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• W2043443184 abstract "Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures. Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures. The plasma membrane changes its structure dynamically in response to a wide variety of extracellular stimuli that alter cell shape. Membrane extension, including the formation of filopodia and lamellipodia, is controlled by the Rho family GTPases Cdc42 and Rac, respectively (1Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3734) Google Scholar). Rac and Cdc42 are involved in forming membrane protrusions essential for phagocytosis and macropinocytosis (2Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3071) Google Scholar), whereas another GTPase, dynamin, has been implicated in producing membrane invaginations and vesicles from the plasma membrane (3Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Dynamin is a multidomain GTPase; it consists of a GTPase domain followed by a central domain lacking homology to any other proteins, a pleckstrin homology domain, an effector domain, and a C-terminal proline-rich motif (4Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar). Dynamin-1 (neuron-specific), dynamin-2 (ubiquitously expressed), and dynamin-3 (expressed only in the testis, brain, and lung), constitute the dynamin family (5Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (341) Google Scholar, 6Sontag J.M. Fykse E.M. Ushkaryov Y. Liu J.P. Robinson P.J. Sudhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 7Kamitani A. Yamada H. Kinuta M. Watanabe M. Li S.A. Matsukawa T. McNiven M. Kumon H. Takei K. Biochem. Biophys. Res. Commun. 2002; 294: 261-267Crossref PubMed Scopus (33) Google Scholar). These proteins are essential for clathrin-dependent and also caveolae-mediated endocytosis (reviewed in Refs. 8Nabi I.R. Le P.U. J. Cell Biol. 2003; 161: 673-677Crossref PubMed Scopus (600) Google Scholar and 9Pelkmans L. Helenius A. Traffic. 2002; 3: 311-320Crossref PubMed Scopus (595) Google Scholar). In addition, association of dynamin with Src homology 3 (SH3) 1The abbreviations used are: SH3, Src homology 3; FBP17, formin-binding protein 17; CIP4, Cdc42-interacting protein 4; FCH, Fer/CIP4 homology; RBD, Rho family protein-binding domain; EGF, epidermal growth factor; CTB, cholera toxin subunit B; EGFP, enhanced green fluorescent protein; DiIC16(3), 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GST, glutathione S-transferase; M-amphiphysin-2, muscle amphiphysin-2. domain-containing molecules has been shown to modulate endocytosis since the C-terminal proline-rich motif provides an SH3 domain-binding site (10Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (355) Google Scholar). The dynamin-binding molecules amphiphysin, endophilin, intersectin, and PACSIN/syndapin have been reported to be involved in modulating dynamin-dependent endocytosis (4Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar, 11Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (424) Google Scholar). Formin-binding protein 17 (FBP17) consists of an N-terminal Fer/Cdc42-interacting protein 4 (CIP4) homology (FCH) domain followed by the first coiled-coil domain, a proline-rich motif, the second coiled-coil domain, a Rho family protein-binding domain (RBD), and a C-terminal SH3 domain. FBP17 was originally isolated as a molecule that binds to the proline-rich region of formin (12Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Crossref PubMed Scopus (194) Google Scholar). FBP17 is fused to mixed-lineage leukemia in acute myeloid leukemia (13Fuchs U. Rehkamp G. Haas O.A. Slany R. Konig M. Bojesen S. Bohle R.M. Damm-Welk C. Ludwig W.D. Harbott J. Borkhardt A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8756-8761Crossref PubMed Scopus (74) Google Scholar). However, its function remains unclear. Based on domain structure, it is evident that FBP17 is closely related to CIP4. CIP4 localizes to microtubules presumably via its N-terminal FCH domain, binds to Cdc42 via the central region corresponding to the RBD of FBP17, and associates with Wiskott-Aldrich syndrome protein via a C-terminal SH3 domain (14Tian L. Nelson D.L. Stewart D.M. J. Biol. Chem. 2000; 275: 7854-7861Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). A CIP4 homolog, Rapostlin, consists of an N-terminal FCH domain, an RBD, and a C-terminal SH3 domain similar to CIP4. Rapostlin has been identified as an effector that binds to the Rho family GTPase Rnd2 (15Fujita H. Katoh H. Ishikawa Y. Mori K. Negishi M. J. Biol. Chem. 2002; 277: 45428-45434Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Rapostlin shares 93% amino acid identity with FBP17, indicating that FBP17 is likely to be an ortholog