Vinculin Nucleates Actin Polymerization and Modifies Actin Filament Structure
2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês
10.1074/jbc.m109.021295
ISSN1083-351X
AutoresKuo‐Kuang Wen, Peter A. Rubenstein, Kris A. DeMali,
Tópico(s)Advanced Fluorescence Microscopy Techniques
ResumoVinculin links integrins to the actin cytoskeleton by binding F-actin. Little is known with respect to how this interaction occurs or affects actin dynamics. Here we assess the consequence of the vinculin tail (VT) on actin dynamics by examining its binding to monomeric and filamentous yeast actins. VT causes pyrene-labeled G-actin to polymerize in low ionic strength buffer (G-buffer), conditions that normally do not promote actin polymerization. Analysis by electron microscopy shows that, under these conditions, the filaments form small bundles at low VT concentrations, which gradually increase in size until saturation occurs at a ratio of 2 VT:1 actin. Addition of VT to pyrene-labeled mutant yeast G-actin (S265C) produced a fluorescence excimer band, which requires a relatively normal filament geometry. In higher ionic strength polymerization-promoting F-buffer, substoichiometric amounts of VT accelerate the polymerization of pyrene-labeled WT actin. However, the amplitude of the pyrene fluorescence caused by actin polymerization is quenched as the VT concentration increases without an effect on net actin polymerization as determined by centrifugation assays. Finally, addition of VT to preformed pyrene-labeled S265C F-actin causes a concentration-dependent decrease in the maximum amplitude of the pyrene fluorescence band demonstrating the ability of VT to remodel the conformation of the actin filament. These observations support the idea that vinculin can link adhesion plaques to the cytoskeleton by initiating the formation of bundled actin filaments or by remodeling existing filaments. Vinculin links integrins to the actin cytoskeleton by binding F-actin. Little is known with respect to how this interaction occurs or affects actin dynamics. Here we assess the consequence of the vinculin tail (VT) on actin dynamics by examining its binding to monomeric and filamentous yeast actins. VT causes pyrene-labeled G-actin to polymerize in low ionic strength buffer (G-buffer), conditions that normally do not promote actin polymerization. Analysis by electron microscopy shows that, under these conditions, the filaments form small bundles at low VT concentrations, which gradually increase in size until saturation occurs at a ratio of 2 VT:1 actin. Addition of VT to pyrene-labeled mutant yeast G-actin (S265C) produced a fluorescence excimer band, which requires a relatively normal filament geometry. In higher ionic strength polymerization-promoting F-buffer, substoichiometric amounts of VT accelerate the polymerization of pyrene-labeled WT actin. However, the amplitude of the pyrene fluorescence caused by actin polymerization is quenched as the VT concentration increases without an effect on net actin polymerization as determined by centrifugation assays. Finally, addition of VT to preformed pyrene-labeled S265C F-actin causes a concentration-dependent decrease in the maximum amplitude of the pyrene fluorescence band demonstrating the ability of VT to remodel the conformation of the actin filament. These observations support the idea that vinculin can link adhesion plaques to the cytoskeleton by initiating the formation of bundled actin filaments or by remodeling existing filaments. Cell migration is critical for embryonic development, adult homeostasis, inflammatory responses, and wound healing. To migrate, a cell must coordinate a number of different inputs into appropriate cellular responses. The cell must polarize in the direction of migration and extend lamellipodial and/or filopodial protrusions. Nascent adhesions that assemble within the branched actin network of the lamellipodium must link to the underlying actin cytoskeleton. This process allows for the maturation of adhesions to structures that anchor the protrusion. These adhesions also provide the traction forces necessary to pull the cell body forward and break older adhesions at the cell rear. Perturbation of any of these events affects a cell's migratory ability. For example, nascent adhesions that do not form linkages to the actin cytoskeleton cannot effectively anchor the protrusion to the substratum. The result is an extension that folds back upon itself, forming a membrane ruffle that cannot provide the traction forces necessary for migration. How adhesions establish links to the underlying actin cytoskeleton has been an area of intense investigation. Integrin-containing structures are active areas of actin polymerization suggesting that adhesion plaques can initiate actin filament formation (reviewed in Refs. 1DeMali K.A. Burridge K. J. Cell Sci. 2003; 116: 2389-2397Crossref PubMed Scopus (153) Google Scholar, 2Galbraith C.G. Yamada K.M. Galbraith J.A. 