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• W2047399443 abstract "The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins. The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins. Actin cytoskeleton is essential for a wide variety of cellular functions, such as cell motility, phagocytosis, cell division, and muscle contraction. A tremendous number of molecules regulate the function of actin cytoskeleton. Regulation of polymerization and depolymerization of actin is crucial for the function of the actin cytoskeleton. Gelsolin-related proteins and actin depolymerizing factor (ADF) 2The abbreviations used are: ADFactin depolymerizing factorBicineN,N-bis(2-hydroxyethyl)glycineGSNL-1gelsolin-like protein-1Cccritical concentration. 2The abbreviations used are: ADFactin depolymerizing factorBicineN,N-bis(2-hydroxyethyl)glycineGSNL-1gelsolin-like protein-1Cccritical concentration./cofilin are the two major classes of actin filament-severing proteins that enhance actin filament turnover by severing and depolymerizing actin filaments and are involved in a number of cell biological processes (for reviews, see Refs. 1Ono S. Int. Rev. Cytol. 2007; 258: 1-82Crossref PubMed Scopus (211) Google Scholar and 2Southwick F.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6936-6938Crossref PubMed Scopus (34) Google Scholar). actin depolymerizing factor N,N-bis(2-hydroxyethyl)glycine gelsolin-like protein-1 critical concentration. actin depolymerizing factor N,N-bis(2-hydroxyethyl)glycine gelsolin-like protein-1 critical concentration. The actin cytoskeleton is highly differentiated into sarcomeric structures in striated muscle, and polymerization and depolymerization of actin must be precisely regulated in order to assemble and maintain striated myofibrils. Functional significance of ADF/cofilin in organized assembly of actin filaments in striated muscle has been demonstrated (1Ono S. Int. Rev. Cytol. 2007; 258: 1-82Crossref PubMed Scopus (211) Google Scholar). A muscle-specific ADF/cofilin isoform, M-cofilin/cofilin-2, is expressed in mammalian striated muscle (3Ono S. Minami N. Abe H. Obinata T. J. Biol. Chem. 1994; 269: 15280-15286Abstract Full Text PDF PubMed Google Scholar, 4Vartiainen M.K. Mustonen T. Mattila P.K. Ojala P.J. Thesleff I. Partanen J. Lappalainen P. Mol. Biol. Cell. 2002; 13: 183-194Crossref PubMed Scopus (173) Google Scholar). A mutation in the human cofilin-2 gene causes nemaline myopathy (5Agrawal P.B. Greenleaf R.S. Tomczak K.K. Lehtokari V.L. Wallgren-Pettersson C. Wallefeld W. Laing N.G. Darras B.T. Maciver S.K. Dormitzer P.R. Beggs A.H. Am. J. Hum. Genet. 2007; 80: 162-167Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In the nematode Caenorhabditis elegans, UNC-60B, a muscle-specific ADF/co-filin isoform, is required for organized assembly of actin filaments in body wall muscle (6Ono S. Baillie D.L. Benian G.M. J. Cell Biol. 1999; 145: 491-502Crossref PubMed Scopus (107) Google Scholar, 7Ono K. Parast M. Alberico C. Benian G.M. Ono S. J. Cell Sci. 2003; 116: 2073-2085Crossref PubMed Scopus (81) Google Scholar) and cooperates with UNC-78/actin-interacting protein 1 to promote actin filament disassembly (8Ono S. J. Cell Biol. 2001; 152: 1313-1319Crossref PubMed Scopus (83) Google Scholar, 9Mohri K. Ono K. Yu R. Yamashiro S. Ono S. Mol. Biol. Cell. 2006; 17: 2190-2199Crossref PubMed Scopus (41) Google Scholar). Gelsolin-related proteins are also expressed in striated muscle, but their function in muscle is not clearly understood. Gelsolin localizes to the thin filaments in vertebrate striated muscle (10Dissmann E. Hinssen H. Eur. J. Cell Biol. 1994; 63: 336-344PubMed Google Scholar, 11Yin H.L. Albrecht J.H. Fattoum A. J. Cell Biol. 1981; 91: 901-906Crossref PubMed Scopus (169) Google Scholar) and ascidian muscle (12Ohtsuka Y. Nakae H. Abe H. Obinata T. Zool. Sci. 1994; 11: 407-412Google Scholar, 13Ohtsuka Y. Nakae H. Abe H. Obinata