of Rapostlin. Like CIP4, Rapostlin partially localizes to microtubules via its N-terminal FCH domain (15Fujita H. Katoh H. Ishikawa Y. Mori K. Negishi M. J. Biol. Chem. 2002; 277: 45428-45434Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although the FCH domain is thought to be a microtubule-targeting domain (16Greer P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 278-289Crossref PubMed Scopus (189) Google Scholar), we have previously shown that the FCH domain of Fer tyrosine kinase is not required for localization of Fer to microtubules (17Kogata N. Masuda M. Kamioka Y. Yamagishi A. Endo A. Okada M. Mochizuki N. Mol. Biol. Cell. 2003; 14: 3553-3564Crossref PubMed Scopus (63) Google Scholar). In addition, the dynamin-associating molecule PACSIN/syndapin, which contains an N-terminal FCH domain, does not localize to microtubules (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar). Since both FBP17 and PACSIN contain an N-terminal FCH domain, a coiled-coil domain, and a C-terminal SH3 domain, it seems probable that FBP17 is also involved in dynamin-regulated endocytosis. In this study, we demonstrate that FBP17 induces the plasma membrane to form tubular structures and that FBP17 associates with a C-terminal proline-rich motif of dynamin. Transferrin, epidermal growth factor (EGF), and cholera toxin subunit B (CTB) are taken up along with FBP17-induced tubules in COS-1 cells expressing FBP17, suggesting that FBP17 is involved in dynamin-mediated endocytosis. Plasmids—Full-length FBP17 and PACSIN-1 cDNAs were amplified by PCR from a human brain cDNA library (Clontech). PCR-amplified DNAs encoding FBP17 and its truncated mutants FBP17-N1 (amino acids 1-250), FBP17-N2 (amino acids 1-377), FBP17-C1 (amino acids 291-616), FBP17-dFCH (amino acids 80-616) were inserted into pCA-EGFP, a protein expression vector tagged at its N terminus with enhanced green fluorescent protein (EGFP) and derived from vector pCAGGS (19Yoshizaki H. Ohba Y. Kurokawa K. Itoh R.E. Nakamura T. Mochizuki N. Nagashima K. Matsuda M. J. Cell Biol. 2003; 162: 223-232Crossref PubMed Scopus (338) Google Scholar). A DNA fragment encoding a modified form of FBP17 referred to as FBP17-P597L, in which Leu is substituted for Pro597 in the SH3 domain, was amplified by PCR-based mutagenesis and ligated into pCA-EGFP. Full-length PACSIN-1 cDNA was likewise inserted into pCA-EGFP. pCXN2-FLAG-FBP17-N2 is derived from pCAGGS and expresses N-terminally FLAG-tagged FBP17-N2. A cDNA encoding the SH3 motif of FBP17 (amino acids 534-616) and a cDNA encoding a nonfunctional SH3 domain (P597L) of FBP17 were amplified by PCR and ligated into pGEX-4T3 (Amersham Biosciences, Little Chalfont, Buckinghamshire, United Kingdom). Full-length dynamin-1 and dynamin-2 cDNAs were obtained by PCR from a human heart cDNA library and ligated into pCA-EGFP and pCXN2-FLAG. Full-length dynamin-3 cDNA was amplified by PCR using KIAA0820 (a kind gift from Kazusa DNA Research Institute, Chiba, Japan) as a template and ligated into pEGFP-C1 (Clontech). cDNA encoding a GTPase-deficient mutant of dynamin-1 with Ala substituted for Lys44 (K44A) was amplified by PCR-based mutagenesis and ligated into pERed-NLS. pERed-NLS-dynamin-1-K44A expresses both FLAG-tagged dynamin-1-K44A and internal ribosomal entry signal-driven DsRed-Express fused with a nuclear localization signal (Clontech). pBluescript (Stratagene, La Jolla, CA) containing nucleotides 809-1851 of FBP17 was used to produce both antisense and sense riboprobes for in situ hybridization. All of the DNA fragments amplified by PCR were ligated into the pCR4blunt-TOPO vector (Invitrogen) and confirmed by sequencing with an Applied Biosystems ABI Prism 3700 sequencer. Reagents and Antibodies—DiIC16(3), Alexa 546-conjugated transferrin, Texas Red-conjugated EGF, Alexa 555-conjugated CTB, Alexa 546-conjugated goat anti-mouse IgG, and Alexa 488-conjugated goat anti-rabbit IgG were purchased from Molecular Probes, Inc. (Eugene, OR). Anti-β-tubulin antibody, anti-vimentin antibody, rhodamine-conjugated phalloidin, and anti-FLAG antibody M2 were from Sigma. Anti-KDEL antibody was from Stressgen Biotech Corp. Anti-dynamin-2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-Sepharose, protein G-Sepharose, and glutathione-Sepharose from Amersham Biosciences. The digoxigenin-labeled riboprobe synthesis kit and the random-primed DNA labeling kit were from Roche Diagnostics (Basel, Switzerland). [α-32P]dCTP (EasyTides™) was from PerkinElmer Life Sciences. Anti-green fluorescent protein (GFP) antibody was developed in our laboratory, and anti-FBP17 antibody was produced by immunizing rabbits with a keyhole limpet hemocyanin-coupled synthetic peptide (CAQDRESPDGSYTEEQSQES) corresponding