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Hence, many of the known mechanisms for initiating filament formation involve recruitment of the Arp2/3 complex, which initiates the formation of branched actin filaments (55Mullins R.D. Stafford W.F. Pollard T.D. J. Cell Biol. 1997; 136: 331-343Crossref PubMed Scopus (195) Google Scholar). It is surprising then that the earliest detectable forms of actin-associated adhesions are interconnected by short actin bundles, not branched filaments (10Zimerman B. Volberg T. Geiger B. Cell Motil. Cytoskeleton. 2004; 58: 143-159Crossref PubMed Scopus (138) Google Scholar). These observations suggest that our current understanding for how nascent adhesions initiate filament formation is incomplete. The earliest detectable actin-associated adhesions are "dots or doublets of dots" and are highly enriched in integrins, paxillin, and vinculin (10Zimerman B. Volberg T. Geiger B. Cell Motil. Cytoskeleton. 2004; 58: 143-159Crossref PubMed Scopus (138) Google Scholar), suggesting that one of these molecules has the capability to initiate actin filament formation from such a plaque. Vinculin has long been implicated in linking adhesion plaques to the actin cytoskeleton by virtue of the ability of its tail to bind (11Johnson R.P. Craig S.W. Nature. 1995; 373: 261-264Crossref PubMed Scopus (321) Google Scholar) and bundle F-actin (12Jockusch B.M. Isenberg G. Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 3005-3009Crossref PubMed Scopus (167) Google Scholar). The interaction of vinculin with actin has been extensively studied from the perspective of vinculin (11Johnson R.P. Craig S.W. Nature. 1995; 373: 261-264Crossref PubMed Scopus (321) Google Scholar, 13Pardo J.V. Siliciano J.D. Craig S.W. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 1008-1012Crossref PubMed Scopus (332) Google Scholar, 14Pardo J.V. Siliciano J.D. Craig S.W. J. 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Biochem. 1997; 247: 1136-1142Crossref PubMed Scopus (88) Google Scholar, 22Menkel A.R. Kroemker M. Bubeck P. Ronsiek M. Nikolai G. Jockusch B.M. J. Cell Biol. 1994; 126: 1231-1240Crossref PubMed Scopus (141) Google Scholar, 23Goldmann W.H. Guttenberg Z. Tang J.X. Kroy K. Isenberg G. Ezzell R.M. Eur. J. Biochem. 1998; 254: 413-419Crossref PubMed Scopus (20) Google Scholar). Studies of recombinant proteins identified two regions of the vinculin tail (VT) 2The abbreviations used are: VTvinculin tailDTTdithiothreitolGSTglutathione S-transferase. that bind F-actin independently (21Hüttelmaier S. Bubeck P. Rüdiger M. Jockusch B.M. Eur. J. Biochem. 1997; 247: 1136-1142Crossref PubMed Scopus (88) Google Scholar, 17Janssen M.E. Kim E. Liu H. Fujimoto L.M. Bobkov A. Volkmann N. Hanein D. Mol. Cell. 2006; 21: 271-281Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), but mapping these sites onto the VT crystal structure reveals that these peptides do not correspond to distinct sites (25Bakolitsa C. de Pereda J.M. Bagshaw C.R. Critchley D.R. Liddington R.C. Cell. 1999; 99: 603-613Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Upon binding actin, vinculin undergoes a conformational change that promotes dimerization suggesting that vinculin self-association may be important for its bundling activities (15Johnson R.P. Craig S.W. J. Biol. Chem. 2000; 275: 95-105Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). vinculin tail dithiothreitol glutathione S-transferase. Less is known with respect to the effect of vinculin on actin filament formation and structure. This lack of knowledge stems from the fact that many of the early studies showed vinculin to have no effect on actin dynamics (26Rosenfeld G.C. Hou D.C. Dingus J. 