ANALYSIS OF CYTOPLASMIC DYNEIN STRUCTURE AND FUNCTION

核通过细胞质的迁移发生在从真菌到人类的生物体中，并且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，丝状真菌构巢曲霉核迁移的分子遗传学。到目前为止，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，这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在一起。还有一些nud基因产物既不是动力蛋白也不是动力肌动蛋白的成分，但可能是在核迁移中起调节作用或未知结构作用的蛋白质。其中一种nud基因产物NUDF特别令人着迷，因为它的人类同源物LIS 1的缺乏会导致无脑畸形，这是一种遗传疾病，其中神经元无法正确地从大脑的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，并且被描述为某些经历核分裂的癌细胞中细胞迁移的重要组成部分，我们提出无脑回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。本申请的主要目的是通过分析NUDF蛋白对细胞质动力蛋白马达、动力蛋白偶联机制以及与马达在体内和体外相互作用的微管轨道的功能的影响来确定NUDF蛋白如何影响核迁移，使用曲霉菌的野生型和裸突变株。

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.