F-actin and Myosin II Binding Domains in Supervillin
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m305311200
ISSN1083-351X
AutoresYu Chen, Norio Takizawa, Jessica L. Crowley, Sang Wook Oh, Cheryl L. Gatto, Taketoshi Kambara, Osamu Satō, Xiang‐dong Li, Mitsuo Ikebe, Elizabeth J. Luna,
Tópico(s)Force Microscopy Techniques and Applications
ResumoDetergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density (∼1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids ∼280-342, ∼344-422, and ∼700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains. Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density (∼1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids ∼280-342, ∼344-422, and ∼700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains. Compartmentalized signaling involving cholesterol-rich, liquid-ordered membrane domains occurs during cell activation triggered by receptor cross-linking, growth factors, or other extracellular stimuli (1Anderson R.G. Jacobson K. Science. 2002; 296: 1821-1825Crossref PubMed Scopus (1006) Google Scholar, 2Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. 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During this procedure, the ∼50-nm liquid-ordered domains present in unactivated cells (22Pralle A. Keller P. Florin E.L. Simons K. Horber J.K. J. Cell Biol. 2000; 148: 997-1008Crossref PubMed Scopus (845) Google Scholar, 23Varma M. Leavitt J. Mutat. Res. 1988; 199: 437-447Crossref PubMed Scopus (8) Google Scholar) coalesce into detergent-resistant membranes (DRMs) 1The abbreviations used are: DRMs, detergent-resistant membranes; DRM-H, DRMs with buoyant densities of ∼1.15-1.18 g/ml; DRM-L, DRMs with buoyant densities of ∼1.09-1.13 g/ml; α-H340, affinity-purified antibody directed against amino acids 1-340 of human supervillin; GST, glutathione S-transferase; HMM, heavy meromyosin; LMM, light meromyosin; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; S1, myosin subfragment-1; S2, myosin subfragment-2; SV, supervillin; EGFP, enhanced green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; MS/MS, tandem mass spectrometry; PSD, post-source decay. that represent a subset of the endogenous raft lipids and proteins (2Pike L.J. J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5164) Google Scholar). Both lipid rafts and DRMs contain resident integral membrane proteins, such as caveolin, stomatin, and flotillin, and signaling proteins, including heterotrimeric G proteins and members of the Src family of protein-tyrosine kinases (24Galbiati F. Razani B. Lisanti M.P. Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). Raft-associated integral membrane and signaling proteins in bovine neutrophil plasma membranes sediment in sucrose gradients as both "light" (DRM-L) and "heavy" (DRM-H) fractions with buoyant densities of ∼1.09-1.13 and ∼1.15-1.18 g/ml, respectively (25Nebl T. Pestonjamasp K.N. Leszyk J.D. Crowley J.L. Oh S.W. Luna E.J. J. Biol. Chem. 2002; 277: 43399-43409Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The neutrophil DRM-H fraction also contains a subset of cytoskeletal proteins, including actin, myosin II, fodrin, α-actinins 1 and 4, vimentin, myosin 1G, and the actin-binding protein supervillin. Most of the integral, signaling, and cytoskeletal proteins in the DRM-H fraction continue to co-sediment with each other after solubilization of raft lipids with octylglucoside, indicating that these proteins are associated through interactions exclusive of those with the bilayer. Supervillin, myosin II, and myosin 1G remain bound to the bilayer after a high pH carbonate extraction, suggesting that these proteins are more proximal to the membrane than are the other DRM-H cytoskeletal proteins. Thus, the DRM-H fraction consists of an actin- and fodrin-based membrane skeleton that is associated with a subset of lipid raft signaling domains, possibly through interactions with supervillin and myosin. Similar raft-associated membrane skeletons may be present in many cell types. Supervillin is a constituent of total lipid rafts from Jurkat T cells (26von Haller P.D. Donohoe S. Goodlett D.R. Aebersold R. Watts J.D. Proteomics. 