Research Article

IntroductionBin-amphiphysin-Rvs167 (BAR) domain-containing superfamilymembers, including extended Fes-CIP4 homology (EFC)/FCH-BAR (F-BAR) domain-containing subfamily members, such asFCHo2, FBP17, Toca-1, CIP4, PACSINs/syndapins and others,sculpt membranes according to the curvature of their three-dimensional structures (Henne et al., 2010; Peter et al., 2004;Shimada et al., 2007; Shimada et al., 2010). The positively chargedsurface of the domain binds to the negatively charged inner surfaceof the plasma membrane, thereby bending the membrane accordingto its protein structures. The various BAR domain surfacecurvatures are thought to correlate with the diverse invaginationsand protrusions of cells. The BAR domain proteins engaged in theformation of clathrin-coated pits, filopodia and spines have beenwell characterized, in contrast to those participating in formingother subcellular structures (Doherty and McMahon, 2009; Itohand De Camilli, 2006; Suetsugu et al., 2010; Takenawa andSuetsugu, 2007).

PACSINs (protein kinase C and casein kinase substrate inneurons proteins) and syndapins, which include three paralogs(Modregger et al., 2000), consist of an F-BAR domain and an SH3domain. The SH3 domain binds to dynamin and the actin-nucleatingprotein N-WASP (Itoh et al., 2005; Qualmann and Kelly, 2000;Simpson et al., 1999; Tsujita et al., 2006). PACSIN1/syndapin-I isimplicated in synaptic vesicle recycling in the brain (Qualmann etal., 1999), and PACSIN3 expression is specific to muscles(Modregger et al., 2000). Although PACSIN2/syndapin-II isexpressed ubiquitously (Modregger et al., 2000), its functionsremain to be elucidated.

The membrane tubulation activity of the F-BAR domain isautoinhibited in full-length PACSIN1 (Wang et al., 2009). We

found that this autoinhibition is also conserved in PACSIN2, sincefull-length PACSIN2 exhibited weaker membrane tubulation abilitythan the F-BAR domain fragment (Shimada et al., 2010). Thecrystal structure of full-length PACSIN1 suggested that theautoinhibition is mediated by an interaction between the SH3 andF-BAR domains (Rao et al., 2010).

The F-BAR domain of PACSIN2 possesses a deeper concavesurface than those of FBP17 and CIP4 (Shimada et al., 2010; Wanget al., 2009). Correspondingly, the membrane tubules induced bythe PACSIN2 F-BAR domain have smaller diameters (~50 nm)than those induced by the F-BAR domains of FCHo2, FBP17,CIP4 and Toca-1 (Shimada et al., 2010; Wang et al., 2009).Caveolae are flask-shaped invaginations with diameters ofapproximately 50 nm to 100 nm (Mundy et al., 2002; Palade andBruns, 1968; Parton and Simons, 2007; Rothberg et al., 1992;Yamada, 1955). This diameter size might correspond to that of thetubules induced by the PACSIN2 F-BAR domain.

Dynamin is also localized at the neck of caveolae (Henley et al.,1998; Oh et al., 1998), and is involved in the fission of caveolaeduring their endocytosis. The recently identified cavin familyproteins, including polymerase I and transcript release factor(PTRF)/cavin-1, reportedly promote caveola biogenesis (Hill etal., 2008). Caveolin-1 is suggested to induce the formation ofmembrane tubules in the absence of PTRF (Verma et al., 2010).

In the present study, we analyzed the function of PACSIN2-induced tubulation in caveolae. We found that the cellular tubulesinduced by the F-BAR domain of PACSIN2 contained caveolin-1,but less PTRF. Surprisingly, caveolin-1 interacted directly with theF-BAR domain of PACSIN2 to release its autoinhibition, thusallowing membrane tubulation. Abnormally-shaped caveolin-1-containing plasma membrane invaginations were also observed in

SummaryCaveolae are flask-shaped invaginations of the plasma membrane that are associated with tumor formation, pathogen entry andmuscular dystrophy, through the regulation of lipids, signal transduction and endocytosis. Caveolae are generated by the fusion ofcaveolin-1-containing vesicles with the plasma membrane, which then participate in endocytosis via dynamin. Proteins containingmembrane-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 domaincontained 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 membranesculpting by caveolin-1 in caveola morphology and recruits dynamin-2 for caveola fission.

Key words: EFC domain, F-BAR domain, Caveolae, Membrane sculpting

Accepted 23 February 2011Journal of Cell Science 124, 2032-2040 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.086264

Essential role of PACSIN2/syndapin-II in caveolaemembrane sculptingYosuke Senju1, Yuzuru Itoh1, Kazunori Takano1,*, Sayaka Hamada1 and Shiro Suetsugu1,2,‡

1Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,Tokyo 113-0032, Japan2PRESTO, Japan Science and Technology Agency, Kawaguchi-shi, Saitama 332-0012, Japan*Present address: Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan‡Author for correspondence (suetsugu@iam.u-tokyo.ac.jp)

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PACSIN2-knockdown cells. The cellular tubulation observed in cellsexpressing the F-BAR domain or with PACSIN2 knockdown mightbe caused by the lack of recruitment of dynamin-2 to caveolae.