T. Biochim. Biophys. Acta. 1998; 1383: 219-231Crossref PubMed Scopus (11) Google Scholar), suggesting that actin severing activity of gelsolin is inhibited or that actin filaments are protected from severing. In Drosophila melanogaster and C. elegans, mutations of Flightless-1, a gelsolin-related protein with N-terminal leucine-rich repeats (14Campbell H.D. Schimansky T. Claudianos C. Ozsarac N. Kasprzak A.B. Cotsell J.N. Young I.G. de Couet H.G. Miklos G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11386-11390Crossref PubMed Scopus (119) Google Scholar), cause disorganization of actin filaments in striated muscle (15Deng H. Xia D. Fang B. Zhang H. Genetics. 2007; 177: 847-860Crossref PubMed Scopus (28) Google Scholar, 16Miklos G.L. De Couet H.G. J. Neurogenet. 1990; 6: 133-151Crossref PubMed Scopus (26) Google Scholar). However, significance of actin severing activity of Flightless-1 has not been demonstrated. Gelsolin strongly severs actin filaments, caps the barbed ends and nucleates actin polymerization in a calcium-dependent manner (17McGough A.M. Staiger C.J. Min J.K. Simonetti K.D. FEBS Lett. 2003; 552: 75-81Crossref PubMed Scopus (156) Google Scholar, 18Silacci P. Mazzolai L. Gauci C. Stergiopulos N. Yin H.L. Hayoz D. Cell. Mol. Life Sci. 2004; 61: 2614-2623Crossref PubMed Scopus (306) Google Scholar, 19Sun H.Q. Yamamoto M. Mejillano M. Yin H.L. J. Biol. Chem. 1999; 274: 33179-33182Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). The gelsolin family proteins have repeats of homologous domains of 100–120 amino acids, which are designated as gelsolin-like (G) domains. Many gelsolin-related proteins, including gelsolin, villin, Flightless-1, and adseverin/scinderin, have six G domains, whereas Physarum fragmin, Dictyostelium severin, and vertebrate CapG have three G domains (see Refs. 1Ono S. Int. Rev. Cytol. 2007; 258: 1-82Crossref PubMed Scopus (211) Google Scholar and 17McGough A.M. Staiger C.J. Min J.K. Simonetti K.D. FEBS Lett. 2003; 552: 75-81Crossref PubMed Scopus (156) Google Scholar, 18Silacci P. Mazzolai L. Gauci C. Stergiopulos N. Yin H.L. Hayoz D. Cell. Mol. Life Sci. 2004; 61: 2614-2623Crossref PubMed Scopus (306) Google Scholar, 19Sun H.Q. Yamamoto M. Mejillano M. Yin H.L. J. Biol. Chem. 1999; 274: 33179-33182Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Thus, it has been speculated that gelsolin-related proteins with six G domains have evolved from gene duplication of the three-G-domain proteins (20Yin H.L. Janmey P.A. Schleicher M. FEBS Lett. 1990; 264: 78-80Crossref PubMed Scopus (38) Google Scholar). However, recently, unconventional gelsolin-related proteins with two, four, or five G domains have been discovered (21Gloss A. Rivero F. Khaire N. Muller R. Loomis W.F. Schleicher M. Noegel A.A. Mol. Biol. Cell. 2003; 14: 2716-2727Crossref PubMed Google Scholar, 22Kawamoto S. Suzuki T. Aki T. Katsutani T. Tsuboi S. Shigeta S. Ono K. FEBS Lett. 2002; 516: 234-238Crossref PubMed Scopus (23) Google Scholar, 23Xiang Y. Huang X. Wang T. Zhang Y. Liu Q. Hussey P.J. Ren H. Plant Cell. 2007; 19: 1930-1946Crossref PubMed Scopus (84) Google Scholar). Actin binding protein29 (ABP29) from Lilium pollen has only two G domains; nevertheless it has severing, nucleating, and capping activities (23Xiang Y. Huang X. Wang T. Zhang Y. Liu Q. Hussey P.J. Ren H. Plant Cell. 2007; 19: 1930-1946Crossref PubMed Scopus (84) Google Scholar). However, biochemical properties of other unconventional gelsolin-related proteins are not clearly understood. C. elegans has three genes that encode gelsolin-related proteins. fli-1 encodes a homolog of Flightless-1 (24Goshima M. Kariya K. Yamawaki-Kataoka Y. Okada T. Shibatohge M. Shima F. Fujimoto E. Kataoka T. Biochem. Biophys. Res. Commun. 1999; 257: 111-116Crossref PubMed Scopus (53) Google Scholar). FLI-1 is widely expressed in many tissues, and fli-1 mutations cause a number of developmental defects (15Deng H. Xia D. Fang B. Zhang H. Genetics. 