Furukawa Ruth, Maselli Andrew, Thomson Susanne A M, Lim Rita W L, Stokes John V, Fechheimer Marcus
Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Cell Sci. 2003 Jan 1;116(Pt 1):187-96. doi: 10.1242/jcs.00220.
The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.
肌动蛋白细胞骨架对钙的变化敏感，钙会影响收缩性、肌动蛋白切割蛋白、肌动蛋白交联蛋白和钙调蛋白调节的酶。为了剖析钙调控对单个蛋白质活性的作用，以区别于钙对其他过程的影响，制备了这些蛋白质的钙不敏感形式，并将其导入活细胞中，以取代同一蛋白质的钙敏感形式。在存在微摩尔游离钙的情况下，盘基网柄菌34 kDa蛋白对肌动蛋白丝的交联和成束作用受到抑制。利用定点诱变制备了钙结合EF手结构域发生突变的34 kDa蛋白的修饰形式（34 kDa deltaEF2），并在大肠杆菌中表达。使用[（45）Ca]CaCl2进行平衡透析表明，野生型蛋白能够结合一个钙离子，解离常数为2.4 microM。这种钙结合在34 kDa deltaEF2蛋白中不存在。34 kDa deltaEF2蛋白的肌动蛋白结合活性与野生型相当，但在体外对钙不敏感。野生型和34 kDa deltaEF2蛋白在34 kDa基因缺失和34 kDa/α辅肌动蛋白双基因缺失的盘基网柄菌突变株中表达，以检验肌动蛋白交联的钙调节在体内很重要这一假设。34 kDa deltaEF2未能为这两种34 kDa基因缺失菌株提供对控制细胞大小和正常生长很重要的34 kDa蛋白的功能。此外，在表达34 kDa deltaEF2的细胞中，34 kDa蛋白和肌动蛋白的分布异常。因此，对于对细胞结构和生长很重要的肌动蛋白细胞骨架的动态行为而言，交联肌动蛋白结构的形成和/或溶解的钙调节是必需的。