to amino acids 506-525 of FBP17. Cell Culture and Transfection—293T cells (a gift from B. J. Meyer, University of Connecticut, Storrs, CT) and COS-1 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Cells were transfected using LipofectAMINE 2000 (Invitrogen). Cell Membrane Staining—COS-1 cells cultured on glass-bottom dishes were washed three times with DMEM without phenol red, incubated with DMEM containing 10 μg/ml DiIC16(3) for 10 min, rinsed twice with phosphate-buffered saline (PBS), fixed with 2% formaldehyde in PBS, and examined by confocal fluorescence imaging. Transferrin, EGF, and CTB Uptake—COS-1 cells expressing EGFP-FBP17 were serum-starved in DMEM for 1 h and incubated with 25 μg/ml Alexa 546-conjugated transferrin for 20 min at either 37 °C or 4 °C and with 1 ng/ml Texas Red-conjugated EGF or 1 μg/ml Alexa 555-conjugated CTB at 37 °C. After rinsing three times with PBS and reducing surface labeling using 50 mm deferoxamine mesylate-containing buffer (150 mm NaCl, 2 mm CaCl2, and 25 mm sodium acetate/acetic acid, pH 4.5), the cells were fixed with 2% formaldehyde in PBS and subjected to fluorescence imaging. Images of fluorescence-conjugated transferrin, EGF, or CTB were obtained with an Olympus IX-71 epifluorescence microscope equipped with a cooled charge-coupled camera (CoolSNAP-HQ, Roper Scientific, Trenton, NJ). Immunoblotting and Immunoprecipitation—Immunoblotting and immunoprecipitation were performed as described previously (17Kogata N. Masuda M. Kamioka Y. Yamagishi A. Endo A. Okada M. Mochizuki N. Mol. Biol. Cell. 2003; 14: 3553-3564Crossref PubMed Scopus (63) Google Scholar). Briefly, 293T cells were washed with PBS and lysed with buffer containing 150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 1.5 mm MgCl2, 1% Triton X-100, and protease inhibitor mixture (Roche Diagnostics). Pre-cleared cell lysates were immunoprecipitated with antibodies as indicated together with protein A- or G-Sepharose. Precipitates were subjected to SDS-PAGE followed by immunoblotting with antibodies as indicated. Proteins that reacted with the primary antibody recognized by the peroxidase-conjugated secondary antibody and that were species-matched were visualized with the ECL system (Amersham Biosciences) and a LAS-1000 image analyzer (Fuji Film, Tokyo, Japan). Tissues from BALB/c mice were rinsed with PBS and homogenized in lysis buffer (62.5 mm Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and bromphenol). The homogenates were centrifuged at 100,000 × g for 10 min. The pellets were fractionated by SDS-PAGE and immunoblotted with anti-FBP17 antibody. Immunohistochemistry and Electron Microcopy—Human testis fixed with Bouin's solution and embedded in paraffin was sectioned, deparaffinized, and immunostained with anti-FBP17 antibody. Immunoreactivity detected by the peroxidase-conjugated secondary antibody was visualized with 1 mg/ml diaminobenzidine. Sections were counter-stained with hematoxylin. For electron microscopy, COS-1 cells expressing EGFP-FBP17 were fixed with 2.5% glutaraldehyde and postfixed in 1% OsO4 followed by embedding in epoxy resin. Ultrathin sections on nickel grids were immersed in target retrieval solution (DakoCytomation, Kyoto, Japan). After washing with distilled water and drying, sections were stained with both uranyl acetate and lead citrate and examined with a Hitachi H-800 electron microscope. Northern Blot Analysis and in Situ Hybridization—A multiple-tissue Northern blot membrane (Human Brain II, Clontech) was prehybridized, followed by high stringency hybridization with an [α-32P]dCTP-labeled FBP17 probe and washing with buffer containing appropriate concentrations of SSC and SDS. RNA hybridized with the radiolabeled probe was detected with a BAS-5000 imaging system (Fuji Film). In situ hybridization was performed as described previously (20Ohnishi J. Ohnishi E. Jin M. Hirano W. Nakane D. Matsui H. Kimura A. Sawa H. Nakayama K. Shibuya H. Nagashima K. Takahashi T. Mol. Endocrinol. 2001; 15: 747-764Crossref PubMed Scopus (37) Google Scholar). Briefly, mouse testis and human brain were embedded in Tissue-Tec OCT compound and frozen in liquid nitrogen. Cryostat sections were fixed in 4% paraformaldehyde, prehybridized, and hybridized overnight at 72 °C with digoxigenin-labeled riboprobe in hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's solution, and 500 μg/ml tRNA). The sections were washed with 0.2× SSC and incubated with anti-digoxigenin antibody. The signal was visualized with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solution containing 0.24 mg/ml levamisole. Confocal Microscopy and Fluorescence Imaging—Cells cultured on glass-bottom dishes and transfected with pCA-EGFP-FBP17 