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However, the interaction of vinculin with G-actin and the effect of vinculin on actin filament dynamics have not been explored. In this study, we have assessed the interaction of vinculin with pyrene-labeled wild-type and mutant yeast actins. We show that the VT can promote the formation of an actin nucleus from which filaments arise and alter the assembly and structure of actin filaments. These findings provide novel insights into how adhesion plaques may be linked to the actin cytoskeleton. The actins were purified from the lysates of frozen yeast cells by a combination of DNase I affinity chromatography, DE52 anion-exchange chromatography, and polymerization/depolymerization cycling as described previously (30Cook R.K. Root D. Miller C. Reisler E. Rubenstein P.A. J. Biol. Chem. 1993; 268: 2410-2415Abstract Full Text PDF PubMed Google Scholar). Wild-type actin was obtained from yeast cakes purchased at a local market, whereas S265C actin was obtained from cells we had previously generated carrying the mutant actin (31Feng L. Kim E. Lee W.L. Miller C.J. Kuang B. Reisler E. Rubenstein P.A. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Covalent attachment of N-(1-pyrenyl)maleimide (Sigma) with actins was performed as described in Feng et al. (31Feng L. Kim E. Lee W.L. Miller C.J. Kuang B. Reisler E. Rubenstein P.A. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To conjugate actin at Cys-374 with Oregon Green 488 maleimide or N′-(3-maleimidypropionyl)biocytin, the maleimide derivatives were resuspended in DMSO and incubated with yeast F-actin at a molar ratio of 3:1 and 5:1, respectively, for 3 h at room temperature. The F-actin was separated from the reactants by centrifugation, and the resulting pellet was resuspended in G-buffer. Rabbit skeletal muscle actin was purified from acetone powder as previously described (32Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Purified unmodified and modified actins were stored as the Ca2+-bound form in calcium G-buffer (10 mm Tris-HCl, pH 7.5, 0.2 mm CaCl2, 0.2 mm ATP, and 1 mm DTT) at 4 °C and used within 4 days of purification. Escherichia coli carrying the plasmid pGEX-4T1, which encodes amino acids 881–1066 of chick vinculin, was obtained from David Critchley (University of Leicester). For expression of this vinculin fragment, known as the vinculin tail (VT), the cells were grown to early log phase and induced for 1 h. at 37 °C with 1 mm IPTG. Bacteria were then suspended in lysis buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm DTT) plus the same set of protease inhibitors used for actin purification (30Cook R.K. Root D. Miller C. Reisler E. Rubenstein P.A. J. Biol. Chem. 1993; 268: 2410-2415Abstract Full Text PDF PubMed Google Scholar). Lysozyme (Sigma) and DNase I (Worthington) at final concentrations of 0.2 μg/ml and 0.02 μg/ml, respectively, were added to the cell suspension, which was then incubated for 10 min at room temperature with agitation. Following sonication of the cells, the insoluble debris was removed by centrifugation for 40 min at 4 °C in a Ti60 rotor at 40,000 rpm in a Beckman ultracentrifuge. The supernatant solution was filtered through four layers of gauze and passed over a GST-Sepharose column equilibrated with lysis buffer. The column was washed with HTND buffer (10 mm Tris-HCl, pH 8, 500 mm NaCl, and 1 mm DTT) followed by washes with LTND substituting 25 mm NaCl for 500 mm NaCl. The GST-VT was eluted from the column with LTND containing 10 mm reduced glutathione (Sigma), and the GST was cleaved from the GST-VT with 40 units of human thrombin (Calbiochem) for 1 h at room temperature. The VT peptide, 881–1066, was separated from the GST and thrombin by passing the solution over a DE52 DEAE column equilibrated with 10 mm Tris-HCl, pH 8.0, and 25 mm NaCl and collecting the flow-through fractions. Protein concentration was determined by measuring the A280 (ϵ = 18,470 m−1cm−1). Either pyrene-labeled or unlabeled yeast calcium G-actin in Ca-G-buffer was converted to the magnesium form 2 min prior to use by dilution of the concentrated stock actin at least 10-fold into Mg2+-G-buffer (10 mm Tris-HCl, pH 7.5, 0.2 mm MgCl2, 0.2 mm ATP, 0.1 mm EGTA, and 1 mm DTT). For assessing the actin-VT interaction in Mg2+ G-buffer, the actin was diluted with G-buffer to a concentration of 1 μm, and the baseline fluorescence was determined. VT was added, and the resulting change in fluorescence