The Biochemical Basis for the Mechanics of Cytokinesis

描述（由申请人提供）：细胞质分裂是细胞形状变化的一个戏剧性例子，在此过程中，收缩环的机械收缩导致细胞在有丝分裂结束时分离。细胞分裂是正常细胞增殖的必要条件，在癌症等增生性疾病中具有重要的医学意义。在盘状盘齿龙中，已经确定了具有互补细胞分布的两个肌动蛋白交联蛋白之间的遗传相互作用。Cortexillin-I定位于收缩环，而dynacortin在皮质上富集，但被排除在收缩环之外。这意味着细胞已经进化出不同的肌动蛋白交联蛋白，它们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区域皮层粘弹性。在本提案中，为了确定动皮素如何控制粘弹性，将使用纯化蛋白和各种平衡和动力学技术研究其肌动蛋白交联机制。dynacortin的细胞作用将通过多种体内试验进行研究，包括在野生型细胞中的显性作用、抑制cortexillin-I和挽救dynacortin丧失功能突变体。由于细胞分裂是一个机械过程，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cortexillin-I控制区域粘弹性。我们正在使用激光跟踪微流变学来测量间期的粘弹性模量，并区分野生型和基因工程菌株，其中dynacortin， cortexillin-I和其他活性已经改变。事实上，在初步实验中，dynacortin和cortexillin-I是皮质粘弹性的重要调节剂。参与皮质形状控制的新基因将通过基因抑制cortexillin-I和基因增强myosin-II来确定。肌球蛋白ii是位于收缩环上的主要机械力发生器。一种新的蛋白，DdERM，被确定为皮质肌动蛋白- 1抑制因子，被假设将皮质肌动蛋白束缚在质膜上，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ezrin-radixin-moesin （ERM）和纤蛋白的融合。因此，由于其在皮质功能和细胞形状控制中的作用及其不寻常的结构域结构，该分子引起了相当大的兴趣。

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.