ANALYSIS OF CYTOPLASMIC DYNEIN STRUCTURE AND FUNCTION

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。我们的实验室一直在研究一种简单的真核生物，丝状真菌中性曲霉的核迁移的分子遗传学。迄今为止，我们已经确定了14个“nud”（核分布）基因，其产物参与核迁移，并克隆和表征了其中的7个。它们都是进化保守的。有些是分子运动细胞质动力蛋白的组分。另一些则是动力蛋白的组成部分，动力蛋白是一种蛋白质复合物，被认为是将动力蛋白马达与膜结合的货物结合起来。还有一些nud基因产物既不是动力蛋白的成分，也不是动力蛋白的成分，但可能是在核迁移中起调节作用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，因为缺乏它的人类同源物，LIS 1，会导致无脑畸形，这是一种遗传性疾病，其中神经元不能正确地从大脑的室旁增殖区迁移到大脑皮层。由于核迁移发生在神经元中，并且在某些经历核分裂的癌细胞中被描述为细胞迁移的重要组成部分，我们提出无脑畸形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。本应用程序的主要目的是通过分析其对细胞质动力蛋白马达、动力蛋白偶联机制的功能的影响，以及在体内和体外与马达相互作用的微管轨道的影响，确定NUDF蛋白如何影响核迁移，使用野生型和突变的曲霉菌菌株。

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.