2007; 177: 847-860Crossref PubMed Scopus (28) Google Scholar). Viln-1 (C10H11.1) encodes a villin-like protein with six G domains and a C-terminal villin headpiece, but its function is currently under investigation. K06A4.3 encodes a gelsolin-related protein with four G domains. Because K06A4.3 was most closely related to conventional gelsolin among the three genes, this gene has been designated as gsnl-1 (gelsolin-like protein-1) in this study. We are particularly interested in the function of gsnl-1, because biochemical properties of a gelsolin-related protein with four G domains have not been characterized, and mRNA of gsnl-1 is enriched in body wall muscle (25Fox R.M. Watson J.D. Von Stetina S.E. McDermott J. Brodigan T.M. Fukushige T. Krause M. Miller 3rd, D.M. Genome Biology. 2007; 8: R188Crossref PubMed Scopus (62) Google Scholar), suggesting that the GSNL-1 protein is a strong candidate of a muscle-specific regulator of actin reorganization. Our biochemical analysis indicates that GSNL-1 is a novel gelsolin-like actin severing and capping protein but, unlike gelsolin, stays bound to the side of actin filaments, binds to G-actin in a 1:1 molar ratio, and does not nucleate actin polymerization. These results provide a new aspect of functional diversity of gelsolin-related proteins. Proteins—Rabbit muscle actin was purified from acetone powder as described (26Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (959) Google Scholar). Pyrene-labeled rabbit muscle G-actin was prepared as described (27Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (717) Google Scholar). Alexa488-labeled rabbit muscle G-actin (1.5 labels/molecule on amines) was purchased from Invitrogen. Rhodamine-labeled rabbit muscle G-actin (0.5 labels/molecule on amines) was purchased from Cytoskeleton, Inc. Gelsolin was purified from newborn calf serum (N4637, Sigma) as reported by Kurokawa et al. (28Kurokawa H. Fujii W. Ohmi K. Sakurai T. Nonomura Y. Biochem. Biophys. Res. Commun. 1990; 168: 451-457Crossref PubMed Scopus (92) Google Scholar) with slight modifications. After gelsolin was eluted from DEAE-cellulose, it was further purified by anion exchange chromatography using Mono Q (4.6/100PE column, Amersham Biosciences). Gelsolin was dialyzed against F-buffer (0.1 m KCl, 20 mm Hepes-NaOH, pH 7.5, 2 mm MgCl2) containing 50% glycerol and stored at -20 °C. Bacterially expressed C. elegans UNC-60B was prepared as described previously (29Ono S. Benian G.M. J. Biol. Chem. 1998; 273: 3778-3783Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Bacterially expressed chicken CapZ (a gift of Dr. Takashi Obinata, Chiba University, Chiba, Japan) was prepared as described previously (30Soeno Y. Abe H. Kimura S. Maruyama K. Obinata T. J. Muscle Res. Cell Motil. 1998; 19: 639-646Crossref PubMed Scopus (53) Google Scholar). Expression and Purification of GSNL-1—A full-length cDNA clone for GSNL-1 (yk1613a08) was obtained from Dr. Yuji Kohara (National Institute of Genetics, Mishima, Japan). A full-length coding region of the GSNL-1 cDNA was amplified with PCR and cloned into pGEX-2T using an infusion cloning kit (BD Biosciences). The sequence of the insert was verified by DNA sequencing. The Escherichia coli strain BL21(DE3) was transformed with pGEX-GSNL-1 and cultured in M9ZB (18.7 mm NH4Cl, 22 mm KH2PO4, 42.3 mm Na2HPO4, 1% Tryptone, 85.6 mm NaCl, 1 mm MgSO4, and 0.4% glucose) containing 50 μg/ml ampicillin at 37 °C until A600 reached 0.6 cm-1. Then the culture was cooled to room temperature, and expression was induced by adding 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 2 h at room temperature. The cells were harvested by centrifugation at 5000 × g for 10 min and homogenized by a French Pressure cell at 360–580 kg/cm2 in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 1.4 mm KH2PO4, 8 mm Na2HPO4). The homogenates were centrifuged at 20,000 × g for 15 min, and