2001; 1: 1010-1021Crossref PubMed Google Scholar) and is purified from HEK293 cells as part of a dodecyl maltoside-insoluble complex that also includes integrin β2, α-actinin, β-actin, P2X7 ATP receptors, laminin α3, phosphatidylinositol 4-kinase, and receptor protein-tyrosine phosphatase-β (27Kim M. Jiang L.H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (336) Google Scholar). A muscle-specific isoform of supervillin, called archvillin, co-isolates with dystrophin and the raft-organizing protein, caveolin 3, in a low buoyant density fraction from skeletal muscle (28Oh S.W. Pope R.K. Smith K.P. Crowley J.L. Nebl T. Lawrence J.B. Luna E.J. J. Cell Sci. 2003; 116: 2261-2275Crossref PubMed Scopus (45) Google Scholar). Supervillin, so-named because of C-terminal similarities to the microvillar protein villin, binds directly and specifically to F-actin (29Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 30Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (105) Google Scholar) and localizes to sites of cell-cell and cell-substrate adhesion in epithelial cells (30Pestonjamasp K.N. Pope R.K. Wulfkuhle J.D. Luna E.J. J. Cell Biol. 1997; 139: 1255-1269Crossref PubMed Scopus (105) Google Scholar, 31Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar). Archvillin localizes at costameres, specialized adhesion sites in muscle (28Oh S.W. Pope R.K. Smith K.P. Crowley J.L. Nebl T. Lawrence J.B. Luna E.J. J. Cell Sci. 2003; 116: 2261-2275Crossref PubMed Scopus (45) Google Scholar). The shared N terminus of supervillin and archvillin is novel, lacking any similarity to the S1 domain in villin and gelsolin that is involved in actin filament severing activity (32Janmey P.A. Stossel T.P. Allen P.G. Chem. Biol. 1998; 5: R81-R85Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 33Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar, 34Way M. Gooch J. Pope B. Weeds A.G. J. Cell Biol. 1989; 109: 593-605Crossref PubMed Scopus (168) Google Scholar). Instead, the supervillin N terminus contains functional nuclear localization sequences and F-actin binding and bundling activities (31Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar). Overexpression of supervillin or its N terminus disrupts stress fibers and vinculin-containing focal adhesions (31Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar), suggesting a role in the regulation of cell-substratum interactions. Dysfunction caused by overexpression of supervillin is supported by the increased levels of this protein found in many carcinoma cell lines (35Pope R.K. Pestonjamasp K.N. Smith K.P. Wulfkuhle J.D. Strassel C.P. Lawrence J.B. Luna E.J. Genomics. 1998; 52: 342-351Crossref PubMed Scopus (40) Google Scholar) and by the demonstration that increased levels of supervillin can activate signaling through the androgen receptor (36Ting H.J. Yeh S. Nishimura K. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 661-666Crossref PubMed Scopus (86) Google Scholar). To understand better the role of supervillin at the membrane, we are mapping functional domains. Here we report the presence and localizations of a binding site for myosin II and three binding sites for filamentous actin within the supervillin N terminus. These F-actin-binding sites support filament bundling and cross-linking in vitro and thus can account for the observed aberrations in F-actin distributions documented in vivo (31Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar). The myosin II-binding site, which is also present in archvillin, selectively recognizes nonmuscle and smooth muscle myosin II isoforms, as opposed to skeletal muscle myosin II. This selectivity is consistent with the co-localization of archvillin with nonmuscle myosin II at