for 24 h were fixed with 2% formaldehyde and permeabilized with 0.1% Triton X-100. For anti-vimentin antibody immunostaining, cells were fixed with methanol. The permeabilized cells were incubated with anti-β-tubulin, anti-KDEL, or anti-vimentin antibody followed by Alexa 546-conjugated goat anti-mouse IgG to visualize tubulin, the endoplasmic reticulum, and vimentin. Actin was visualized with rhodamine-conjugated phalloidin. Fluorescent images of EGFP and of Alexa 546 or rhodamine were obtained with an Olympus BX50EI confocal microscope controlled by Fluoview (Olympus, Tokyo) as described previously (21Nagashima K. Endo A. Ogita H. Kawana A. Yamagishi A. Kitabatake A. Matsuda M. Mochizuki N. Mol. Biol. Cell. 2002; 13: 4231-4242Crossref PubMed Scopus (84) Google Scholar). Time-lapse fluorescence imaging was performed as described previously (21Nagashima K. Endo A. Ogita H. Kawana A. Yamagishi A. Kitabatake A. Matsuda M. Mochizuki N. Mol. Biol. Cell. 2002; 13: 4231-4242Crossref PubMed Scopus (84) Google Scholar). Briefly, COS-1 cells expressing EGFP-FBP17 were cultured on a collagen-coated glass-bottom dish in DMEM/nutrient mixture F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mml-glutamine, and 10 mm HEPES without phenol red. The cells were imaged using an Olympus IX-71 inverted microscope with a 75-watt xenon arc lamp equipped with a CoolSNAP-HQ cooled charge-coupled camera and two shutters, controlled by MetaMorph Version 5.0 software (Roper Scientific). To localize EGFP-tagged proteins, we obtained a fluorescent image every 20 s. Time-lapse images were converted to video format with MetaMorph Version 5.0 software. Quantitative Analysis of the Effect of Dynamin-1-K44A on Internalization of FBP17-induced Tubules—To examine the effect of dynamin-1-K44A on FBP17-induced tubule formation, COS-1 cells were cotransfected with either pCA-EGFP-FBP17 or pCA-EGFP-FBP17-P597L and pERed-NLS-dynamin-1-K44A. Both EGFP images and DsRed images were obtained using an Olympus IX-71 epifluorescence microscope. EGFP intensity, which reflects the intracellular accumulation of all tubular invaginations that were not processed in the endocytic pathway, was calculated by measuring the total intensity of the cell divided by the total cell area using MetaMorph Version 5.0 software. Data obtained from 50 cells were averaged, and statistical significance was evaluated by Student's t test. FBP17 Forms Tubular Invaginations in Living Cells—FBP17 has an N-terminal FCH domain. It has been suggested that this domain is a microtubule-targeting domain (16Greer P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 278-289Crossref PubMed Scopus (189) Google Scholar). Indeed, a previous study revealed that Rapostlin, a rat ortholog of human FBP17, partially localizes to microtubules (15Fujita H. Katoh H. Ishikawa Y. Mori K. Negishi M. J. Biol. Chem. 2002; 277: 45428-45434Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We therefore examined the localization of EGFP-tagged FBP17 in COS-1 cells (Fig. 1A) and found that it was located in cylindrical fiber structures of the cytoplasm. It was not present in the cytoskeleton, including microtubules, actin stress fibers, and intermediate filaments, or in the endoplasmic reticulum of COS-1 cells (Supplemental Fig. 1). When FLAG-tagged FBP17 was expressed in the cells, similar cylindrical fiber-like immunostaining was observed in the cytoplasm (Supplemental Fig. 2B). We examined whether the cylindrical fiber structures are tubes. COS-1 cells transfected with a plasmid expressing EGFP-FBP17 were examined with an electron microscope. The open ends and the blind ends of the tubular structures induced by FBP17 were in the plasma membrane and cytoplasm, respectively (Fig. 1B). To examine how cylindrical fiber-like structure developed in the living cells, we monitored EGFP-FBP17 in COS-1 cells by time-lapse fluorescence microscopy. The fibers arose from the cell periphery, grew toward the center of the cell, and sometimes contracted back toward the periphery (Fig. 1C and Supplemental Video 1). We further noticed that some EGFP-FBP17-marked tubules were internalized instead of being contracted (Fig. 1D and Supplemental Video 2). The GFP-expressing plasmid used as a negative control did not induce any tubule formation. These results indicate that FBP17 induces tubular plasma membrane invaginations. FBP17 Is Expressed in the Brain and Testis—We developed an anti-FBP17 antibody and examined the expression of FBP17 in human and mouse organs and tissues. Immunoblot analysis revealed that FBP17 was expressed in mouse testis and brain (Fig. 2A). We then examined the localization of FBP17 in the seminiferous tubules of human testis by immunohistochemistry. Germ cells were immunoreactive to anti-FBP17 antibody, whereas Sertoli's cells were negative. Of the germ cells, the secondary spermatocytes in the inner layer of the tubules exhibited strong immunoreactivity, whereas spermatogonia in the outermost layer and spermatozoa in the innermost layer showed no immunoreactivity (Fig. 2B). This suggests that the expression of FBP17 is correlated with the maturation of germ cells. More detailed examination of the maturation stages revealed that germ cells, from secondary spermatocytes to elongated spermatids, were immunoreactive. 