the supernatants were applied to a glutathione Uniflow (Clontech) column (1.5-ml bed volume). After washing with phosphate-buffered saline, 15 units of thrombin (Roche Applied Science) was added to cleave the N-terminal glutathione S-transferase tag, and GSNL-1 eluted from the column. GSNL-1 was dialyzed against F-buffer containing 50% glycerol and stored at -20 °C. Protein concentration was determined with a BCA protein assay (23225, Pierce). Actin Filament Severing and Capping Assays with Fluorescence Microscopy—Observation of actin filament severing activity by fluorescence microscopy was performed as described previously (31Ichetovkin I. Han J. Pang K.M. Knecht D.A. Condeelis J.S. Cell Motil. Cytoskeleton. 2000; 45: 293-306Crossref PubMed Scopus (79) Google Scholar, 32Ono S. Mohri K. Ono K. J. Biol. Chem. 2004; 279: 14207-14212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 33Yamashiro S. Gimona M. Ono S. J. Cell Sci. 2007; 120: 3022-3033Crossref PubMed Scopus (24) Google Scholar) with slight modifications. Previously, we used anti-biotin antibody (Invitrogen) to immobilize biotin-labeled actin on the glass surface. However, this antibody has been discontinued by the company, and we found that several other commercially available anti-biotin antibodies were not very efficient in tethering actin filaments. Instead, unlabeled actin (1.6 μm) and Alexa488-labeled actin (0.4 μm) were co-polymerized and attached to a glass coverslip using heavy meromyosin (MH01, Cytoskeleton Inc.). Other procedures were the same as our previous reports. Capping activity of GSNL-1 was monitored as described previously (32Ono S. Mohri K. Ono K. J. Biol. Chem. 2004; 279: 14207-14212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, Alexa488-labeled actin filaments were incubated with GSNL-1 in a perfusion chamber for 3 min, then 0.4 μm rhodamine-labeled G-actin (AR05, Cytoskeleton Inc.) was infused and allowed to elongate from free barbed ends for 3 min. Unincorporated actin was washed with anti-bleaching buffer containing 0.2 μm cytochalasin D, and micrographs of Alexa488- and rhodamine-actin from the same field were taken. Filaments were observed by epifluorescence using a Nikon TE2000 inverted microscope with a 60× Plan Apo objective (oil, NA = 1.4), and images were captured by a SPOT RT Monochrome CCD camera (Diagnostic Instruments) and processed by IPLab (BD Biosciences Bioimaging) and Adobe Photoshop CS3. Light Scattering and Fluorescence Assays for Actin Depolymerization and Polymerization—Kinetics of actin depolymerization induced by gelsolin or GSNL-1 were monitored by light-scattering measurements. 10 μm actin was polymerized in the presence of 100 nm CapZ for 2 h at room temperature in F-buffer containing 0.1 mm CaCl2. CapZ-F-actin was diluted to 0.5 μm actin in F-buffer containing 0.1 mm CaCl2 or 5 mm EGTA in the presence of 1 μm latrunculin A (Biomol) and various concentrations of gelsolin or GSNL-1. Intensity of light scattering was monitored at a wavelength of 400 nm and at an angle of 90° with a PerkinElmer Life Sciences LS50B fluorescence spectrophotometer. Kinetics of actin polymerization were monitored by measuring fluorescence of pyrene-labeled actin. 20 μm pyrene-labeled G-actin (20% labeled) was diluted to 2.5 μm with G buffer (0.2 mm ATP, 0.2 mm CaCl2, 0.2 mm dithiothreitol, 2 mm Tris-HCl, pH 8) in the presence of gelsolin or GSNL-1. After 5 min, salt and buffer were adjusted to final concentrations of 0.1 m KCl, 2 mm MgCl2, 0.1 mm CaCl2, 20 mm Hepes-NaOH, pH 7.5, and actin was diluted to 2 μm. Fluorescence of pyrene (excitation at 366 nm and emission at 384 nm) was monitored for 20 min with a PerkinElmer Life Sciences LS50B fluorescence spectrophotometer. To determine the critical concentration for actin polymerization, varying concentrations (0.1–1 μm) of pyrene-labeled G-actin (20% labeled) was polymerized for 18 h at room temperature in the presence of a