ResultsEndogenous PACSIN2 colocalizes with caveolin-1 atcaveolaeWe examined the localization of endogenous PACSIN2, by labelingHeLa cells with anti-PACSIN2 and anti-caveolin-1 antibodies (Fig.1A,B). A proportion of the endogenous PACSIN2 or caveolin-1puncta (44±13% or 48±14%, respectively) also exhibited caveolin-1 or PACSIN2 staining. Importantly, exogenous green fluorescentprotein (GFP)–PACSIN2 and caveolin-1–DsRed exhibited 90±5%colocalization, when these two proteins were coexpressed in HeLacells (Fig. 1C). Similar colocalization of endogenous PACSIN2and endogenous caveolin-1 was observed in other cells, such as

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Hs578T, SK-BR-3 and BT549 cells (supplementary material Fig.S1).

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 waslabeled with anti-caveolin-1 and anti-PACSIN2 antibodies, followedby immunogold. The PACSIN2 label was found in close proximityto the caveolin-1 label (Fig. 1D,E). The PACSIN2 label was alsoobserved at structures lacking the caveolin-1 label, which weresmaller than caveolae (Fig. 1E). Thin-section electron microscopyrevealed anti-PACSIN2 antibody staining in the necks of the flask-like invaginations typical of caveolae (Fig. 1F). Staining signalswere also detected on the relatively flat plasma membrane and inthe cytosol (Fig. 1F). No signals were identified in the absence ofantibody (supplementary material Fig. S2). These data suggest thatPACSIN2 functions in caveolae at the plasma membrane.

Fig. 1. Localization of endogenous PACSIN2 at caveolae.(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 isshown. The average percentages of PACSIN2 or caveolin-1 punctawith 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) inHeLa cells. The percentage of GFP puncta with DsRed in ten cellsis shown (±s.d.). (D,E)A plasma membrane sheet from non-transfected HeLa cells was stained with an anti-caveolin-1 antibodywith a 10-nm-gold-conjugated antibody (arrows in enlarged imagesin E) and an anti-PACSIN2 antibody with a 5-nm-gold-conjugatedantibody (arrowheads in E). (F)Thin-section electron micrographof non-transfected HeLa cells stained with anti-PACSIN2 antibody.Control images without anti-PACSIN2 antibody are shown insupplementary material Fig. S2.

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Overexpression of the PACSIN2 F-BAR domain alterslocalization of caveolin-1The canonical F-BAR-domain-containing proteins, such as FBP17and Toca-1, induce plasma membrane invagination whenoverexpressed in cells. These invaginations are often observed asthe mesh-like localization of the overexpressed F-BAR domainproteins. The overexpressed F-BAR domain fragment of PACSIN2exhibited a similar mesh-like localization in HeLa cells (Fig. 2).We confirmed that this mesh-like structure corresponded to theplasma membrane invaginations, by labeling the cells withmembrane-staining FM dye and the culture medium withsulforhodamine (supplementary material Fig. S3).

We then examined the localization of various membrane markerproteins to the PACSIN2 F-BAR-induced mesh-like structures,and found that caveolin-1 colocalized with PACSIN2. In cells withlow expression of the PACSIN2 F-BAR domain (Fig. 2A, left cellin large panel), the F-BAR domain and caveolin-1 colocalizedalong the tubules induced by F-BAR domain expression. However,caveolin-1 accumulated into several larger assemblies, whichformed at the convergence of the PACSIN2-induced invaginationsin cells with high F-BAR-domain expression (Fig. 2A, right cell inlarge panel). This caveolin-1 accumulation was not observed incells expressing PACSIN2 F-BAR domains with tubulation-

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defective mutations (M124T/M125T) (Shimada et al., 2010) or inthose expressing FBP17 (supplementary material Fig. S4A,B).Importantly, the clathrin distribution was not altered by expressionof the PACSIN2 F-BAR domain (supplementary material Fig.S4C). PTRF was only partially localized at tubules induced by thePACSIN2 F-BAR domain (supplementary material Fig. S4D).

Interestingly, the overexpressed PACSIN1 F-BAR domain hada weak effect on caveolin-1 localization, whereas the overexpressedPACSIN3 F-BAR domain had a similar effect on caveolin-1localization as the PACSIN2 F-BAR domain (Fig. 2B,C).

