The Biochemical Basis for the Mechanics of Cytokinesis

细胞分裂机制的生化基础

• 批准号: 7104818
• 负责人: DOUGLAS N ROBINSON
• 金额: $ 32.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104818
• 关键词: actins; bacterial proteins; biochemistry; cell cycle; cell cycle proteins; cell growth regulation; cell morphology; crosslink; cytoskeletal proteins; myosins; protein protein interaction; protein structure function; tissue /cell culture; viscosity

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.

描述（由申请人提供）：细胞因子是细胞形状变化的一个戏剧性例子，在此过程中，收缩环的机械收缩会导致有丝分裂结束时细胞分离。细胞因子对于正常细胞增殖至关重要，并且具有医学兴趣，因为它在癌症等高增殖性疾病中的作用。 在迪斯特尔迪斯特尔（Dictyostelium discoideum）中，已经在两个具有互补细胞分布的肌动蛋白交联蛋白之间鉴定出遗传相互作用。 Cortexillin-I本地化在收缩环上，而DynAcortin则具有皮质富集，但被排除在收缩环之外。这意味着细胞已经与互补的细胞分布一起演化了不同的肌动蛋白交联蛋白，这些细胞分布会策划细胞形状的变化，而这些不同的肌动蛋白交联蛋白可以控制区域皮质粘弹性。 在此提案中，为了确定DynAcortin如何控制粘弹性，将使用纯化的蛋白质以及各种平衡和动力学技术来研究其肌动蛋白交联机制。将使用多种体内测定方法研究DynAcortin的细胞作用，包括野生型细胞中的主要作用，抑制皮质己糖蛋白I和营救功能功能丧失突变体。由于细胞因子是一种机械过程，肌动蛋白细胞骨架是该细胞粘弹性的主要因素，因此我们假设Dy​​nAcortin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相间的粘弹性模量，并分裂野生型和基因工程菌株，在这些菌株中，dynacortin，cortexillin-i和其他活动已改变。实际上，在初步实验中，dynAcortin和Cortexillin-I是皮质粘弹性的重要调节剂。 使用遗传抑制皮质己糖蛋白I和肌球蛋白II的遗传增强，将鉴定出与皮质形状控制有关的新基因。肌球蛋白-II是位于收缩环的主要机械力发生器。一种新型的蛋白质DDER被鉴定为皮质辛抑制剂，假设将皮质肌动蛋白绑在质膜上，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）和脂肪的融合。因此，由于其在皮质功能和细胞形状控制及其异常结构域中的作用，该分子具有相当大的兴趣。