was followed until a steady state was obtained. For all assays, the excitation wavelength was 365 nm. For WT actin, single wavelength kinetic assays were carried out with an emission wavelength of 386 nm. For studies involving S265C actin, emission spectra were recorded between 375 and 550 nm. For VT-actin co-polymerization assays, VT was added to 1 μm G-actin, labeled or unlabeled in Mg2+ G-buffer. For pyrene assays, the increase in fluorescence was allowed to reach steady state after which 2 mm MgCl2 and 50 mm KCl were added to induce actin polymerization. The subsequent change in fluorescence was recorded as a function of time. All fluorescence assays were performed in a final volume of 120 μl in a microcuvette housed in a thermostatted sample compartment of either a Fluorolog-3 or FluoroMax-3 spectrometer (Jobin Yvon-Spex). To quantitate the amount of actin filaments or bundles formed and the amount of actin-bound VT, the appropriate concentration of VT was combined with 1 μm Mg2+ G-actin in Mg2+ buffer as above. The sample, either before or after addition of MgCl2 and KCl to induce polymerization, was subjected to centrifugation in a TLA.1 rotor at 25 °C using a Beckman TL-100 centrifuge. To assess filament formation, centrifugation was for 15 min at 80,000 rpm. For assessment of bundle formation, centrifugation was at 20,000 rpm for 30 min. The supernatant fractions were carefully removed, and the pellets were resuspended in 30 μl of distilled water. The entire pellet and one-fourth of the supernatant fractions were resolved by SDS-PAGE using 12% acrylamide gels. The protein bands were visualized by Coomassie Blue staining, and the density of the bands was quantitated using an Epson Perfection 2450 photo scanner and ImageJ software (National Institutes of Health) ensuring that the intensities of the bands were within the linear response range of the instrument. Band densities for actin and VT were normalized based on their relative molecular weights, and the resulting data were utilized to calculate VT/actin ratios in the pellet fractions. To visualize actin filaments and filament bundles, a 3-μl sample from an actin-VT solution was deposited on a carbon-coated Formvar grid (400 mesh) and negatively stained with 1% uranyl acetate. The sample was then observed using a JEOL 1230 transmission electron microscope housed in the University of Iowa Central Microscopy Facility. Two types of experiments were performed to examine the actin-VT interaction. In one, the increase in pyrene fluorescence following VT addition was followed as a function of time. In the second, VT-induced actin bundling was followed as a function of the amount of material pelleted in a co-sedimentation assay as described above. For the G-actin co-sedimentation study, we utilized two different types of data from the same experiment. One was the percentage of total actin pelleted (Fig. 3A), and the other the molar ratio of VT/actin in the pelleted material (Fig. 3B). We then generated Fig. 3C, a graph of VT-bound versus VT total using the Equation 1, where T = total. [VT]actin-bound=[VT]precipitated[actin]precipitated×[actin]precipitated[actin]T×[actin]T(Eq. 1) The data in Fig. 3C were then fitted to a quadric binding equation (Equation 2) in which the value of F was set at 1. [VT]bound=F×([actin]T+[VT]T+Kd)-([actin]T+[VT]T+Kd)-4×[actin]T×[VT]T2(Eq. 2) In this analysis, to obtain a good fit, we needed to assume that each actin monomer contains two independent VT binding sites. Thus, the total actin concentration shown is the concentration of sites rather than the actual actin concentration. Kd values were then obtained as previously described (33Wen K.K. McKane M. Houtman J.C. Rubenstein P.A. J. Biol. Chem. 2008; 283: 9444-9453Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For the pyrene assays we used the same basic quadratic equation to analyze the increase in fluorescence over time. However, we multiplied the right side of Equation 2 by the total change in fluorescence for each VT concentration used (represented by F). For the quenching-based assay in Fig. 9, we used the same quadratic equation but, instead of net fluorescence change, we used the value of 1 − F. The flow cell was essentially prepared as described by Kuhn and Pollard (34Kuhn J.R. Pollard T.D. Biophys. J. 2005; 88: 1387-1402Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Cells