MICROFILAMENT ORGANIZATION DURING MITOSIS AND CELL TRANS

这项提议的长期目标是了解分子机制。 在有丝分裂过程中调节微丝重组，以及 在培养细胞的致癌转化过程中。我们将专注于我们的 有丝分裂特异的生物学意义的研究 钙调蛋白的磷酸化。我们第一次展示了 非肌肉钙蛋白，一种相对分子质量为mR=83,000的蛋白质 它与肌动蛋白和钙调蛋白结合，与微丝分离 在有丝分裂期间，显然是磷酸化的结果。我们相信 这一过程可能有助于所看到的形状和结构的变化 在进行有丝分裂的细胞中，因为caldesmon抑制肌动蛋白ATPase， 因为钙调蛋白与原肌球蛋白一起调节肌动蛋白 明胶的截留和封端活性。我们将在生物化学方面 表征有丝分裂特异的钙调蛋白磷酸化如何改变 微丝组装和肌动球蛋白在体外的相互作用。活体内 本课程将探讨钙调蛋白有丝分裂特异性磷酸化的功能。 通过微量注射磷酸化钙调蛋白，阻断 磷酸化或去磷酸化，以及缺乏 磷酸化位点，进入培养细胞。我们将观察是否 显微注射会扰乱有丝分裂期间微丝的组装 和其他细胞周期阶段。我们将检查磷酸化的变化 详细观察细胞周期进程中钙调素的状态 钙调蛋白的磷酸化与微丝的变化相关 集合。因为转化的细胞表现出类似的形态变化 对于在有丝分裂细胞中发现的那些，我们将使用TS大鼠-I细胞来检查， 钙调蛋白在细胞转化过程中是否被磷酸化。最后， 因为我们已经发现cdc2激酶(有丝分裂促进因子)是 在有丝分裂过程中使钙调素磷酸化的两种激酶活性中，我们 将表征cdc2激酶和其他激酶的活性 磷酸化钙离子。我们还将寻找其他 被这些激酶磷酸化的微丝相关蛋白 在有丝分裂期间。因为细胞周期控制与细胞密切相关 转型，拟议的研究将帮助我们不仅了解如何 细胞分裂，但也是为什么癌细胞失去对细胞分裂的控制。

The long term goal of this proposal is to understand molecular mechanisms that regulate microfilament reorganization during mitosis, as well as during oncogenic transformation of cultured cells. We will focus our studies on the biological significance of the mitosis-specific phosphorylation of caldesmon. We have demonstrated, for the first time, that nonmuscle caldesmon, a protein of relative molecular mass Mr = 83,000 which binds to actin and calmodulin, is disassociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. We believe that this process may contribute to the changes of shape and structure seen in cells undergoing mitosis because caldesmon inhibits actomyosin ATPase, and because caldesmon, together with tropomyosin, regulates the actin severing and capping activities of gelsolin. We will biochemically characterize how the mitosis-specific phosphorylation of caldesmon changes microfilament assembly and actomyosin interactions in vitro. In vivo functions of mitosis-specific phosphorylation of caldesmon will be explored through microinjection of phosphorylated caldesmon, antibodies that block phosphorylation or dephosphorylation, and mutant caldesmons lacking the phosphorylation sites, into cultured cells. We will observe whether the microinjection will perturb the assembly of microfilaments during mitosis and other cell cycle stages. We will examine changes in the phosphorylation state of caldesmon during cell cycle progression in detail to see whether the phosphorylation of caldesmon correlates with changes in microfilament assembly. Because transformed cells show morphological alterations similar to those found in mitotic cells, we will examine, using ts Rat-I cells, whether caldesmon is phosphorylated during cell transformation. Finally, because we have found that cdc2 kinase (mitosis promotion factor) is one of two kinase activities that phosphorylate caldesmon during mitosis, we will characterize how cdc2 kinase and the other kinase activity phosphorylate caldesmon. We will also search for other microfilament-associated proteins that are phosphorylated by these kinases during mitosis. Because cell cycle control is intimately related to cell transformation, the proposed studies will help us understand not only how cells divide, but also why cancer cells lose control of cell division.