Binding of caveolin-1 to PACSIN2 induces membranetubulationThe substantial colocalization of PACSIN2 with caveolin-1 upontheir coexpression suggested a direct interaction between these twoproteins. PACSIN2 consists of the F-BAR domain and the SH3domain (Fig. 3A). Caveolin-1 consists of two cytoplasmic regionsconnected by a region embedded in the membrane (Fig. 3A). Weprepared several purified fragments of caveolin-1, and analyzedtheir binding to purified full-length PACSIN2 in a pull-down assay.The N-terminal cytoplasmic region of caveolin-1 (residues 1–100or 61–100) interacted with PACSIN2 (Fig. 3B). However, theassociation was not observed for the C-terminal cytoplasmic regionof caveolin-1 (Fig. 3B). We then mapped the binding region ofPACSIN2 to caveolin-1. Similarly to the finding that the PACSIN2F-BAR domain alone was sufficient for colocalization withcaveolin-1 (Fig. 2), the F-BAR domain alone was sufficient forbinding to caveolin-1 (Fig. 3C). The binding of caveolin-1 to theF-BAR domain was stronger than that to full-length PACSIN2(Fig. 3C). The PACSIN2 C-terminal region, including the SH3domain (residues 301–486), did not interact with caveolin-1 in apull-down assay with purified proteins (supplementary materialFig. S5A). The association of PACSIN2 and caveolin-1 in cellswas confirmed by immunoprecipitation analysis with an anti-caveolin-1 antibody (supplementary material Fig. S5B).

We then examined the binding of the PACSIN1 and PACSIN3F-BAR domains to caveolin-1. Consistent with the weak effect ofthe PACSIN1 F-BAR domain expression on caveolin-1 localization,the PACSIN1 F-BAR domain had weak affinity to caveolin-1. Bycontrast, the PACSIN3 F-BAR domain bound well to caveolin-1(Fig. 3D).

Because the F-BAR domain binds to the membrane, weexamined the binding surface of the PACSIN2 F-BAR domain forcaveolin-1 with those of the F-BAR domains bearing either theR50D or M124T/M125T mutation, on the membrane-bindingconcave surface, and the F-BAR domain bearing the R245Emutation, on the convex surface (Fig. 3E). The binding was notaltered by the R50D or M124T/M125T mutation, but it wasweakened by the R245E mutation (Fig. 3C and supplementarymaterial Fig. S5C). Therefore, the PACSIN2 F-BAR domainappears to bind to both caveolin-1 and the membranesimultaneously, for the induction of caveolin-1-localized tubules.Interestingly, the amino acid residues conserved between PACSIN2and PACSIN3, but not conserved in PACSIN1, were mapped onthe convex surface of PACSIN2 (Fig. 3E and supplementarymaterial Fig. S6).

We then addressed the effect of caveolin-1 fragment binding tothe F-BAR domain in the liposome association of the PACSIN2 F-BAR domain. The caveolin-1 1–100 amino acid (aa) fragment isknown to form oligomers, which precipitated upon centrifugation(supplementary material Fig. S5D) (Sargiacomo et al., 1995).

Fig. 2. Overexpression of PACSIN2 F-BAR domain induces abnormalcaveolin-1 localization. (A)GFP-PACSIN2 F-BAR domain (green) wasoverexpressed in HeLa cells. Endogenous caveolin-1 was visualized byindirect immunofluorescence (red). Actin filaments were visualized byphalloidin staining (blue). The merged image is on the left, and the individualimages are on the right. Scale bar: 50mm. (B,C)GFP–PACSIN1 (B) or GFP–PACSIN3 (C) F-BAR domain was overexpressed and analyzed as in A.

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Therefore, the binding of the caveolin-1 fragment to liposomescould not be examined (supplementary material Fig. S5D).However, in the presence of the PACSIN2 F-BAR domain, but inthe absence of liposomes, the caveolin-1 fragment did notprecipitate after centrifugation, confirming the physical interactionbetween the PACSIN2 F-BAR domain and the caveolin-1 fragment.The presence of the caveolin-1 1–100 aa fragment did not affectthe liposome binding of the PACSIN2 F-BAR domain

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(supplementary material Fig. S5D). The membrane tubulationinduced by the F-BAR domain was not affected by the presence ofthe caveolin-1 fragment (data not shown).

To confirm the caveolin-1 binding to PACSIN2 on the tubulatedmembrane, we expressed the caveolin-1 fragment with PACSIN2in cells. The caveolin-1 fragment was found on the PACSIN2-induced tubular structures, indicating the interaction of PACSIN2with the caveolin-1 fragment on the tubulated membrane

