrane sheets of these dynasore-treated cells were prepared and then stained with these antibodies. The caveolin-1 signals in control siRNA cells colocalized with the dynamin-2 signals in the plasma membrane sheets. By contrast, these signals were not colocalized in the plasma membrane sheets of the PACSIN2 siRNA cells; the dynamin signals were observed at noncaveolin-1-associated structures in these cells (Fig. 5F,G). The distribution of caveolin-1 signals on the plasma membrane was abnormal in the PACSIN2 siRNA cells, as observed by thin-section electron micrography (Fig. 4C, Fig. 5G).
We then examined whether PACSIN2 is involved in caveolamediated endocytosis. We examined cholera toxin B (CTxB) incorporation into control and PACSIN2 siRNA cells, as well as PACSIN2 siRNA cells expressing wild-type or mutants of PACSIN2. CTxB is known to bind to GM1 gangliosides, which are enriched at caveolae. The incorporated CTxB accumulated at the center of the cells, whereas the unincorporated CTxB remained at the cell periphery or at the plasma membrane. The CTxB incorporation was observed in control cells, and it was significantly decreased in cells treated with PACSIN2 siRNA (supplementary material Fig. S10). The expression of the F-BAR domain fragment of PACSIN2 induced tubular membrane invaginations with CTxB localization, supporting the idea that the F-BAR-domain-induced tubules are related to caveolae. The incorporation was restored by the expression of wild-type PACSIN2, but not by the expression of either the R245E or M124T/M125T mutant of PACSIN2 (supplementary material Fig. S10).

Discussion
These results provide several lines of evidence indicating that PACSIN2 mediates the shape formation of caveolae. First, the caveolin-1-associated plasma membrane was not flask-shaped in PACSIN2 siRNA cells (Fig. 4). Second, the presence of PACSIN2 at the neck of caveolae appeared to correspond to the membrane tubulating ability in vivo (Fig. 1). Therefore, the membranesculpting activity of the PACSIN2 F-BAR domain seems to be responsible for caveola morphology.
PACSIN2 appears to recruit dynamin-2 for caveola fission, because fewer caveolin-1 spots containing dynamin-2 were detected in PACSIN2 siRNA cells, and the PACSIN2-induced tubules were antagonized by dynamin-2 ( Fig. 5 and supplementary material Fig.  S9). Elongated tubules that contained caveolin-1 were observed in the PACSIN2 siRNA cells (Fig. 4). Because PACSIN2 was knocked down, the elongated tubules were probably formed by proteins other than PACSIN2 in the absence of a sufficient amount of dynamin. Caveolin-1 reportedly binds directly to dynamin (Yao et al., 2005). Thus, an insufficient amount of dynamin-2 might induce deeper invaginations without membrane scission.
Cavins are believed to facilitate the membrane shape formation of caveolae, by suppressing the formation of caveolin-1-localized long tubules (Hansen et al., 2009;Hill et al., 2008;Verma et al., 2010). The limited participation of PTRF in the tubulation induced by the PACSIN2 F-BAR domain appears to be consistent with this inhibition of tubulation by PTRF (supplementary material Fig. S4E).
PACSIN2 was first identified as a casein kinase and protein kinase C (PKC) substrate (Plomann et al., 1998), and PKC is enriched at caveolae (Smart et al., 1995). This might also provide a key to regulate the caveola invagination mediated by PACSIN2. The detailed time course of the recruitment of these proteins should be examined in the future, to clarify the regulation of caveola formation.
Our present results reveal that the PACSIN2 F-BAR domain protein induces membrane tubulation for caveola sculpting and fission. In addition, caveolin-1 was found to regulate the sculpting ability of PACSIN2. Caveolae constitute platforms for various receptors, channels, and signal transduction. PACSIN2 therefore appears to be a fundamental protein that is involved in cellular homeostasis and disease (e.g. in generation of tumors), through the regulation of caveolae. Interestingly, the PACSIN3 F-BAR domain was associated with caveolin, whereas the PACSIN1 F-BAR domain was not. The different affinities of the PACSIN/Syndapin paralogs to caveolin-1 might reflect the tissue-specific variations of caveolae function.

Liposome preparation, co-sedimentation assay and tubulation assay
Liposomes were prepared from total bovine brain lipids (Folch fraction 1; Avanti Polar Lipids) (Michelsen et al., 1995). Lipids were dried under nitrogen gas in glass test tubes and were resuspended in XB (10 mM HEPES, pH 7.9, 100 mM KCl, 2 mM MgCl 2 , 0.2 mM CaCl 2 and 5 mM ethylene glycol tetraacetic acid/EGTA), containing 100 mM sucrose, by mixing with a vortexer, and then hydrated at 37°C for 1 hour. This preparation yielded a mixture of unilamellar liposomes with various diameters (0.1-2 mm) and large, multilamellar vesicles, as determined by thinsection electron microscopy. The majority of the liposomes were unilamellar. Liposome tubulation was examined by negative staining, as described previously (Shimada et al., 2010). Proteins without GST were used in tubulation assays. The co-sedimentation assay was performed as follows. The proteins were incubated with PACSIN2 in caveolae liposomes (1 mg/ml) in 50 ml XB for 20 minutes at room temperature (RT), and were then centrifuged at 78,000 g for 30 minutes at 25°C in a TLA-100 rotor (Beckman). Supernatants and pellets were subjected to SDS-PAGE.