2Y. Kamioka, H. Sawa, and N. Mochizuki, unpublished data. We next asked whether FBP17 is expressed in the brain. Expression was examined by both Northern blot analysis and in situ hybridization. FBP17 mRNA was detected as a transcript of ∼6.0 kb in all regions of the human brain (Fig. 2C). It was also detected in situ in mouse testis and in the cortex of the cerebrum and in the granular layer of the cerebellum of human brain (Fig. 2D). FBP17-induced Invagination Originating from the Plasma Membrane—To examine whether FBP17-induced tubules are continuous with the plasma membrane, we used DiIC16(3). DiIC16(3) is a lipophilic fluorescent probe used for plasma membrane staining (22Mukherjee S. Maxfield F.R. Traffic. 2000; 1: 203-211Crossref PubMed Scopus (195) Google Scholar). COS-1 cells expressing EGFP-tagged FBP17 were stained for DiIC16(3) and imaged for fluorescence (Fig. 3). EGFP-FBP17 expression and DiIC16(3) staining overlapped, supporting the idea that FBP17-generated tubules arise from the plasma membrane. The C-terminal SH3 Domain Is Not Required for FBP17-induced Tubular Invagination—To investigate the mechanism by which tubular invagination is induced by FBP17, we constructed a series of deletion or point mutants of EGFP-tagged FBP17 (Fig. 4A). We confirmed that the EGFP-tagged mutants were correctly constructed, as they had the expected molecular mass in immunoblots probed with anti-GFP antibody (Fig. 4B). Full-length FBP17 and an SH3 domain mutant (FBP17-P597L) formed tubular structures (Fig. 4C), as did a derivative (FBP17-N2) with a deletion of the second coiled-coil region, the RBD, and the SH3 domain. In contrast, removal of the FCH domain in FBP17-dFCH or of both the FCH domain and the first coiled-coil domain in FBP17-C1 abolished tube formation. In addition, FBP17-N1, a derivative containing only the FCH domain and the first coiled-coil domain, was incapable of inducing tube formation. These results indicate that the FCH domain, the first coiled-coil domain, and the proline-rich region are essential for FBP17-induced tube formation. We also tested whether PACSIN-1, a molecule structurally related to FBP17, forms tubules like FBP17. Although PACSIN-1, like FBP17, contains an FCH domain followed by a coiled-coil domain and a C-terminal SH3 domain, it did not generate tubular structure, in agreement with a previous report (23Wasiak S. Quinn C.C. Ritter B. de Heuvel E. Baranes D. Plomann M. McPherson P.S. J. Biol. Chem. 2001; 276: 26622-26628Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We hypothesized that the self-assembly might contribute to the tube formation by FBP17. To test for self-assembly of FBP17, we expressed EGFP-tagged FBP17 and FLAG-tagged FBP17-N2 in 293T cells and examined their possible association by immunoprecipitation. Both EGFP-tagged full-length FBP17 and EGFP-tagged FBP17-N2 were co-immunoprecipitated with FLAG-tagged FBP17-N2 (Fig. 4D, second and fourth lanes), whereas EGFP-tagged FBP17-N1, FBP17-C1, and FBP17-SH3 were not co-immunoprecipitated. These results indicate that the FCH domain followed by a coiled-coil domain and polyproline regions are required for self-assembly. FBP17 Associates with Dynamin via Its SH3 Domain—PACSIN-1 and its rat ortholog, syndapin, are involved in clathrin-mediated endocytosis through an association with dynamin via their C-terminal SH3 domains (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1062Crossref PubMed Scopus (252) Google Scholar). We tested whether FBP17 associates with dynamin because FBP17 contains an SH3 domain in its C terminus like PACSIN. EGFP-tagged full-length FBP17 was co-immunoprecipitated with FLAG-tagged dynamin-1, as was PACSIN-1 (Fig. 5A). To examine whether the association of FBP17 with dynamin-1 depends on the SH3 domain, we used the derivative of FBP17 with a nonfunctional SH3 domain (FBP17-SH3-P597L) and FBP17-N2 lacking the SH3 domain. As expected, EGFP-tagged full-length FBP17 and FBP17-SH3 were co-immunoprecipitated with FLAG-tagged dynamin-1 (Fig. 5B, lanes 3 and 5), whereas FBP17-N2 and FBP17-SH3-P597L were not (lanes 4 and 6), indicating that the association of FBP17 with