ADF/COFILIN IN EARLY VERTEBRATE DEVELOPMENT

描述：ADF是由Bamburg在他职业生涯开始时发现的， 他一直专注于此，几乎完全是如此。ADF是一种小单体， 是一类肌动蛋白结合蛋白的典型代表，具有奇怪的 肌动蛋白结合活性包括切断肌动蛋白细丝和结合 肌动蛋白单体。这些蛋白质存在于所有真核生物中，并且 在所有几个研究病例中都是必不可少的，包括酵母和 网柄网柄菌属。细胞中含有大量的它们，摩尔比为 与肌动蛋白的比例约为1：10。它们受磷脂酰肌醇、pH的调节 和磷酸化。最值得注意的是，班伯格发现磷酸化 在体外调节ADF的肌动蛋白结合活性， 在发育中的肌肉细胞中，磷酸化以一种从 在那里发生的肌动蛋白组装。因此，ADF是最好的 对调节肌动蛋白组装的蛋白质的调节。班堡有 鉴定了磷酸化位点为丝氨酸，并准备了替代 构成地模仿开和关状态的突变体(Glu和Ala， )。 在这项应用的前期工作中，Bamburg分离和分析了 非洲爪蛙ADF的cDNAs和Abs及其重组ADF的制备 在体外具有完全的功能。他发现人类社会的发展变化 控制肌动蛋白结合活性的磷酸化。AB本地化 研究表明，ADF与丝状肌动蛋白位于相同的位置。目标1是 确定ADF在肌动蛋白重组过程中是否重要 卵子发生。 目的2是确定激活ADF的磷酸酶的性质 在受精后对其进行脱磷。战略将是增加 位于上游的各种第二信使的集中 单独的磷酸酶。此外，磷酸酶也将被提纯 生物化学地从受精卵中提取。 目的3是确定ADF是否在肌动蛋白重组事件中起重要作用 在受精后，使用抑制性抗体和活性或 不活跃的ADF突变体。 目标4是确定ADF在细胞质分裂过程中如何影响肌动蛋白细丝， 在新的初步数据中发现了这一点。方法将是相似的 在《目标3》中。 目标5是确定小G蛋白Rho、rac1和CDc42是否 对于皮质旋转和胞质分裂很重要，如果是，如果它们的影响 是通过ADF介导的。再一次，占主导地位的负面的和合乎宪法的 活动形式将被显微注射，并观察 生物现象。将检测对ADF磷酸化的影响， 以及抑制性抗ADF单抗阻断这些效应的能力。

DESCRIPTION: ADF was discovered by Bamburg at the start of his career and he has focused on it, almost exclusively. ADF is a small monomer that typifies a class of actin-binding proteins by having a curious mixture of actin-binding activities including severing actin filaments and binding actin monomers. These proteins are found in all eukaryotes and are essential in all of several studied cases, including yeast and Dictyostelium. Cells contain a large amount of them, at a molar ratio of about 1:10 relative to actin. They are regulated by phosphoinositides, pH and phosphorylation. Most notably, Bamburg has found that phosphorylation regulates the actin-binding activity of ADF in vitro, and that phosphorylation occurs in developing muscle cells in a manner predicted from the actin assembly that occurs there. ADF is therefore the best case of regulation of a protein that regulates actin assembly. Bamburg has identified the phosphorylation site as a Serine and prepared substitution mutants that constitutively mimic the on and off states (Glu and Ala, respectively). In preliminary work for this application, Bamburg has isolated and analyzed cDNA's and Ab's for Xenopus ADF along with preparing recombinant ADF that is fully functional in vitro. He finds developmental changes in the phosphorylation that controls actin-binding activity. Ab localization studies show ADF in the same places as filamentous actin. Aim 1 is to determine whether ADF is important for actin reorganization during oogenesis. Aim 2 is to determine the nature of the phosphatase that activates ADF by dephosphorylating it after fertilization. The strategy will be to increase the concentration of various second messengers that lie upstream of individual phosphatases. Also, the phosphatase will be purified biochemically from fertilized eggs. Aim 3 is to determine if ADF is important in actin reorganization events that follow fertilization, using injection of inhibitory Abs, and active or inactive ADF mutants. Aim 4 is to determine how ADF influences actin filaments during cytokinesis, which has been found in new preliminary data. The approach will be similar to that in Aim 3. Aim 5 is to determine if the small G-proteins rho, rac1 and cdc42 are important for cortical rotation and cytokinesis and if so, if their effects are mediated through ADF. Again, dominant negative and constitutively active forms will be microinjected, with observation of changes in the biological phenomena. Effects on phosphorylation of ADF will be examined, along with the ability of inhibitory anti-ADF Abs to block these effects.