Schwartz M A, Luna E J
Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1988 Jul;107(1):201-9. doi: 10.1083/jcb.107.1.201.
We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.
我们之前已经表明（施瓦茨，M. A.，和E. J. 卢纳。1986年。《细胞生物学杂志》102卷：2067 - 2075页），肌动蛋白以正协同性与盘基网柄菌的质膜结合。即使在远低于溶液中聚合临界浓度的情况下，肌动蛋白也会在膜表面聚合。能阻止溶液中肌动蛋白聚合的低盐缓冲液也会阻止肌动蛋白与膜结合。为了进一步探究肌动蛋白聚合与膜结合之间的关系，我们制备了四种化学修饰的肌动蛋白，它们在溶液中似乎无法聚合。其中三种衍生物也失去了与膜结合的能力。第四种衍生物（EF肌动蛋白），其组氨酸 - 40用乙氧基甲酸酐标记，与膜的结合亲和力降低。结合曲线呈现正协同性，交联实验表明膜结合的肌动蛋白是多聚体。因此，结合和聚合紧密耦合，并且这些膜聚合肌动蛋白的能力得到了显著证明。EF肌动蛋白在溶液中与未处理的肌动蛋白弱共组装，但在膜上共组装良好。未处理的肌动蛋白和EF肌动蛋白的结合相互竞争，表明它们结合到相同的膜位点。希尔图表明肌动蛋白三聚体是紧密结合到膜所需的最小组装状态。对我们数据的最佳解释是一个模型，其中肌动蛋白寡聚体通过与聚集的膜位点结合而组装，肌动蛋白丝一侧的连续单体与膜结合。由于多价性，预计单个结合亲和力较低，但肌动蛋白 - 膜的总亲和力较高。我们的结果表明，使膜蛋白聚集的细胞外因子可能会为肌动蛋白核的形成创造位点，从而触发细胞内的肌动蛋白聚合。
