MECHANISMS AND FUNCTIONS OF SUBCELLULAR MOTILITY

亚细胞运动的机制和功能

基本信息

批准号：
3305705
负责人：
WILLIAM M SAXTON
金额：
$ 13.44万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 1996-07-31
项目状态：
已结题

项目摘要

Cytoplasmic motility processes that direct the movement of specific molecules, supramolecular structures, and organelles to particular locations in the cell are essential in eukaryotes. Defining the mechanisms behind these processes is important in understanding both how cells function normally, and how they malfunction in medical conditions such as cancer, aneuploidy, infertility, and paralysis. It has become evident that many cellular components move by being pulled along microtubules by forceproducing "motor" proteins. My research focuses on kinesin, a recently discovered motor protein that is capable of moving plastic beads along microtubules in vitro. My long term goals are 1) to define kinesin's biological functions, and 2) to learn the molecular details of how kinesin accomplishes those functions in the cell. To achieve these goals, we are studying kinesin in Drosophila using genetics, molecular genetics, biochemistry, and cytology. With collaborators, I have characterized Drosophila kinesin, prepared antibodies against the force-producing element, the kinesin heavy chain, and cloned and characterized the Drosophila kinesin heavy chain gene. Recently, I have isolated 13 lethal mutations in the kinesin heavy chain gene (khc). To determine how the heavy chain works in vivo, we will determine what structural features of the heavy chain are required for viability. The genetic lesions in each of the 13 mutations in hand and in another 40 that we propose to isolate will be located by DNA heteroduplex mismatch cleavage, and identified by partial sequence analysis. I expect this work to define the location of important functional sites in the kinesin heavy chain, and to illuminate some of the biochemistry operative at those sites. The information gained in this study will allow development of refined molecular models of kinesin structure/function and subsequent tests of those models by in vitro mutagenesis. We have begun to identify what kinesin's biological functions are by analysis of the effects of loss of kinesin function caused by heavy chain mutations. The data suggest that kinesin function is critical in neuronal tissue, and that its primary role there may be in axonal transport. This hypothesis will be tested by examining the structure and function of neurons in khc mutant and control larvae, using electron microscopy and electrophysiology. The kinesin heavy chain is a maternally loaded protein, so determining what kinesin's functions are in oocytes and embryos will be done by 1) germline clonal analysis of khc null alleles, and 2) studying dominant interactions of khc alleles with other mutations known to cause problems with microtubule-based motility processes. Preliminary results indicate that kinesin participates in meiotic chromosome segregation. I expect further analysis to identify other cellular processes that depend on kinesin function, and to define how kinesin participates in those processes.

指导特定运动的细胞质运动过程分子，超分子结构和细胞器细胞中的位置在真核生物中至关重要。定义机制这些过程的背后对于理解细胞的方式很重要正常功能，以及它们如何在医疗状况（例如癌症，非整倍性，不育症和瘫痪。已经很明显许多细胞成分通过沿着微管拉动而移动强生产“运动”蛋白。我的研究重点是动力素，一个最近发现的运动蛋白能够移动塑料珠沿着微管体外。我的长期目标是1）定义运动动力生物学功能，以及2）学习驱动蛋白的分子细节在单元格中完成这些功能。为了实现这些目标，我们正在研究果蝇中的动力素遗传学，分子遗传学，生物化学和细胞学。和合作者，我已经表征了果蝇驱动蛋白，准备了抗体针对产生力的元素，驱动蛋白重链，并克隆并表征了果蝇驱动蛋白重链基因。最近，我在驱动蛋白重链基因（KHC）中已分离出13个致命突变。为了确定重链在体内的工作原理，我们将确定什么重链的结构特征是可行性所必需的。这手中的13个突变中的每个突变中的每一个中的遗传病变我们建议通过DNA HeteroDuplex不匹配定位分离物切割，并通过部分序列分析确定。我希望这项工作定义重要功能位点在驱动蛋白重量中的位置链，并在这些站点阐明一些生物化学操作员。这项研究中获得的信息将允许开发精致驱动蛋白结构/功能的分子模型以及随后的测试这些模型通过体外诱变。我们已经开始确定动力蛋白的生物学功能是什么分析由重链引起的驱动蛋白功能损失的影响突变。数据表明，动力素功能在神经元中至关重要组织，其主要作用在轴突运输中可能存在。这假设将通过检查 KHC突变体和对照幼虫中的神经元，使用电子显微镜和电生理学。驱动蛋白重链是一种母体负荷的蛋白质，因此，确定卵母细胞中的动力蛋白功能和胚胎的功能将是通过1）KHC NULL等位基因的种系克隆分析和2）研究 KHC等位基因与已知引起的其他突变的主要相互作用基于微管的运动过程问题。初步结果表明驱动蛋白参与减数分裂染色体分离。我期望进一步的分析确定其他依赖的蜂窝过程动力蛋白功能，并定义运动蛋白如何参与其中过程。