Immunoprecipitation
Subconfluent HeLa cells in a 15 cm dish were washed with PBS, and suspended in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 10% glycerol, 2% Triton X-100, 5 mM NaF, 5 mM EDTA, 1 mg/ml aprotinin and 1 mg/ml leupeptin. The resulting cell lysate was sonicated and centrifuged at 15,000 r.p.m. at 4°C. The supernatant was incubated with 1 mg of anti-caveolin-1 (7C8) antibody or control IgG. After incubation for 1 hour at 4°C, Dynabeads protein A (Invitrogen) were added and further incubated for 1 hour at 4°C. The beads were then washed and subjected to western blot analysis.

Sulforhodamine or FM-dye labeling of HeLa cells
HeLa cells were seeded on glass-bottom dishes (Asahi Glass, 3911-035), transfected with the vector expressing the GFP-tagged F-BAR domain of PACSIN2, and cultured overnight. Sulforhodamine (Invitrogen, S359) was then added to the medium at a 0.1 mM final concentration. After 10 minutes, cells were visualized by confocal microscopy (FV1000D, Olympus) at 25°C. FM-dye labeling was performed basically as described previously (Terebiznik et al., 2002). HeLa cells were seeded on glassbottom dishes, transfected, and cultured overnight. The cells were then chilled on ice for 15 minutes, washed with HBSS supplemented with 10 mM glucose, and incubated with 10 mM FM4-64 on ice for 1 minute. After the medium was removed, the cells were washed twice with ice-cold HBSS supplemented with 10 mM glucose for 5 minutes, and then the cells were observed by confocal microscopy at 25°C.

Immunocytochemistry
The cells were fixed in 3.7% formaldehyde in PBS for 5 minutes, followed by permeabilization with TBS supplemented with 1% BSA and 0.1% Triton X-100 for 5 minutes and blocking with TBS supplemented with 1% BSA for 2 hours. The anti-PACSIN2 antibody was affinity purified from the serum of rabbits immunized with the F-BAR domain protein of PACSIN2. The rabbit monoclonal anti-caveolin-1 antibody was purchased from Cell Signaling Technology. The mouse monoclonal anti-Myc (clone PL14) and the anti-caveolin-1 (7C8) antibodies were purchased from MBL International, and Santa Cruz Biotechnology, respectively. The anticlathrin heavy chain (clone 23) and anti-dynamin-2 antibodies were obtained from BD Transduction Laboratories. After washing, cells were stained with Alexa Fluor 488 and/or Alexa Fluor 568 labeled secondary antibodies and Alexa Fluor 633 phalloidin. Fluorescence images were obtained by confocal microscopy (Olympus Fluoview 1000D) at room temperature. A 100ϫ oil-immersion objective (NA 1.45; Olympus) was used.
The spots of certain protein staining that were recognized as particles of higher signals than the surrounding areas in each image were counted. Subsequently, if the spots contained other protein staining signals that were higher than the area surrounding the spots, then the spots were considered to be colocalization spots of the two proteins. This analysis was performed manually, with the aid of the Cell Counter Plug-in and Channel Tools of the ImageJ program (NIH).

Cholera toxin incorporation assay
HeLa cells were labeled with Alexa-Fluor-555-labeled Cholera toxin B (CTxB) (Invitrogen) on ice, as described previously (del Pozo et al., 2005). Briefly, cells were chilled on ice for 15 minutes, labeled with 1 mg/ml CTxB on ice for 15 minutes, washed twice with ice-cold PBS, and incubated with DMEM + 10% FCS for 60 minutes. The cells were then fixed with 4% paraformaldehyde in PBS. The localization of CTxB close to the coverslip, including the basal plasma membrane, was then visualized by confocal microscopy. The cells with CTxB at the cell periphery without significant CTxB accumulation at the cell center were considered as cells without CTxB incorporation.

Electron microscopy
Cells on plastic coverslips (Thermanox, Nalge Nunc International) were fixed in 2% paraformaldehyde and 2% glutaraldehyde for 5 minutes at room temperature. The cells were permeabilized with 0.1% saponin and 1% bovine serum albumin (BSA) in Tris-buffered saline for 20 minutes. The cells were blocked overnight with 1% BSA in Tris-buffered saline and then incubated with the anti-PACSIN2 antibody and a Nanogold rabbit Fab fragment (Nanoprobes). The stained cells were postfixed with 1% glutaraldehyde in phosphate-buffered saline and blocked, and then the gold signal was enhanced by Goldenhance (Nanoprobes). After the gold-enhancement reaction, the cells were washed, stained with 1% osmium tetroxide and 1% potassium ferrocyanide, washed, dehydrated in 50%, 60%, 70%, 80%, 90%, 95.5% and 100% ethanol, placed in propylene oxide, and embedded in the epoxy resin Quetol-812, according to the manufacturer's instructions (Nissin EM). After polymerization of the resin, 80 nm sections were prepared and counterstained for 10 minutes in uranyl acetate and for 2 minutes in lead citrate. Dried sections were examined by transmission electron microscopy.
Plasma membrane sheets were prepared and observed as described previously (Vinten et al., 2001), and were stained with an anti-PACSIN2 rabbit polyclonal antibody and an anti-caveolin-1 (7C8) mouse monoclonal antibody, or an anticaveolin-1 rabbit monoclonal antibody and anti-dynamin-2 mouse monoclonal antibody, followed by 5-nm-or 10-nm-gold-conjugated antibodies.

Statistical analysis
All statistical analyses were performed using Microsoft Excel. Significance was assessed by the Student's t-test. All of the images are representative of at least three independent experiments.