dynamin-1 is dependent upon the SH3 domain of FBP17. Dynamin-2 is ubiquitously expressed, whereas dynamin-1 is expressed exclusively in neurons, and dynamin-3 is restricted to the testis, brain, and lung (5Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (341) Google Scholar). FBP17 is mostly expressed in the brain and testis (Fig. 2A). Therefore, we tested whether dynamin-2 and dynamin-3, in addition to dynamin-1, associate with FBP17 by pull-down assays using the glutathione S-transferase (GST)-fused SH3 domain of FBP17 (Supplemental Fig. 2A). EGFP-tagged dynamin-1, -2, and -3 bound to the GST-fused SH3 domain of FBP17 but not to GST alone or GST fused to the nonfunctional SH3 domain of FBP17 (FBP17-SH3-P597L). These results demonstrate that FBP17 associates with dynamin family proteins in an SH3 domain-dependent manner. FBP17 Co-localizes with Dynamin—We proceeded to examine the co-localization of FBP17 with dynamin in COS-1 cells. EGFP-FBP17 expressed in COS-1 cells co-localized with FLAG-tagged dynamin expressed in the same cells (Fig. 6, A-C), whereas an SH3 mutant incapable of associating with dynamin did not co-localize with dynamin (Fig. 6, D and E). Although dynamin-1 expressed alone in COS-1 cells exhibited a diffuse staining pattern (Fig. 6F), dynamin-1 coexpressed with FBP17 exhibited a tubular pattern (Fig. 6B), indicating that dynamin-1 co-localizes with FBP17. We further examined the co-localization of FBP17 with endogenous dynamin-2 in COS-1 cells. Endogenous dynamin-2 was detected as small dots using anti-dynamin-2 antibody (Fig. 6I), whereas it co-localized with FBP17 in COS-1 cells expressing EGFP-FBP17 (Fig. 6, G, H, and J-L). These results suggest that FBP17 may be involved in endocytic signaling via dynamin. Involvement of FBP17 in Dynamin-mediated Endocytosis—To assess the consequence of the association of FBP17 with dynamin, we compared the localization of the molecules processed in the endocytic pathways with that of FBP17. Transferrin and EGF are endocytosed in a clathrin-dependent manner. Alexa 546-labeled transferrin was internalized along with FBP17 in COS-1 cells expressing FBP17 at 37 °C (Fig. 7A), whereas it was taken up as vesicles in parental COS-1 cells (Fig. 7D). We further examined whether the transferrin distribution follows the endocytic pathway. Whereas transferrin was observed as internalized vesicles near the nucleus at 37 °C (Fig. 7D), it was not when incubated at 4 °C (Fig. 8A). Although COS-1 cells expressing FBP17 exhibited the tubular structure, transferrin was not observed along these tubules at 4 °C (Fig. 8, B and C). These results suggest that FBP17-induced tubules involve transferrin uptake in an endocytosis-dependent manner. Similarly, Texas Red-labeled EGF was found at the tubular structure in COS-1 cells expressing FBP17, although EGF was found in a vesicular pattern in parental COS-1 cells (Fig. 7, B and E). CTB has been used as a marker for caveolae-mediated endocytosis (25Parton R.G. Richards A.A. Traffic. 2003; 4: 724-738Crossref PubMed Scopus (495) Google Scholar). Caveolin-mediated endocytosis is dependent upon dynamin (2Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3071) Google Scholar). We therefore examined the involvement of FBP17 in caveolin-mediated internalization of CTB in COS-1 cells expressing FBP17. Internalized CTB localized to FBP17-marked tubules, whereas CTB internalized in parental cells exhibited a vesicular pattern (Fig. 7, C and F). These data suggest that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner.Fig. 8Transferrin uptake is mediated by endocytosis in COS-1 cells expressing FBP17. COS-1 cells expressing EGFP-tagged FBP17 were incubated with Alexa 546-conjugated transferrin at 4 °C instead of 37 °C. A, parental COS-1 cells were incubated with Alexa 546-conjugated transferrin at 4 °C and imaged as described in the legend to Fig. 7. Note that there was no vesicular uptake of transferrin at 4 °C, in contrast to 37 °C. Bar = 10 μm. B, COS-1 cells expressing FBP17 were imaged after incubation with Alexa 546-conjugated transferrin at 4 °C. The EGFP image (green) and the Alexa 546 image (red) are shown. Bar = 10 μm. C, the boxed area in B was enlarged. Note that transferrin did not co-localize with FBP17-induced tubules.