Fig. 3. Binding of PACSIN2 F-BAR domain to caveolin-1 and membrane tubulation. (A)Domain structures of PACSIN2 and caveolin-1. NPF, the Asn-Pro-Phe (NPF) motif. (B)Pull-down assay of purified full-length PACSIN2 (1mM) with glutathione S-transferase (GST)-caveolin-1 fragments (2mM) 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-downassay of purified caveolin-1 (aa 1–100) and PACSIN2 F-BAR domain. Concentration-dependent binding of full-length PACSIN2 or wild-type (WT), R50E, orR245E PACSIN2 F-BAR domain with GST–caveolin-1 (aa 1–100) (2mM) was analyzed. Bound proteins were visualized by western blotting. After washing, a10% portion of the reaction mixture was blotted with 5% of the initial reaction mixture of 5mM PACSIN2 protein. (D)Pull-down assay of purified F-BAR domainsof PACSIN1, PACSIN2 and PACSIN3 (1mM) with glutathione S-transferase (GST)-caveolin-1 fragments (1mM) 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 conservedin PACSIN1 are colored magenta. Left, bottom view (concave side); right, side view. Because PACSIN2 F-BAR domain forms a dimer, the same amino acidresidues are present symmetrically.(F) Competitive binding of the GST–SH3 domain (aa 387–486) of PACSIN2 (2mM) with the PACSIN2 F-BAR domain (1mM)in the presence or absence of 10mM caveolin-1 fragment (aa 1–100) and/or dynamin-1 PxxP peptide (200mM). 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 (1mM), in the absence orpresence of the caveolin-1 aa 1–100 fragment (10mM) and/or dynamin-1 PxxP peptide (200mM). Liposomes incubated with caveolin-1 fragment alone (15mM)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 withsmaller diameters (<200 nm) lacked tubulation. Note that the diameter of tubules induced by PACSIN2 is ~50–100 nm (Shimada et al., 2010). Therefore, theliposomes with smaller diameters (<200 nm) could not have tubulation. The significance to the liposomes incubated with PACSIN2 alone was calculated using theStudent’s t-test. *P<0.05.

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(supplementary material Fig. S7). Consistently, the cellulartubulation induced by the expression of the PACSIN2 F-BARdomain was decreased by the knockdown of caveolin-1, suggestingthat caveolin-1 enhances the membrane localization of PACSIN2F-BAR domain in cells (supplementary material Fig. S8).

Caveolin-1 inhibits the intramolecular interaction ofPACSIN2We next addressed the autoinhibition of PACSIN2 through theintramolecular interaction between its SH3 and F-BAR domains,by pull-down assay. We found that this interaction was decreasedremarkably by incubation with the caveolin-1 fragment (aa 1–100)(Fig. 3F). The SH3 domain of PACSIN2 is also known to bind todynamin, and the binding to dynamin is also suggested to affectthe intramolecular interaction. The intramolecular interaction wasfurther decreased by incubation with the dynamin-derived PxxPpeptide and the caveolin-1 fragment (Fig. 3F).

We also examined the tubulation induced by full-lengthPACSIN2 in vitro. We found that the tubulation was markedlystrengthened in the presence of the caveolin-1 fragment (Fig.3G,H). The tubulation occurred more efficiently in the presence ofboth the caveolin-1 fragment and PxxP peptide (Fig. 3G,H). Thecaveolin-1 fragment alone, or the PxxP peptide alone, had noeffect on liposome morphology (Fig. 3G,H). Therefore, themembrane interaction might be stimulated by the release of theSH3 domain from the F-BAR domain, thus allowing membranetubulation in the presence of caveolin-1.

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We then examined whether this mechanism functioned in cells.We expressed dynamin-2 proline-rich peptide or the caveolin-1fragment with PACSIN2 in cells, and the enhancement ofPACSIN2-induced tubulation was observed, supporting thedisruption of the intramolecular interaction by these two fragments(supplementary material Fig. S7). The overexpression of full-lengthcaveolin-1 with PACSIN2 also enhanced PACSIN2-inducedtubulation (supplementary material Fig. S7).

We also co-expressed the wild-type or dominant-negative mutantof dynamin-2 (K44A) with the wild-type PACSIN2 in HeLa cells,and quantified the tubulation formation. PACSIN2-expressing cellswith the dynamin-2 K44A mutant exhibited more tubulation thanthe cells expressing PACSIN2 alone (supplementary material Fig.S9). The PACSIN2-expressing cells with wild-type dynamin-2displayed less tubulation than the cells expressing PACSIN2 alone(supplementary material Fig. S9). The co-expression of anotherSH3 binding protein, N-WASP, minimally affected the tubulation(supplementary material Fig. S9). These results suggested that thetubules induced by PACSIN2 could be activated by K44A dynamin-2 or dynamin-2 proline-rich peptide, but were antagonized bywild-type dynamin-2, presumably as a result of its membranescission activity.

Knockdown of PACSIN2 alters the morphology ofcaveolin-1-associated membranesThe results mentioned above indicated that PACSIN2 mediatesmembrane tubulation upon binding to caveolin-1, thus sculpting

Fig. 4. Electron microscopy of caveolae in cells treated with PACSIN2 siRNA. (A)Lysates from HeLa cells treated with control or PACSIN2 siRNA weresubjected to western blotting with the indicated antibodies. Approximate molecular masses (kDa) are shown on the right. (B,C)Thin-section electron micrograph of(B) control or (C) PACSIN2 siRNA cells labeled with an anti-caveolin-1 antibody, followed by a colloidal gold-conjugated secondary antibody.(D,E)Representative traces of plasma membranes with anti-caveolin-1 staining from (D) control and (E) PACSIN2 siRNA cells. (F,G)Distributions of (F) neckwidth and (G) depth of plasma membrane invaginations with caveolin-1 signals, quantified from (D,E). The numbers of signals analyzed for (F) and (G) wereapproximately 100 for each experiment. The neck width is 62±2 nm (control cells) or 79±3 nm (PACSIN2 RNAi cells) and the p-value was calculated by theStudent’s t-test. P<0.05. The depth is 106±6 nm (control cells) or 189±9 nm (PACSIN2 siRNA cells) and the P-value was calculated by the Student’s t-test.P<0.05. The data shown are the mean values from five (control) or three (PACSIN2 siRNA) experiments with s.e.m.