were coated with avidin (NeutrAvidin, Invitrogen) by allowing 15 μl of 50 nm avidin dissolved in Tris-buffered saline (50 mm Tris-HCl, pH 7.5, and 150 mm NaCl) to enter the cell via capillary action. The loaded cell was incubated in a humid chamber for at least 30 min at room temperature, and the excess avidin was removed by washing the cell with Tris-buffered saline with 1% bovine serum albumin and then TIRF buffer (10 mm imidazole, pH 7, 50 mm KCl, 1 mm MgCl2, 1 mm EGTA, 100 mm DTT, 0.2 mm ATP, 15 μm glucose, 0.5% methylcellulose, 20 μg/ml catalase, 100 μg of glucose oxidase). Calcium-actin mixtures containing 52% unlabeled actin, 33% Oregon Green 488 maleimide-actin, and 15% N′-(3-maleimidypropionyl)biocytin-actin were converted to Mg-actin as described above. For monitoring actin polymerization, the Mg-actin mixture was diluted to a final concentration of 2 μm in TIRF buffer. In a separate tube, VT was diluted to 5.0 μm in a modified TIRF buffer containing 0.2 mm MgCl2. The two samples were combined resulting in a mixture containing 1.0 μm actin and 2.5 μm VT. A 10-μl aliquot was immediately loaded into the cell by capillary action, and the cell was mounted on a Leica AM TIRF MC imaging system. Images were acquired every 10 s and analyzed using the ImageJ software package. Migration of many vertebrate cells requires both actin polymerization and adhesion to the substratum. The earliest detectable actin-associated adhesions are vinculin-rich and interconnected by short actin bundles (10Zimerman B. Volberg T. Geiger B. Cell Motil. Cytoskeleton. 2004; 58: 143-159Crossref PubMed Scopus (138) Google Scholar). This observation suggests that vinculin, itself, may trigger bundle formation. We thus wished to assess the effect of vinculin on actin monomer and filament dynamics using the vinculin tail, VT, as a probe because it contains the actin-binding sites for the whole protein. We first determined if VT would bind to the actin monomer. For this work, we converted G-actin to the expected Mg2+ form, the predominant species found in the cell and labeled the active -SH of Cys-374 with pyrene-maleimide. If the two proteins interact, and this interaction alters the environment of the pyrene, its fluorescence properties will change providing an assay for the interaction. We thus combined VT with Mg-G-actin in a low ionic strength solution that will prevent polymerization of pure actin (Mg2+ G-buffer). Fig. 1A shows that this combination resulted in a biphasic time-dependent change in fluorescence consisting of a rapid first phase increase and a slower subsequent phase until a new steady state was reached after ∼20 min. Fig. 1B shows that this VT-induced change was saturable with respect to the amount of VT added to a constant amount of actin. The biphasic fluorescence increase observed in Fig. 1A suggested that, following an initial actin-VT interaction, some subsequent assembly step, possibly polymerization, might be occurring despite the fact that the proteins were combined in G-buffer. The polymerization-dependent increase in fluorescence of a pyrene at Cys-374 is, in fact, a commonly used assay for actin polymerization. To assess this possibility, aliquots of the VT-actin solutions at saturating VT concentrations were removed after the fluorescence increase had reached a steady state and visualized by electron microscopy after negative staining. Fig. 2 (B and C) shows the appearance of actin filament bundles that require VT to form (Fig. 2A). No evidence of bundle or filament formation was observed when we used actin with calcium rather than magnesium-bound to the high affinity divalent cation binding site (data not shown). We next wished to determine the dependence of actin filament formation and bundling in G-buffer on VT concentration. Increasing amounts of VT were added to a fixed amount of actin, and the mixtures were then centrifuged at speeds sufficient to pellet actin bundles but not individual filaments. Aliquots of the pellet and supernatant fractions were analyzed by SDS-PAGE, and the Coomassie Blue-stained bands were quantitated by densitometry. Supplemental Fig. S1 shows an increasing appearance of VT and actin in the pellet fraction as a function of added VT. Fig. 3A shows that the amount of pelleted actin reaches a plateau at a VT concentration somewhat lower than what is required to achieve the