ADF/COFILIN IN EARLY VERTEBRATE DEVELOPMENT

描述：ADF是班伯格在职业生涯开始时发现的， 他几乎完全专注于此。 ADF是一种小单体， 典型的一类肌动蛋白结合蛋白，有一个奇怪的混合物， 肌动蛋白结合活性，包括切断肌动蛋白丝和结合 肌动蛋白单体。 这些蛋白质存在于所有真核生物中， 在所有几个研究的情况下，包括酵母和 网骨藻 细胞含有大量的它们，摩尔比为 大约是肌动蛋白的1：10。 它们受磷酸肌醇、pH 和磷酸化。 最值得注意的是，Bamburg发现磷酸化 在体外调节ADF的肌动蛋白结合活性， 磷酸化发生在发育中的肌肉细胞中， 肌动蛋白在那里组装。 因此，ADF是 对调节肌动蛋白装配的蛋白质的调节。 班贝格有 将磷酸化位点鉴定为丝氨酸，并制备取代物 组成型模拟开和关状态的突变体（Glu和Ala， 分别）。 在这项应用的初步工作中，班伯格分离并分析了 非洲爪蟾ADF的cDNA和Ab沿着制备重组ADF， 在体外完全发挥作用。 他发现了 磷酸化控制肌动蛋白结合活性。 抗体定位 研究表明ADF与丝状肌动蛋白位于相同的位置。 目标1： 确定ADF是否对肌动蛋白重组重要， 卵子发生 目的2是确定激活ADF的磷酸酶的性质， 在受精后使其去磷酸化。 战略将是增加 各种第二信使的浓度， 单个磷酸酶。 此外，磷酸酶将被纯化， 从受精卵中分离出来 目的3是确定ADF在肌动蛋白重组事件中是否重要 受精后，使用注射抑制性抗体，和活性或 失活ADF突变体。 目的4是确定ADF如何影响胞质分裂过程中的肌动蛋白丝， 这是在新的初步数据中发现的。 方法将类似 在Aim 3中。 目的5是确定小G蛋白rho、rac1和cdc42是否是 重要的皮质旋转和胞质分裂，如果是这样，如果他们的影响 通过ADF进行调解。 同样，显性否定和本质上 活性形式将被显微注射，并观察 生物现象。 将检查对ADF磷酸化的影响， 沿着抑制性抗ADF Ab阻断这些作用的能力。

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.