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对于真核生物至关重要。定义机制这些过程背后对于理解细胞如何功能正常，以及它们在医疗条件下如何发生故障，例如癌症、非整倍体、不孕症和瘫痪。已经很明显的是许多细胞成分通过沿着微管的拉动而移动产生力量的“运动”蛋白质。我的研究重点是驱动蛋白最近发现能够移动塑料珠的运动蛋白在体外沿着微管。我的长期目标是 1) 定义驱动蛋白生物功能，2) 了解驱动蛋白如何发挥作用的分子细节在细胞中完成这些功能。为了实现这些目标，我们正在研究果蝇中的驱动蛋白遗传学、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，制备了抗体针对产生力的元件，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) 中分离出 13 个致死突变。为了确定重链在体内如何发挥作用，我们将确定什么重链的结构特征是生存所需的。这手头的 13 个突变和另外 40 个突变中的每一个都有基因损伤我们建议通过 DNA 异源双链体错配来定位分离物切割，并通过部分序列分析进行鉴定。我期待这个作品确定驱动蛋白重链中重要功能位点的位置链，并阐明在这些位点起作用的一些生物化学。本研究中获得的信息将有助于开发完善的驱动蛋白结构/功能的分子模型和后续测试这些模型通过体外诱变。我们已经开始确定驱动蛋白的生物学功能重链引起的驱动蛋白功能丧失的影响分析突变。数据表明驱动蛋白功能在神经元中至关重要组织，其主要作用可能是轴突运输。这假设将通过检查结构和功能来检验 khc 突变体和对照幼虫的神经元，使用电子显微镜和电生理学。驱动蛋白重链是母体负载的蛋白质，因此确定卵母细胞和胚胎中驱动蛋白的功能是什么通过 1) khc 无效等位基因的种系克隆分析和 2) 研究完成 khc 等位基因与已知导致的其他突变的显着相互作用基于微管的运动过程的问题。初步结果表明驱动蛋白参与减数分裂染色体分离。我期望进一步分析以确定依赖于的其他细胞过程驱动蛋白功能，并定义驱动蛋白如何参与这些流程。