maximal change in pyrene fluorescence. This difference is not the result of the pyrene label on actin, as 100 and 5% pyrene-labeled actin behaved similarly in a co-sedimentation assay (data not shown). Rather, the difference reflects two different processes that are being measured. In the fluorescence assay, the maximum fluorescence change is obtained at the maximum exposure of the probed actin to VT (saturation). On the other hand, bundling sufficient to pellet all the actin can be achieved with a number of filament cross-links less than that required for saturable binding of VT. To determine how much of the polymerized actin induced by VT was bundled, we repeated the co-sedimentation assay at higher speeds that will quantitatively pellet unbundled actin filaments. The resulting curve (data not shown) is virtually identical to that obtained at lower speeds indicating that bundling occurs early in the polymerization process. Together, these data indicate that small amounts of VT cannot cause a propagated cooperative effect resulting in large amounts of actin polymerization. For a number of actin-bundling proteins, large amounts of bundling can be achieved by the formation of a small number of filament-filament cross-links (35Esue O. Harris E.S. Higgs H.N. Wirtz D. J. Mol. Biol. 2008; 384: 324-334Crossref PubMed Scopus (43) Google Scholar, 36Xu J. Tseng Y. Wirtz D. J. Biol. Chem. 2000; 275: 35886-35892Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 37Tseng Y. An K.M. Esue O. Wirtz D. J. Biol. Chem. 2004; 279: 1819-1826Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 38Tseng Y. Fedorov E. McCaffery J.M. Almo S.C. Wirtz D. J. Mol. Biol. 2001; 310: 351-366Crossref PubMed Scopus (114) Google Scholar, 39Bretscher A. Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 6849-6853Crossref PubMed Scopus (132) Google Scholar). To determine if such were the case with our VT-G-actin interaction, we determined the molar ratio of VT/actin in the pellet at each VT concentration. Fig. 3B shows that, at the lowest VT concentration, the ratio was almost equimolar and that it increased to a ratio of 2:1 at saturation. We used the information obtained in Fig. 3 (A and B) to generate a graph of VT-bound versus VT total (Fig. 3C). This analysis reveals that maximal binding of VT to 1.0 μm actin occurs at a VT concentration of ∼5–7.5 μm. Although we observed VT-dependent actin filament formation in G-buffer, the ionic strength difference between it and F-buffer (G-buffer plus 2 mm MgCl2 and 50 mm KCl) may have resulted in an aberrant filament structure. To examine this possibility, we utilized a mutant yeast actin S265C that we had previously characterized (31Feng L. Kim E. Lee W.L. Miller C.J. Kuang B. Reisler E. Rubenstein P.A. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This residue is at the tip of a hydrophobic loop between actin subdomains 3 and 4. Pyrene labeling of this actin results in the incorporation of 2 mol of probe per mol of actin, one at Cys-374 and one at Cys-265. Excitation of F-actin made with this doubly labeled protein results in the appearance of a new pyrene excimer band centered at 485 nm. This band results from the interaction of a pyrene at residue 265 of one monomer with that at Cys-374 of another monomer in the apposing strand of the filament and is observed only in the context of F-actin. Fig. 4A shows that introduction of VT into a solution of pyrene-labeled S265C Mg-G-actin in G-buffer results in the formation of the pyrene excimer band. Fig. 4B shows that the amplitude of this band depends on the VT/actin ratio and reaches a plateau at 5–7.5 μm. This concentration is near that required to reach a plateau of the fluorescence in the pyrene-labeled WT actin studies in Fig. 1B (10 μm). This result thus indicates that the filaments formed in G-buffer, described above, have very close to if not the same structure as those formed by pure actin alone in F-buffer and shows that the formation of this excimer depends on substantial binding of VT along the filament surface. We determined what effect VT would have on Mg-actin polymerization in F-buffer, which much more closely approximates the ionic strength conditions present in the cell. We thus examined the effect of preincubation of increasing VT concentrations on the polymerization kinetics of a f
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