constant concentration of GSNL-1 or CapZ in F-buffer containing 0.1 mm CaCl2 or 5 mm EGTA. Final fluorescence intensity of pyrene (excitation at 366 nm and emission at 384 nm) was measured. F-actin Sedimentation Assay—Varying concentrations of gelsolin or GSNL-1 were added to 5 μm F-actin in 100 mm KCl, 2 mm MgCl2, 0.2 mm dithiothreitol, 20 mm Hepes-NaOH, pH 7.5, containing 0.1 mm CaCl2 or 0.1 mm EGTA. 1 mm EGTA was used for gelsolin. After incubation for 1 h at room temperature, the mixtures were ultracentrifuged at 436,000 × g for 15 min at 20 °C. Supernatant and pellet fractions were adjusted to the same volumes and subjected to SDS-PAGE and staining with Coomassie Brilliant Blue R-250. Gels were scanned by a UMAX Powerlook III scanner at 300 dots per inch, and band intensity was quantified using Image J. Actin Monomer Binding Assays—Nondenaturing polyacrylamide gel electrophoresis was performed as described (34Safer D. Anal. Biochem. 1989; 178: 32-37Crossref PubMed Scopus (72) Google Scholar). G-actin and GSNL-1 were incubated in G-buffer for 30 min at room temperature. The samples were supplemented with 0.25 volume of a loading buffer (50% glycerol, 0.05% bromphenol blue) and electrophoresed using a Bicine/triethanolamine buffer system. The proteins were visualized by staining with Coomassie Brilliant Blue R-250 (National Diagnostics). To analyze stoichiometry of actin and GSNL-1, a mixture of 10 μm actin and 10 μm GSNL-1 was applied to nondenaturing polyacrylamide gel electrophoresis, and protein composition of each band was examined by SDS-PAGE. Core regions of the observed six bands were excised and cut into small pieces. After washing with deionized water, 50 μl of SDS sample buffer (2% SDS, 80 mm Tris-HCl, 5% β-mercaptoethanol, 15% glycerol, 0.05% bromphenol blue) was added. The gel pieces were then extensively sonicated and heated at 98 °C for 5 min. The extracted proteins were resolved by SDS-PAGE, and relative band density of actin and GSNL-1 was compared with a standard (a 1:1 mixture of actin and GSNL-1). The change in the fluorescence of pyrene-labeled G-actin was used to detect binding of GSNL-1 to G-actin. A dissociation constant (Kd) for binding of GSNL-1 with G-actin was determined by a modification of the method that was developed for 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled actin by Carlier et al. (35Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (817) Google Scholar). Varying concentrations of GSNL-1 (0.05–2 μm) were incubated with 1 μm G-actin (20% pyrene-labeled) in G-buffer at room temperature for 30 min. Then the pyrene fluorescence (F) (excitation at 366 nm and emission at 384 nm) was measured, and relative fluorescence (E) was calculated as E=(F-F0)(Fmax-F0)(Eq. 1) where F0 is fluorescence of 1 μm actin alone, and Fmax is the maximal fluorescence when GSNL-1 saturated binding to 1 μm actin. Second, the data were fitted to Equation 2, E=12c+12z-12(c+z)2-4z(Eq. 2) where z=[GSNL-1]total[actin]total(Eq. 3) and c=1+Kd[actin]total(Eq. 4) curve fitting was performed with SigmaPlot 10 (SYSTAT Software, Inc.). GSNL-1 Has Four Gelsolin-like Domains—The C. elegans gene K06A4.3 encodes a 55-kDa protein that has four gelsolin-like (G) domains (Fig. 1A). Therefore, we designated this gene as gsnl-1. Previously, Der f 16, an allergen from the house dust mite, has been reported to have four G domains (22Kawamoto S. Suzuki T. Aki T. Katsutani T. Tsuboi S. Shigeta S. Ono K. FEBS Lett. 2002; 516: 234-238Crossref PubMed Scopus (23) Google Scholar). However, gelsolin-related proteins typically have three or six G domains, and a protein with four G domains has not been biochemically characterized. Sequence alignment by the ClustalW2 method (36Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (21916) Google Scholar) indicates that GSNL-1 is highly homologous to Der f 16 (31% identity) and that the first three G domains (G1-G3) are very similar