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the membranes of caveolae. Therefore, we performed RNAinterference (RNAi) of PACSIN2 and analyzed the morphology ofthe caveolin-1-associated membranes by electron microscopy. HeLacells treated with small interfering RNA (siRNA) to knock downPACSIN2 contained significantly less PACSIN2 (Fig. 4A). Theamounts 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 treatedwith PACSIN2 siRNA. These cells were labeled with an anti-caevolin-1 antibody and then with a colloidal gold-labeledsecondary antibody. Portions of the plasma membrane withcaveolin-1 in PACSIN2 siRNA cells showed non-flask-shapedinvaginations (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 invaginationswas measured (Fig. 4F). The average diameter at the neck was62±2 nm in control siRNA cells, whereas it was 79±3 nm inPACSIN2 siRNA cells (values are mean ± s.e.m.; P<0.05 byStudent’s t-test), clearly indicating the function of PACSIN2 at thecaveolae neck. The depths of the caveolin-associated invaginations

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were also measured (Fig. 4G). The depths were 106±6 nm incontrol cells and 189±9 nm in PACSIN2 siRNA cells. Although thechange in the depth of caveolae was not very large, for observationsby light microscopy, the increase in the depth might suggest thefailure of proteins, such as dynamin, to interact with caveolae.

PACSIN2 recruits dynamin-2 to caveolin-1 spots forcaveolae endocytosisFinally, we addressed the possible defects in dynamin recruitmentto caveolae in PACSIN2 siRNA cells. The caveolae associated withdynamin-2 are believed to execute endocytosis, and thus thequantification of dynamin-2 localization at caveolin-1 spots wouldbe difficult. Therefore, we examined the localization of thedynamin-2 K44A mutant at caveolin-1 spots, in control andPACSIN2 siRNA cells. Many caveolin-1 spots associated withdynamin K44A were observed in the control siRNA cells, whereasvery few were detected in the PACSIN2 siRNA cells (Fig. 5A,B).

To confirm the defect in dynamin-2 recruitment upon RNAi ofPACSIN2, the cells were briefly treated with an inhibitor ofdynamin, dynasore, to prevent the scission of caveolae into vesicles.The cells were then stained with anti-caveolin-1 and anti-dynamin-

Fig. 5. Role of PACSIN2 in recruitment of dynamin-2 tocaveolae. (A)Localization of the K44A mutant of dynamin-2 with endogenous caveolin-1 in cells treated with controland PACSIN2 siRNA. (B)Percentages of K44A dynamin-2puncta associated with endogenous caveolin-1 in control andPACSIN2 siRNA cells. Values are the means (±s.e.m.) fromten cells. Statistical significance was calculated using theStudent’s t-test. *P<0.05. (C,D)Localization of endogenousdynamin-2 with endogenous caveolin-1 in cells treated withcontrol and PACSIN2 siRNA after treatment with dynasore(40mM) for 5 minutes. Rectangles indicate the area ofenlargement in D. (E)Percentages of caveolin-1 punctaassociated with endogenous dynamin-2 in control and cellstreated with PACSIN2 siRNA. Values are the means (±s.e.m.) from ten cells. Statistical significance wascalculated using the Student’s t-test. *P<0.05. (F,G)Aplasma membrane sheet from control (F) or PACSIN2 siRNA(G) HeLa cells was stained with an anti-dynamin-2 antibodywith a 10-nm-gold-conjugated antibody and an anti-caveolin-1 antibody with a 5-nm-gold-conjugated antibody.Two representative images are shown.

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2 antibodies. The number of caveolin-1 spots colocalized withdynamin-2 significantly decreased upon PACSIN2 RNAi (Fig.5C–E). The defect in dynamin-2 recruitment to caveolae inPACSIN2 siRNA cells was then examined by electron microscopy.The plasma membrane sheets of these dynasore-treated cells wereprepared and then stained with these antibodies. The caveolin-1signals in control siRNA cells colocalized with the dynamin-2signals in the plasma membrane sheets. By contrast, these signalswere not colocalized in the plasma membrane sheets of thePACSIN2 siRNA cells; the dynamin signals were observed at non-caveolin-1-associated structures in these cells (Fig. 5F,G). Thedistribution of caveolin-1 signals on the plasma membrane wasabnormal in the PACSIN2 siRNA cells, as observed by thin-sectionelectron micrography (Fig. 4C, Fig. 5G).