the sarcolemma in differentiating and mature skeletal muscle (28Oh S.W. Pope R.K. Smith K.P. Crowley J.L. Nebl T. Lawrence J.B. Luna E.J. J. Cell Sci. 2003; 116: 2261-2275Crossref PubMed Scopus (45) Google Scholar). Overexpression of the myosin II-binding sequence disrupts the co-localization of nonmuscle myosin IIB with actin filament bundles in COS7 cells, suggesting a role for supervillin, and by extension, archvillin, in the organization of actin and myosin II at liquid-ordered membrane domains. Glutathione-Sepharose™, PreScission™ Protease, and DEAE-Sephacryl™ were purchased from Amersham Biosciences. Chicken gizzards were from Pel-Freez Biologicals (Rogers, AR). Chemical reagents were from Sigma, Calbiochem-Novabiochem, Fisher, or VWR International Inc. (Grove, IL). EGFP-Supervillin—Bovine supervillin cDNA (NCBI Nucleotide Database accession number AF025996) was used as a template to generate by PCR chimeric cDNAs from forward and reverse primers with 5′ restriction enzyme sites for cloning into pGEM-T (Promega Corp., Madison, WI), pGEM-T Easy (Promega), or pCR2.1-TOPO® (Invitrogen) TA vectors. Primer and/or vector cloning sites were used to transfer supervillin sequences into the pGEX-6P-1 vector (Amersham Biosciences) for expression as fusion proteins with glutathione S-transferase (GST) and into pEGFP vectors (Clontech, Palo Alto, CA) for mammalian cell expression (Table I). The construction of EGFP-SV-(1-830) has been described previously (31Wulfkuhle J.D. Donina I.E. Stark N.H. Pope R.K. Pestonjamasp K.N. Niswonger M.L. Luna E.J. J. Cell Sci. 1999; 112: 2125-2136Crossref PubMed Google Scholar).Table IPCR primers for EGFP-tagged bovine supervillin constructsConstructpEGFP vectorForward primerReverse primerSV-(1-174)N35′ - GAAGATCTTGAATGAAAAGAAAAGAAAGAATTGCCC - 3′5′ - GGAATTCAGCCCCGAGAGCTCAGTCCT - 3′SV-(1-342)C15′ - GAAGATCTTGAATGAAAAGAAAAGAAAGAATTGCCC - 3′5′ - GGAATTCCGGTCCTGGAGGCGC - 3′SV-(171-571)C15′ - GAAGATCTGAGCTCTCGGGGCTCAGG - 3′5′ - GGAATTCGCATAGATGGCCTCTTTATGGTTG - 3′SV-(171-342)C15′ - GAAGATCTGAGCTCTCGGGGCTCAGG - 3′5′ - GGAATTCGCATAGATGGCCTCTTTATGGTTG - 3′SV-(343-571)C15′ - GAAGATCTTCACACACGCAGCCCGTCACC - 3′5′ - GGAATTCATATCTTCCAGGGGTCTGGAAACT - 3′SV-(343-830)N25′ - GCACGGTACCGCATATGTCCCACACGCAGCCCGTCA - 3′5′ - TCCCCCCGGGAAGCTTCGATATCTTCCAGGGGTCTGGAAAC - 3′SV-(570-830)N25′ - GCACGGTACCGCATATGTATGCCCTTCCGAGGAAAGGAA G - 3′5′ - TCCCCCCGGGAAGCTTCGATATCTTCCAGGGGTCTGGAAAC - 3′ Open table in a new tab GST-Supervillin—Supervillin sequences in pEGFP-N3 and pEGFP-C1 vectors were transferred in-frame by ligation of inserts removed by digestion with BglII and EcoRI to pGEX-6P-1 cut with BamHI and EcoRI. Constructs in pEGFP-N2 (Table I) were transferred similarly from TA vectors to pGEX-6P-1 after re-PCR with primers containing appropriately situated 5′ BglII and EcoRI sites plus the gene-specific sequences shown in Table I. DNA Sequencing—All PCR products in TA vectors were verified by sequencing at the Iowa State University DNA Sequencing and Synthesis Facility (Ames, IA) or at the University of Massachusetts Nucleic Acid Facility (Worcester, MA). Expression constructs were checked by sequencing through the cloning sites. GST Fusions—GST fusion proteins were expressed after induction in BL21 cells and purified with glutathione-Sepharose™ (37Swaffield J.C. Johnston S.A. Susubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1996: 16.0.5-16.4.11Google Scholar). After cleavage of GST with PreScission™ Protease, the proteins were further purified by chromatography on DEAE-Sephacryl™ and elution with 0-0.2 m NaCl. The supervillin fragments were dialyzed against dialysis buffer (100 mm KCl, 2 mm MgCl2, 1 mm DTT in either 40 mm PIPES, pH 7.0, or 40 mm MOPS, pH 7.5). Dialyzed proteins were frozen quickly in liquid nitrogen and stored at -80 °C until use. Muscle Proteins—G-actin was prepared from an acetone powder of rabbit skeletal muscle (38Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). G-actin either was used directly in viscosity measurements or was column-purified (39MacLean-Fletcher S. Pollard T.D. Biochem. Biophys. Res. Commun. 