to the corresponding domains of human gelsolin, a six-G-domain protein, and CapG, a three-G-domain protein with no severing activity (Fig. 1B). Two regions of gelsolin, LDDYLNG in G1 and GFKHVV in a linker between G1 and G2 (boxed in Fig. 1B), are important for its actin filament severing activity. CapG does not sever actin filaments because these sequences are not conserved (37Southwick F.S. J. Biol. Chem. 1995; 270: 45-48Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). GSNL-1 has very similar sequence to gelsolin in these two regions (boxed in Fig. 1B), suggesting that GSNL-1 possesses severing activity. Interestingly, the ClustalW2 method aligned the fourth G domain (G4) of GSNL-1 with the sixth G domain (G6) of gelsolin (Fig. 1B). Indeed, individual comparison of GSNL-1 G4 with gelsolin G4, G5, or G6 indicated that GSNL-1 G4 is most closely related to gelsolin G6 (data not shown). One notable difference between gelsolin and GSNL-1 was that gelsolin has a flexible linker of ∼50 amino acids between G3 and G4 that acts like a hinge during calcium activation (38Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), whereas GSNL-1 has only a very short linker of ∼10 amino acids between G3 and G4 (Fig. 1B), suggesting that gelsolin and GSNL-1 undergo different conformational changes upon calcium activation. Thus, GSNL-1 is an unconventional gelsolin-related protein that apparently lacks two G domains that are equivalent to G4 and G5 of gelsolin. GSNL-1 Severs Actin Filaments in a Calcium-dependent Manner—To elucidate whether GSNL-1 possesses actin filament severing activity, a microscopic perfusion assay (31Ichetovkin I. Han J. Pang K.M. Knecht D.A. Condeelis J.S. Cell Motil. Cytoskeleton. 2000; 45: 293-306Crossref PubMed Scopus (79) Google Scholar, 32Ono S. Mohri K. Ono K. J. Biol. Chem. 2004; 279: 14207-14212Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 33Yamashiro S. Gimona M. Ono S. J. Cell Sci. 2007; 120: 3022-3033Crossref PubMed Scopus (24) Google Scholar) was employed. Alexa488-labeled F-actin was attached to a heavy meromyosin-coated coverslip, and GSNL-1 was infused and incubated for 3 min. Incubation of actin filaments with buffer only in the presence (Fig. 2A, a and b) or absence (Fig. 2A, k and l) of Ca2+ caused no major alteration in the morphology of the actin filaments. In the presence of 100 μm Ca2+, GSNL-1 caused fragmentation of actin filaments (Fig. 2A, c and d, e and f, g and h, i and j). Fragmentation was detected at 5 nm GSNL-1 (Fig. 2A, c and d). As the concentrations of GSNL-1 were increased, actin filaments became shorter and nearly completely disassembled at 30 nm GSNL-1 (Fig. 2A, i and j). Similar actin filament severing activity of GSNL-1 was observed when calcium concentration was decreased to 1 μm (data not shown). However, when calcium was removed by EGTA, 5–30 nm GSNL-1 did not sever actin filaments (Fig. 2A, m and n, o and p, q and r, s and t). Thus, these microscopic assays demonstrate that GSNL-1 severs actin filaments in a calcium-dependent manner. Allen et al. (39Allen P.G. Laham L.E. Way M. Janmey P.A. J. Biol. Chem. 1996; 271: 4665-4670Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) reported that gelsolin, the six-G-domain protein, severs ADP-actin more strongly than ADP-Pi actin filaments. Therefore, we tested whether GSNL-1 also preferentially severs ADP-actin. Inorganic phosphate (Pi) reversibly binds to ADP-actin filaments with a millimolar affinity (40Carlier M.F. Pantaloni D. J. Biol. Chem. 1988; 263: 817-825Abstract Full Text PDF PubMed Google Scholar). Actin filaments in a perfusion chamber were preincubated with a buffer with or without 10 mm potassium phosphate for 5 min, then they were incubated with 40 nm GSNL-1 in the absence of Pi for 2 min. In the absence of GSNL-1, Pi caused no major alteration in the actin filaments (compare Fig. 2B, a and b, and e and f). GSNL-1 only weakly severed the filaments