We then examined whether PACSIN2 is involved in caveola-mediated endocytosis. We examined cholera toxin B (CTxB)incorporation into control and PACSIN2 siRNA cells, as well asPACSIN2 siRNA cells expressing wild-type or mutants ofPACSIN2. CTxB is known to bind to GM1 gangliosides, whichare enriched at caveolae. The incorporated CTxB accumulated atthe center of the cells, whereas the unincorporated CTxB remainedat the cell periphery or at the plasma membrane. The CTxBincorporation was observed in control cells, and it was significantlydecreased in cells treated with PACSIN2 siRNA (supplementarymaterial Fig. S10). The expression of the F-BAR domain fragmentof PACSIN2 induced tubular membrane invaginations with CTxBlocalization, supporting the idea that the F-BAR-domain-inducedtubules are related to caveolae. The incorporation was restored bythe expression of wild-type PACSIN2, but not by the expressionof either the R245E or M124T/M125T mutant of PACSIN2(supplementary material Fig. S10).

DiscussionThese results provide several lines of evidence indicating thatPACSIN2 mediates the shape formation of caveolae. First, thecaveolin-1-associated plasma membrane was not flask-shaped inPACSIN2 siRNA cells (Fig. 4). Second, the presence of PACSIN2at the neck of caveolae appeared to correspond to the membranetubulating ability in vivo (Fig. 1). Therefore, the membrane-sculpting activity of the PACSIN2 F-BAR domain seems to beresponsible for caveola morphology.

PACSIN2 appears to recruit dynamin-2 for caveola fission,because fewer caveolin-1 spots containing dynamin-2 were detectedin PACSIN2 siRNA cells, and the PACSIN2-induced tubules wereantagonized by dynamin-2 (Fig. 5 and supplementary material Fig.S9). Elongated tubules that contained caveolin-1 were observed inthe PACSIN2 siRNA cells (Fig. 4). Because PACSIN2 was knockeddown, the elongated tubules were probably formed by proteinsother than PACSIN2 in the absence of a sufficient amount ofdynamin. Caveolin-1 reportedly binds directly to dynamin (Yao etal., 2005). Thus, an insufficient amount of dynamin-2 might inducedeeper invaginations without membrane scission.

The PACSIN2 F-BAR domain binds to negatively chargedphosphatidylserine at the plasma membrane. Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] is also negatively charged, andfacilitates the membrane binding of the PACSIN2 F-BAR domain(Dharmalingam et al., 2009). Interestingly, PtdIns(4,5)P2 is enrichedin caveolae (Fujita et al., 2009). In addition, the Eps15-homology-domain containing (EHD) protein, which also possesses membrane-deformation ability, reportedly binds to the Asn-Pro-Phe (NPF)motif of PACSIN2 (Braun et al., 2005; Xu et al., 2004). The EHD

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proteins are highly conserved eukaryotic ATPases that areimplicated in caveolae formation, clathrin-independent endocytosisand recycling from endosomes, and they have membrane-tubulationability (Daumke et al., 2007; Verma et al., 2010). An EHD proteinbound to caveolae might cooperate with PACSIN2 in caveolabiogenesis or caveola-mediated endocytosis. In addition, EHDproteins are present in PTRF/cavin-1-rich fractions (Aboulaich etal., 2004). Thus, the EHD proteins might function as bridgesbetween cavins and PACSIN2.

Cavins are believed to facilitate the membrane shape formationof caveolae, by suppressing the formation of caveolin-1-localizedlong tubules (Hansen et al., 2009; Hill et al., 2008; McMahon etal., 2009; Verma et al., 2010). The limited participation of PTRFin the tubulation induced by the PACSIN2 F-BAR domain appearsto be consistent with this inhibition of tubulation by PTRF(supplementary material Fig. S4E).

PACSIN2 was first identified as a casein kinase and proteinkinase C (PKC) substrate (Plomann et al., 1998), and PKC isenriched at caveolae (Smart et al., 1995). This might also providea key to regulate the caveola invagination mediated by PACSIN2.The detailed time course of the recruitment of these proteins shouldbe examined in the future, to clarify the regulation of caveolaformation.

Our present results reveal that the PACSIN2 F-BAR domainprotein induces membrane tubulation for caveola sculpting andfission. In addition, caveolin-1 was found to regulate the sculptingability of PACSIN2. Caveolae constitute platforms for variousreceptors, channels, and signal transduction. PACSIN2 thereforeappears to be a fundamental protein that is involved in cellularhomeostasis and disease (e.g. in generation of tumors), through theregulation of caveolae. Interestingly, the PACSIN3 F-BAR domainwas associated with caveolin, whereas the PACSIN1 F-BARdomain was not. The different affinities of the PACSIN/Syndapinparalogs to caveolin-1 might reflect the tissue-specific variationsof caveolae function.