1980; 96: 18-27Crossref PubMed Scopus (357) Google Scholar) for use in co-sedimentation assays and 125I-labeled F-actin blot overlays (40Chia C.P. Hitt A.L. Luna E.J. Cell Motil. 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Ikebe M. Am. J. Physiol. 2003; 284: C250-C262Crossref PubMed Scopus (18) Google Scholar) or with calcium phosphate-precipitated DNA (49Spector D.L. Goldman R.D. Leinwand L.A. Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 86.2-86.3Google Scholar). For analysis of EGFP-tagged supervillin sequences by F-actin blot overlay and anti-EGFP staining, transfected cells were washed with PBS (pH 7.4) and harvested from 10-cm plates using 0.5 ml/plate of M-PER® Mammalian Protein Extraction Reagent, 3 μm pepstatin, 2 μm leupeptin, 0.2 μg/ml phenylmethylsulfonyl fluoride (PMSF), as described by the manufacturer (Pierce). Proteins were denatured by heating for 5 min at 95 °C in 2× SDS sample buffer and separated on SDS-polyacrylamide gels (50Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar). Protein concentrations were determined by BCA Protein Assays™ (Pierce). Gels were stained for protein with Coomassie Blue or electrotransferred to nitrocellulose (0.45-μm pore size; Schleicher & Schuell) for immunoblot analyses. Nitrocellulose blots were blocked with 5% nonfat powdered milk and probed with primary antibodies for 2 h at room temperature or overnight at 4 °C. Primary antibodies used in this study were diluted as follows: affinity-purified anti-supervillin (α-H340 (25Nebl T. Pestonjamasp K.N. Leszyk J.D. Crowley J.L. Oh S.W. Luna E.J. J. Biol. Chem. 2002; 277: 43399-43409Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar)), ∼1 μg/ml; a murine monoclonal antibody MMS-456S against most forms of myosin II (Babco-Covance, Richmond, CA), 1:1000; a murine monoclonal antibody 5F9F5 against all actin isoforms (Novus Biologicals, Littleton, CO), 1:10; and a murine monoclonal antibody FIL-2 against filamin (Sigma), 1:1000. Interacting antibodies were visualized using anti-mouse IgG, mouse IgM, or rabbit IgG antibodies conjugated to horseradish peroxidase, an ECL substrate kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and Biomax-MS x-ray film (Eastman Kodak Co.). For double labeling with radioactively labeled F-actin, anti-rabbit antibody conjugated to alkaline phosphatase was used with a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate kit (Kirkegaard & Perry Laboratories) for colorimetric detection. F-actin—125I-Labeled actin was prepared, polymerized in the presence of rabbit gelsolin, stabilized with phalloidin, and used at a final concentration of 50 μg/ml in 5% nonfat powered milk (41Luna E.J. Methods Enzymol. 1998; 298: 32-42Crossref PubMed Scopus (12) Google Scholar). In some experiments, actin was labeled with [α-32P]ATP (51Mackay D.J.G. Esch F. Furthmayr H. Hall A. J. Cell Biol. 1997; 138: 927-938Crossref PubMed Scopus (269) Google Scholar), using 1 mg of actin and 1 mCi of [α-32P]ATP. Nitrocellulose blots were exposed to film, or the signal was visualized with a Phosphor Imager SI™ optical scanner and ImageQuant software (Amersham Biosciences). 35S-SV-(1-174)—In vitro transcription and translation of SV-(1-174) was carried out with the TnT T7-coupled reticulocyte lysate system (Promega Corp.) in the presence of ∼20 μCi of [35S]methionine (PerkinElmer Life Sciences). 35S-Labeled SV-(1-174) was stored in 50-μl aliquots at -80 °C until use. For blot overlays with 35S-labeled SV-(1-174), proteins resolved on 8% SDS-PAGE gels were electrotransferred to nitrocellulose and blocked for 1 h at room temperature or overnight at 4 °C with 5% nonfat milk and 0.05% Nonidet P-40 in hybridization buffer (25 mm HEPES-KOH, pH 7.7, 25 mm NaCl, 5.0 mm MgCl2, 1 mm DTT) (52Cavaillès V. Dauvois S. Danielian P.S. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10009-10013Crossref PubMed Scopus (340) Google Scholar). Bound proteins were denatured and renatured by a series of 10-min incubations at room temperature in hybridization buffer, 0.05% Nonidet P-40 plus the following concentrations of guanidine hydrochloride: 6, 6, 3, 1.5, 0.75, 0.375, and 0.187 m. After two additional 10-min washes with hybridization buffer, nonspecific sites on the filter were blocked for 1 h at room temperature with 5% milk, 0.05% Nonidet P-40, hybridization buffer and for another hour at room temperature with 1% milk, 0.05% Nonidet P-40, 1 mm methionine, hybridization buffer. Filters were incubated with 35S-labeled SV-(1-174) (25-50 μl) for 2.5 h at room temperature in 1 ml of H Buffer (20 mm HEPES-KOH, pH 7.7, 75 mm KCl, 0.1 mm EDTA, 2.5 mm MgCl2, 1 mm DTT, 0.05% Nonidet P-40, 1 mm methionine). Filters were washed five times for 1-2 min per wash with H Buffer and then exposed to film or a PhosphorImager screen. Proteins specifically bound to GST-SV-(1-174) were identified from tryptic digests of Coomassie Blue-stained polypeptides (53Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (845) Google Scholar) by Dr. John D. Leszyk, Proteomic Mass Spectrometry Laboratory of the University of Massachusetts Medical School (Shrewsbury, MA). PSD fragment ions were fitted to a generated curve, which was calibrated with PSD fragments from the synthetic peptide, P14R. Data base searches were performed using Protein Prospector (prospector.ucsf.edu). Average peptide masses were searched against the NCBI nonredundant data base using the MS-Fit program and 250-500 ppm mass tolerances with 1 or 2 missed cleavages. PSD fragments were searched against the NCBI nonredundant data base using MS-Tag and 0.5 Da as the parent tolerance and 1.0 Da as the fragment tolerance. Co-sedimentation binding assays were performed either by adding supervillin fragments cleaved from purified GST fusion proteins to pre-polymerized F-actin (Fig. 2A) or by co-polymerizing actin with GST fusion proteins (Fig. 2C). All proteins and G-actin in Buffer A were clarified by centrifugation at 250,000 × g for 21 min at 4 °C immediately before use. In co-polymerization experiments, G-actin, GST, or GST fusion proteins were mixed on ice and co-assembled in a final volume of 250 μl of an actin polymerization buffer (100 mm KCl, 5 mm MgCl2, 1 mm ATP, 0.5 mm EGTA, 10 mm Tris-HCl, pH 8.0). The final concentrations of actin, GST fusion proteins, and GST were 2.3, ∼2, and 3.5 μm, respectively. Assay mixtures were incubated on ice for 1 h and then for 1 h at 18-20 °C. Actin filaments in 150 μl of each assay mixture were centrifuged as above through 50 μl of 10% sucrose in actin polymerizing buffer. Supernatants were collected as the top 100 μl in each tube, the rest of the liquid was carefully removed, and pellets were resuspended to 150 μl with Buffer A. Equal volumes (30 μl) of supernatant and pellet fractions were loaded onto SDS-polyacrylamide gels and immunoblotted with anti-GST antibody or stained with Coomassie Blue. Co-sedimentation experiments with pre-polymerized F-actin (1 mg/ml) and cleaved supervillin fragments (0.18 mg/ml) were performed similarly, except that the polymerization buffer contained 10 mm MOPS, pH 7.5, instead of Tris-HCl. Actin (1.25 mg/ml) was pre-polymerized for 30 min at 28 °C before addition of the supervillin fragments and continued incubation for 10 min at 28 °C. Actin viscosity was measured using a low shear falling ball viscometer (54Fowler V. Taylor D.L. J. Cell Biol. 1980; 85: 361-376Crossref PubMed Scopus (126) Google Scholar, 55Fowler V.M. Luna E.J. Hargreaves W.R. Taylor D.L. Branton D. J. Cell Biol. 1981; 88: 388-395Crossref PubMed Scopus (33) Google Scholar, 56Griffith L.M. Pollard T.D. J. Cell Biol. 1978; 78: 958-965Crossref PubMed Scopus (249) Google Scholar). Varyin
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