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Internalization of FBP17-induced Tubules Is Dependent upon Dynamin—Dynamin-1-K44A is a dominant-negative mutant of dynamin that is defective in GTP hydrolysis and GTP binding and therefore inhibits clathrin-dependent endocytosis and caveolin-mediated internalization (4Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar). We used this mutant to examine the mechanism by which dynamin is involved in the internalization of FBP17-induced tubes. The FBP17-induced tubular structure was internalized (Fig. 1D) and co-localized with dynamin (Fig. 6). Hence, we hypothesized that dynamin-1-K44A perturbs the internalization of the tubular structure. The EGFP intensity of COS-1 cells expressing both EGFP-FBP17 and dynamin-1-K44A was compared with that of COS-1 cells expressing only EGFP-FBP17. COS-1 cells expressing both FBP17 and dynamin-1-K44A were distinguished from those expressing only EGFP-FBP17 by internal ribosomal entry signal-driven red fluorescence in the nucleus. The cells expressing both FBP17 and dynamin-1-K44A were brighter than those expressing only FBP17 (Fig. 9A). The quantitative results are shown in Fig. 9B. Consistently, cells expressing EGFP-FBP17-P597L, which were incapable of associating with dynamin but capable of inducing tubules, were brighter than those expressing wild-type FBP17 (Fig. 9B), suggesting the involvement of endogenous dynamin-2 present in COS-1 cells. These results indicate that the internalization of FBP17-induced tubules is mediated by dynamin. We have demonstrated that FBP17 forms tubular invaginations when expressed in cultured cells. Although Rapostlin, a rat ortholog of human FBP17, partially localizes to microtubules when expressed in HeLa cells (15Fujita H. Katoh H. Ishikawa Y. Mori K. Negishi M. J. Biol. Chem. 2002; 277: 45428-45434Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), FBP17 did not localize to any components of the cytoskeleton such as microtubules, intermediate filaments, and actin fibers (Supplementary Fig. 1). FBP17-induced tubules originated from the plasma membrane and grew toward the cytoplasm, suggesting that FBP17 is involved in endocytosis. FBP17 associates with dynamin in an SH3 domain-dependent manner. Among dynamin-associating molecules, PACSIN/syndapin, like FBP17, has an N-terminal FCH domain and a C-terminal SH3 domain (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar, 24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1062Crossref PubMed Scopus (252) Google Scholar). FBP17 containing a nonfunctional SH3 domain did not associate with dynamin, indicating that the association between FBP17 and dynamin depends on the interaction between the SH3 domain of FBP17 and the proline-rich motif of dynamin. Consistently, dynamin co-localized with the FBP17-induced tubular structure (Fig. 6). Our data are in agreement with results showing that dynamin localizes to the tubular structure formed by muscle amphiphysin-2 (M-amphiphysin-2) (26Lee E. Marcucci M. Daniell L. Pypaert M. Weisz O.A. Ochoa G.C. Farsad K. Wenk M.R. De Camilli P. Science. 2002; 297: 1193-1196Crossref PubMed Scopus (326) Google Scholar). FBP17-induced tubular invagination is strongly reminiscent of that generated by an isoform of amphiphysin, M-amphiphysin-2. GFP-tagged M-amphiphysin-2 induces massive tubulation in Chinese hamster ovary cells (26Lee E. Marcucci M. Daniell L. Pypaert M. Weisz O.A. Ochoa G.C. Farsad K. Wenk M.R. De Camilli P. Science. 2002; 297: 1193-1196Crossref PubMed Scopus (326) Google Scholar). There are common characteristics between M-amphiphysin-2 and FBP17. First, it is noteworthy that the SH3 domain in the C termini of both M-amphiphysin-2 and FBP17 are dispensable for tubulation (Fig. 4C). The BAR domain (Bin1/amphiphysin/Rvs) of M-amphiphysin-2 is probably responsible for membrane targeting via membrane phosphatidylinositol 4,5-phosphates and tubule formation (26Lee E. Marcucci M. Daniell L. Pypaert M. Weisz O.A. Ochoa G.C. Farsad K. Wenk M.R. De Camilli P. Science. 2002; 297: 1193-1196Crossref PubMed Scopus (326) Google Scholar, 27Zhang B. Zelhof A.C. Traffic. 2002; 3: 452-460Crossref PubMed Scopus (72) Google Scholar, 28Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (514) Google Scholar). Thus, the FCH domain of FBP17, like the BAR domain, may function as a membrane-targeting domain and also a plasma membrane-deforming domain. Second, the N termini of amphiphysin-2 and FBP17 are also essential for dimerization (29Ramjaun A.R. Philie J. de Heuvel E. McPherson P.S. J. Biol. Chem. 1999; 274: 19785-19791Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). We found that the N terminus of FBP17 consisting of the FCH domain, a coiled-coil domain, and a proline-rich region was required for self-assembly to form tubular structures. Third, both FBP17 and M-amphiphysin-2 co-localize with dynamin in cultured cells. These results prompted us to examine the involvement of FBP17 in endocytosis since amphiphysin participates in endocytosis. The various SH3 domain-binding partners of dynamin affect vesiculation differently during endocytosis, which depends upon the pinch-off effect of