The Biochemical Basis for the Mechanics of Cytokinesis

描述(申请人提供)：细胞质分裂是细胞形状变化的戏剧性例子，在此过程中，收缩环的机械收缩导致有丝分裂结束时细胞分离。胞质分裂对于正常的细胞增殖是必不可少的，由于它在癌症等增殖性疾病中的作用而具有医学价值。在盘基网柄菌中，已经确定了两个具有互补细胞分布的肌动蛋白交联蛋白之间的遗传相互作用。Cortex-I定位于收缩环，而dynacortin在皮质上丰富，但排除在收缩环之外。这意味着，细胞进化出了独特的肌动蛋白交联蛋白，这些交联蛋白具有互补的细胞分布，协调细胞形状的变化，这些不同的肌动蛋白交联蛋白可能控制局部皮质的粘弹性。在这项提案中，为了确定dynacortin如何控制粘弹性，将使用纯化的蛋白质和各种平衡和动力学技术来研究其肌动蛋白的交联机制。研究dynacortin的细胞作用将使用多种体内检测方法，包括对野生型细胞的显性作用，对皮质素-I的抑制，以及抢救dynacortin功能丧失的突变体。因为胞质分裂是一个机械过程，而肌动蛋白细胞骨架是细胞粘弹性的主要贡献者，我们假设dynacortin和Cortex-I控制局部粘弹性。我们正在使用激光跟踪微观流变学来测量界面的粘弹模数，并将Dynacortin、Cortex-I和其他活性发生变化的野生型和基因工程菌株分开。事实上，在初步实验中，dynacortin和皮质菌素-I是皮质粘弹性的重要调节剂。涉及皮质形状控制的新基因将通过皮质素-I的遗传抑制和肌球蛋白-II的遗传增强来识别。肌球蛋白-II是位于收缩环的主要机械力发生器。一种新的蛋白质，DdERM，被确定为皮质素-I抑制物，被认为是将皮质肌动蛋白拴在质膜上，并有助于皮质粘弹性。DdERM是两类哺乳动物肌动蛋白相关蛋白的融合产物，即Ezrin-Radisin-moesin(ERM)和fimbrin。因此，这种分子因其在皮质功能和细胞形状控制中的作用以及其不寻常的结构域而引起人们的极大兴趣。

DESCRIPTION (provided by applicant): Cytokinesis is a dramatic example of a cell shape change during which the mechanical constriction of the contractile ring leads to cell separation at the end of mitosis. Cytokinesis is essential for normal cell proliferation and is of medical interest for its role in hyperproliferative diseases such as cancer. In Dictyostelium discoideum, genetic interactions have been identified between two actin cross-linking proteins that have complementary cellular distributions. Cortexillin-I is localized to the contractile ring while dynacortin is cortically enriched but excluded from the contractile ring. The implication is that cells have evolved distinct actin cross-linking proteins with complementary cellular distributions that orchestrate cell shape changes, and these different actin cross-linking proteins may control regional cortical viscoelasticity. In this proposal, to ascertain how dynacortin controls viscoelasticity, its actin cross-linking mechanism will be studied using purified proteins and a variety of equilibrium and kinetic techniques. The cellular role of dynacortin will be studied using a variety of in vivo assays including dominant effects in wild type cells, suppression of cortexillin-I and rescue of a dynacortin loss-of-function mutant. Because cytokinesis is a mechanical process and the actin cytoskeleton is the principal contributor to the cell's viscoelasticity, we hypothesize that dynacortin and cortexillin-I control regional viscoelasticity. We are using laser-tracking microrheology to measure the viscoelastic moduli of interphase and dividing wild type and genetically engineered strains where dynacortin, cortexillin-I and other activities have been altered. Indeed, in preliminary experiments, dynacortin and cortexillin-I are significant modulators of cortical viscoelasticity. New genes involved in cortical shape control will be identified using genetic suppression of cortexillin-I and genetic enhancement of myosin-II. Myosin-II is the major mechanical force generator located at the contractile ring. One novel protein, DdERM, which was identified as a cortexillin-I suppressor, is hypothesized to tether the cortical actin to the plasma membrane and contribute to cortical viscoelasticity. DdERM is a fusion of two classes of mammalian actin-associated proteins, ezrin-radixin-moesin (ERM) and fimbrin. Thus, this molecule is of considerable interest because of its role in cortical function and cell shape control and its unusual domain structure.