that were preincubated with Pi (Fig. 2B, c and d), whereas it strongly severed filaments that were preincubated in a buffer without Pi (Fig. 2B, g and h). These results indicate that GSNL-1 preferentially severs ADP-bound actin filaments in a similar manner to gelsolin (39Allen P.G. Laham L.E. Way M. Janmey P.A. J. Biol. Chem. 1996; 271: 4665-4670Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Next, we compared the severing activities of GSNL-1 and gelsolin by measuring the kinetics of actin depolymerization (Fig. 3). CapZ-capped actin filaments were diluted to 0.5 μm actin in the presence of GSNL-1 or gelsolin. Latrunculin A was included in the reactions to sequester actin monomers. Because 0.5 μm actin is below the critical concentration (Cc) at the pointed end, severing should increase the number of pointed ends and accelerate the rate of depolymerization. In the presence of calcium, GSNL-1 increased the light scattering signal at time 0 and gradually decreased the signal over time (Fig. 3A). Because we manually assembled the reactions in a cuvette and set in a fluorometer, there was a delay of 10–15 s before time 0. Therefore, we interpreted that the initial increase is due to rapid association of GSNL-1 to the side of actin filaments before time 0 (see below) and that the following decrease is caused by filament severing in a similar manner to ADF/cofilin (41Yamashiro S. Mohri K. Ono S. Biochemistry. 2005; 44: 14238-14247Crossref PubMed Scopus (37) Google Scholar). In the absence of calcium, 400 nm GSNL-1 did not cause significant alteration in the depolymerization kinetics (Fig. 3B). In contrast, 30 nm gelsolin decreased the initial level of light scattering in the presence of calcium (Fig. 3C, blue curve). By reducing gelsolin to 15 nm, we were able to detect a rapid decrease in light scattering within 10 s (Fig. 3C, red curve). In the presence of EGTA 30 nm gelsolin did not alter the depolymerization kinetics (Fig. 3C, green and purple curves). These results suggest that gelsolin instantaneously severs actin filaments. Our direct comparison of GSNL-1 and gelsolin revealed two major differences; 1) GSNL-1 severs actin filaments more slowly and requires higher concentrations than gelsolin, and 2) GSNL-1 initially associates with the side of filaments and gradually severs them, whereas gelsolin instantaneously severs filaments. GSNL-1 Caps the Barbed Ends of Actin Filaments—Gelsolin caps the barbed ends of actin filaments, thus preventing polymerization and depolymerization from these ends (42Harris H.E. Weeds A.G. FEBS Lett. 1984; 177: 184-188Crossref PubMed Scopus (51) Google Scholar, 43Yin H.L. Hartwig J.H. Maruyama K. Stossel T.P. J. Biol. Chem. 1981; 256: 9693-9697Abstract Full Text PDF PubMed Google Scholar). To determine whether GSNL-1 caps the barbed ends, we examined the effect of GSNL-1 on the Cc of actin. Cc at the barbed end is ∼0.1 μm, whereas Cc at the pointed end is ∼0.6 μm (44Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (578) Google Scholar). Therefore, if the barbed ends are capped, Cc of total actin will be close to the Cc value at the pointed end. When actin alone was polymerized, amounts of F-actin as measured by pyrene fluorescence, linearly increased at above 0.2 μm actin in the presence (Fig. 4A, black circles) or absence of calcium (Fig. 4B, black circles). In the presence of calcium and GSNL-1, the critical concentration was shifted, and the amounts of F-actin increased at above 0.6 μm actin, whereas at below 0.6 μm actin, fluorescence of pyrene remained constant (Fig. 4A, open circles and squares). CapZ, which is known to cap the barbed ends (45Casella J.F." @default.
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• W2047399443 title "Caenorhabditis elegans Gelsolin-like Protein 1 Is a Novel Actin Filament-severing Protein with Four Gelsolin-like Repeats" @default.
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