ANALYSIS OF CYTOPLASMIC DYNEIN STRUCTURE AND FUNCTION

细胞质动力蛋白结构与功能分析

• 批准号: 6519628
• 负责人: N. R MORRIS
• 金额: $ 34.54万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-09 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519628
• 关键词: Aspergillus; cell migration; cell nucleus; dynein ATPase; fungal genetics; intracellular transport; microtubules; molecular cloning; mutant; neurons; protein binding; protein structure function

Nuclear migration through the cytoplasm occurs in organisms from fungi to man and is required for the growth and proper development of most eukaryotes. Our laboratory has been studying the molecular genetics of nuclear migration in a simple eukaryote, the filamentous fungus Aspergillus nidulans. To date we have identified fourteen "nud" (nuclear distribution) genes whose products are involved in nuclear migration and have cloned and characterized seven of them. All are evolutionarily conserved. Some are components of the molecular motor cytoplasmic dynein. Others are components of dynactin, a protein complex thought to couple the dynein motor to membrane-bounded cargo. Still other nud gene products are components of neither dynein or dynactin, but presumably are proteins that play a regulatory or as yet unknown structural role in nuclear migration. One of these nud gene products, NUDF, is particularly fascinating because a deficiency of its human homolog, LIS 1, causes lissencephaly, a genetic disease in which neurons fail to migrate properly from the paraventricular proliferative zone of the brain to the cerebral cortex. Because nuclear migration occurs in neurons and has been described to be an important component of cell migration in certain cancer cells that undergo nucleokinesis, we have proposed that the failure of neuronal migration in lissencephaly is really a failure of nuclear migration. Evidence from a LIS 1 knockout mouse supports this conclusion. The main thrust of this application is to determine how the NUDF protein affects nuclear migration by analyzing its effects on the function of the cytoplasmic dynein motor, the dynactin coupling machinery, and on the microtubule track with which the motor interacts in vivo and in vitro, using wild-type and nud mutant strains of Aspergillus.

通过细胞质的核迁移发生在从真菌到人类的生物体中，是大多数真核生物生长和正常发育所必需的。我们实验室一直在研究一种简单的真核生物--丝状真菌--尼杜拉曲霉的核迁移的分子遗传学。到目前为止，我们已经确定了14个“核分布”基因，其产物与核迁移有关，并克隆了其中7个基因并对其进行了表征。它们在进化上都是保守的。有些是分子马达细胞质动力蛋白的组成部分。其他的是动力蛋白的成分，动力蛋白是一种蛋白质复合体，被认为可以将动力蛋白马达连接到膜结合的货物上。还有其他nud基因产物既不是dynein也不是dynactin的成分，但推测是在核迁移中发挥调节或尚不清楚的结构作用的蛋白质。这些nud基因产物之一NUDF特别令人着迷，因为它的人类同源基因LIS 1的缺失会导致无脑，这是一种遗传性疾病，神经元无法正确地从脑室旁增殖区迁移到大脑皮层。由于核迁移发生在神经元中，并被描述为某些经历核运动的癌细胞中细胞迁移的重要组成部分，我们提出无脑中神经元迁移的失败实际上是核迁移的失败。来自LIS 1基因敲除小鼠的证据支持这一结论。这项应用的主要目的是通过分析NUDF蛋白对胞质动力蛋白马达、动力肌动蛋白偶联机械的功能以及在体内和体外与马达相互作用的微管轨道的影响，来确定NUDF蛋白是如何影响核迁移的，方法是使用曲霉的野生型和NUD突变株。