Materials and MethodsRecombinant proteins and pull-down assaysGlutathione S-transferase (GST)–PACSIN2 (mouse), GST–PACSIN2 F-BAR domain(EFCL; aa 1–339 and EFCS aa 1–306), GST–SH3 domain (aa 301–486 or aa 387–486), GST–PACSIN1 F-BAR domain (mouse) (aa 1–306), GST–PACSIN3 F-BARdomain (mouse) (aa 1–306) and GST–caveolin-1 fragments (mouse) (amino acids1–100, 61–100, and 137–179) were expressed in Escherichia coli, as describedpreviously (Shimada et al., 2010). GST was removed by PreScission Protease (GEHealthcare). Dynamin-1 proline-rich peptide, RSPTSSPTPQRRAPAVPPARPG, wasfrom Bex (Tokyo, Japan). Pull-down assays with the indicated fragments wereperformed in buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mMphenylmethylsulfonyl fluoride (PMSF), 10% glycerol, and 5 mMethylenediaminetetraacetic acid (EDTA)] containing 2% Triton X-100 and theindicated protein concentrations. The purified proteins and glutathione Sepharosebeads were mixed in the buffer for 1 hr at 4°C. After washing, the bound proteinswere visualized by western blotting or Coomassie Brilliant Blue (CBB) stainingafter sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). EFCLwas used as the PACSIN2 F-BAR domain for all of the assays, except for thecomparison with PACSIN1–PACSIN3 (Fig. 3D).

Liposome preparation, co-sedimentation assay and tubulation assayLiposomes were prepared from total bovine brain lipids (Folch fraction 1; AvantiPolar Lipids) (Michelsen et al., 1995). Lipids were dried under nitrogen gas in glasstest tubes and were resuspended in XB (10 mM HEPES, pH 7.9, 100 mM KCl, 2mM MgCl2, 0.2 mM CaCl2 and 5 mM ethylene glycol tetraacetic acid/EGTA),containing 100 mM sucrose, by mixing with a vortexer, and then hydrated at 37°Cfor 1 hour. This preparation yielded a mixture of unilamellar liposomes with variousdiameters (0.1–2 mm) and large, multilamellar vesicles, as determined by thin-section 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. Theco-sedimentation assay was performed as follows. The proteins were incubated with

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liposomes (1 mg/ml) in 50 ml XB for 20 minutes at room temperature (RT), and werethen 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.

Cell cultureHeLa and Hs578T cells were cultured in Dulbecco’s modified Eagle medium(DMEM) supplemented with 10% fetal calf serum (FCS), penicillin, andstreptomycin. SKBR-3 and BT549 cells were cultured in Roswell Park MemorialInstitute (RPMI) medium supplemented with 10% FCS, penicillin, and streptomycin.

ImmunoprecipitationSubconfluent HeLa cells in a 15 cm dish were washed with PBS, and suspended inbuffer 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/mlleupeptin. The resulting cell lysate was sonicated and centrifuged at 15,000 r.p.m. at4°C. The supernatant was incubated with 1 mg of anti-caveolin-1 (7C8) antibody orcontrol 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 washedand subjected to western blot analysis.

TransfectionGgreen fluorescent protein (GFP), Venus and mCherry labelled PACSIN1, PACSIN2and PACSIN3 proteins were prepared by subcloning the mouse Pacsin1, Pacsin2and Pacsin3 cDNA into the pEGFP-C1, pVenus-C1, and pmCherry-C1 vectors(Clontech), in which the GFP in pEGFP-C1 was replaced with Venus and mCherry,respectively (Nagai et al., 2002; Shaner et al., 2004). Venus is a GFP mutant withbrighter fluorescence. Venus was labeled as GFP, for simplicity. The PACSIN2–GFPand PACSIN2–mCherry proteins were respectively expressed by the pEGFP-N3vector (Clontech) and the pmCherry-N3 vector, in which the GFP in pEGFP-N3 wasreplaced with mCherry. The caveolin-1–GFP was expressed by subcloning cDNAencoding mouse caveolin-1 into the pEGFP-N3 or pDsRed-Monomer-N1 vector.The wild-type and K44A dynamin-2 constructs were gifts from Mark A. McNiven(Orth et al., 2002). The wild-type, actin-polymerization-defective VCA, and thepartially active phospho-mimic Y253E were described previously (Sasaki et al.,2000; Suetsugu et al., 2002). The proline-rich region of dynamin-2 (aa 741–870) wassubcloned into the pCMV-Tag3B vector (Stratagene) with a Myc tag. The siRNAsto knockdown caveolin-1 and PACSIN2 were Stealth Select RNAiTM siRNAs,Invitrogen catalog nos. 1299003 and 1299001, respectively, containing three oligosiRNAs that were mixed and transfected simultaneously. The control RNA forsiRNA was Stealth RNAi Negative Control Duplexes (Invitrogen). Transfection wasperformed with the Lipofectamine LTX and PLUS reagents (Invitrogen), accordingto the manufacturer’s protocols. Dynasore (Sigma, D7693) was applied to DMEM+ 10% FCS at 40 mM for 5 minutes before fixation.