dynamin when it hydrolyzes GTP (3Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 30Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (194) Google Scholar). We have demonstrated that FBP17 associated and co-localized with dynamin and that FBP17 involved dynamin-mediated transferrin and EGF endocytosis (Fig. 7). In addition, the internalization of FBP17-induced tubules was regulated by dynamin (Fig. 9). These results are consistent with the observation that amphiphysin-dynamin interaction enhances dynamin-mediated endocytosis in a clathrin-dependent manner (28Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (514) Google Scholar). In contrast, all PACSIN isoforms block clathrin-mediated transferrin endocytosis (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar), and endophilin perturbs dynamin-mediated vesiculation (31Farsad K. Ringstad N. Takei K. Floyd S.R. Rose K. De Camilli P. J. Cell Biol. 2001; 155: 193-200Crossref PubMed Scopus (487) Google Scholar). Thus, dynamin-binding proteins such as syndapin, amphiphysin-2, endophilin, and intersectin appear to be involved at distinct stages of clathrin-mediated vesicle formation (32Simpson F. Hussain N.K. Qualmann B. Kelly R.B. Kay B.K. McPherson P.S. Schmid S.L. Nat. Cell Biol. 1999; 1: 119-124Crossref PubMed Scopus (233) Google Scholar). Accordingly, FBP17 may participate in the recruitment of dynamin, and the recruited dynamin may then pinch off the tubules or vesicles induced by FBP17 in vivo. Given that FBP17 was expressed in the testis (Fig. 2) and that dynamin-2 and dynamin-3 are expressed in the testis (7Kamitani A. Yamada H. Kinuta M. Watanabe M. Li S.A. Matsukawa T. McNiven M. Kumon H. Takei K. Biochem. Biophys. Res. Commun. 2002; 294: 261-267Crossref PubMed Scopus (33) Google Scholar), the association of FBP17 with dynamins in the testis is likely to be involved in spermatogenesis. The expression of Rnd2, which belongs to the Rho family of GTPases and is an RBD partner of Rapostlin, an ortholog of FBP17, is restricted to germ cells at the spermatocyte and spermatid stages (33Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Crossref PubMed Scopus (308) Google Scholar, 34Naud N. Toure A. Liu J. Pineau C. Morin L. Dorseuil O. Escalier D. Chardin P. Gacon G. Biochem. J. 2003; 372: 105-112Crossref PubMed Scopus (30) Google Scholar). The expression of FBP17 determined by immunohistochemistry paralleled Rnd2 expression in germ cells. Hence, the Rnd2-FBP17-dynamin complex may be involved in endocytosis required for sperm maturation in the testis. In conclusion, we have demonstrated that FBP17 forms membrane invaginations originating from the plasma membrane and that FBP17 is likely to be involved in dynamin-dependent endocytosis. We thank Drs. Y. Nishimune and K. Kashima for advice; H. K. Surks and J. T. Pearson for critical reading of the manuscript; and M. Sone, Y. Ohba, N. Irisawa, and M. Sato for technical assistance. Download .zip (2.44 MB) Help with zip files Download .zip (2.44 MB) Help with zip files" @default.
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• W2043443184 title "A Novel Dynamin-associating Molecule, Formin-binding Protein 17, Induces Tubular Membrane Invaginations and Participates in Endocytosis" @default.
• W2043443184 cites W1508658015 @default.
• W2043443184 cites W1540291954 @default.
• W2043443184 cites W1564784444 @default.
• W2043443184 cites W1565336950 @default.
• W2043443184 cites W1569853787 @default.
• W2043443184 cites W1660040913 @default.
• W2043443184 cites W1972812883 @default.
• W2043443184 cites W1978154545 @default.
• W2043443184 cites W1997349805 @default.
• W2043443184 cites W1997719326 @default.
• W2043443184 cites W2015752928 @default.
• W2043443184 cites W2023054999 @default.
• W2043443184 cites W2025929812 @default.
• W2043443184 cites W2026740840 @default.
• W2043443184 cites W2027810531 @default.
• W2043443184 cites W2050291058 @default.
• W2043443184 cites W2053610637 @default.
• W2043443184 cites W2064958741 @default.
• W2043443184 cites W2071877471 @default.
• W2043443184 cites W2082741761 @default.
• W2043443184 cites W2087799476 @default.
• W2043443184 cites W2096476993 @default.
• W2043443184 cites W2108040056 @default.
• W2043443184 cites W2109208017 @default.
• W2043443184 cites W2109593473 @default.
• W2043443184 cites W2113888821 @default.
• W2043443184 cites W2129995506 @default.
• W2043443184 cites W2135606345 @default.
• W2043443184 cites W2138198341 @default.
• W2043443184 cites W2154190090 @default.
• W2043443184 cites W2156727641 @default.
• W2043443184 cites W2157920366 @default.
• W2043443184 cites W2167806590 @default.
• W2043443184 cites W2171753511 @default.
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