Sulforhodamine or FM-dye labeling of HeLa cellsHeLa cells were seeded on glass-bottom dishes (Asahi Glass, 3911-035), transfectedwith the vector expressing the GFP-tagged F-BAR domain of PACSIN2, and culturedovernight. Sulforhodamine (Invitrogen, S359) was then added to the medium at a0.1 mM final concentration. After 10 minutes, cells were visualized by confocalmicroscopy (FV1000D, Olympus) at 25°C. FM-dye labeling was performed basicallyas described previously (Terebiznik et al., 2002). HeLa cells were seeded on glass-bottom dishes, transfected, and cultured overnight. The cells were then chilled on icefor 15 minutes, washed with HBSS supplemented with 10 mM glucose, and incubatedwith 10 mM FM4-64 on ice for 1 minute. After the medium was removed, the cellswere washed twice with ice-cold HBSS supplemented with 10 mM glucose for 5minutes, and then the cells were observed by confocal microscopy at 25°C.

ImmunocytochemistryThe cells were fixed in 3.7% formaldehyde in PBS for 5 minutes, followed bypermeabilization with TBS supplemented with 1% BSA and 0.1% Triton X-100 for5 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 withthe F-BAR domain protein of PACSIN2. The rabbit monoclonal anti-caveolin-1antibody was purchased from Cell Signaling Technology. The mouse monoclonalanti-Myc (clone PL14) and the anti-caveolin-1 (7C8) antibodies were purchasedfrom MBL International, and Santa Cruz Biotechnology, respectively. The anti-clathrin heavy chain (clone 23) and anti-dynamin-2 antibodies were obtained fromBD Transduction Laboratories. After washing, cells were stained with Alexa Fluor488 and/or Alexa Fluor 568 labeled secondary antibodies and Alexa Fluor 633phalloidin. Fluorescence images were obtained by confocal microscopy (OlympusFluoview 1000D) at room temperature. A 100� oil-immersion objective (NA 1.45;Olympus) was used.

The spots of certain protein staining that were recognized as particles of highersignals than the surrounding areas in each image were counted. Subsequently, if thespots contained other protein staining signals that were higher than the areasurrounding the spots, then the spots were considered to be colocalization spots ofthe two proteins. This analysis was performed manually, with the aid of the CellCounter Plug-in and Channel Tools of the ImageJ program (NIH).

Cholera toxin incorporation assayHeLa cells were labeled with Alexa-Fluor-555-labeled Cholera toxin B (CTxB)(Invitrogen) on ice, as described previously (del Pozo et al., 2005). Briefly, cellswere 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 60minutes. The cells were then fixed with 4% paraformaldehyde in PBS. Thelocalization of CTxB close to the coverslip, including the basal plasma membrane,was then visualized by confocal microscopy. The cells with CTxB at the cellperiphery without significant CTxB accumulation at the cell center were consideredas cells without CTxB incorporation.

Electron microscopyCells on plastic coverslips (Thermanox, Nalge Nunc International) were fixed in 2%paraformaldehyde and 2% glutaraldehyde for 5 minutes at room temperature. Thecells 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 anda Nanogold rabbit Fab fragment (Nanoprobes). The stained cells were postfixed with1% glutaraldehyde in phosphate-buffered saline and blocked, and then the goldsignal was enhanced by Goldenhance (Nanoprobes). After the gold-enhancementreaction, the cells were washed, stained with 1% osmium tetroxide and 1% potassiumferrocyanide, 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 ofthe resin, 80 nm sections were prepared and counterstained for 10 minutes in uranylacetate and for 2 minutes in lead citrate. Dried sections were examined bytransmission electron microscopy.

Plasma membrane sheets were prepared and observed as described previously(Vinten et al., 2001), and were stained with an anti-PACSIN2 rabbit polyclonalantibody and an anti-caveolin-1 (7C8) mouse monoclonal antibody, or an anti-caveolin-1 rabbit monoclonal antibody and anti-dynamin-2 mouse monoclonalantibody, followed by 5-nm- or 10-nm-gold-conjugated antibodies.

Statistical analysisAll statistical analyses were performed using Microsoft Excel. Significance wasassessed by the Student’s t-test. All of the images are representative of at least threeindependent experiments.

We thank Mark A. McNiven (Mayo Clinic, Rochester, MN) for thedynamin-2 constructs. We are grateful to Toyoshi Fujimoto for criticalreading of the manuscript. We thank Tadaomi Takenawa, KazuyaTsujita and Tsukasa Oikawa (Kobe University, Kobe, Japan) for thecaveolin-1 and FBP17 expression vectors. This work was supported,in part, by a Grant-in-Aid from the Ministry of Education, Culture,Sports, Science and Technology of Japan and by Grants-in-Aid fromthe Japan Science and Technology Corporation (J.S.T.), the UeharaMemorial Foundation, the Mochida Memorial Foundation for Medicaland Pharmaceutical Research and the Inamori Foundation.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/12/2032/DC1

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