The Role of Motor Proteins in Astral and Spindle Forces

9408249本项目的目的是确定如何产生移动有丝分裂装置的力。被选择的生物体，丝状子囊菌红球线虫，是细胞学上最容易驯化的真菌，也适用于传统和分子遗传技术。它是唯一在体内被证明同时存在星体拉力和纺锤体推动力的生物体；这两种力都有助于B期后期的染色体分离。以前的研究也确定了红球线虫有丝分裂的时间进程和超微结构细节。这项研究的分子靶标是驱动蛋白样蛋白(KLP)，它们是在纺锤体中产生推动力的候选蛋白，以及动力蛋白样蛋白(DLP)，它们被认为在纺锤体中产生牵引力。已知KLP是其他三种真菌(酿酒酵母、裂殖酵母和尼杜拉曲霉)正常有丝分裂所必需的，而最近发现DLP是酿酒酵母有丝分裂正常进行所必需的。红球线虫优越的细胞学和光学特性将被用来观察这些蛋白质在体内有丝分裂中的作用。其策略是克隆编码KLP和DLP的基因，通过基因组中的定点突变破坏每个克隆基因的功能，并通过视频显微镜观察每个突变引起的细胞学表型(如果有)。对于每一种蛋白质，消除或过度生产将改变有丝分裂运动速度的假设将得到检验。然后，激光微束实验将被用来确认蛋白质在有丝分裂力量产生中的可疑作用。此外，每个蛋白质将使用免疫荧光显微镜在有丝分裂装置中进行原位定位，并将使用体外运动分析来确定每个蛋白质产生的力的极性。KLP编码基因Klp1已经被克隆，基因组拷贝被破坏，KLP1缺失的表型效应被证明是有丝分裂纺锤体的异常行为。KLP1在有丝分裂中的作用将被研究，分析将扩展到生物体中的其他KLP和DLP。这项拟议的工作提供了一个难得的机会，可以将特定的马达蛋白与体内显示的已知极性和位置的有丝分裂力量联系起来。通过对突变体进行激光微豆实验来测试功能推断的策略是完全独特的，如果不同的马达蛋白参与有丝分裂的不同方面，结果可能会提供明确的证据，说明哪种马达蛋白负责哪种有丝分裂力量。这将大大有助于我们理解有丝分裂的机制，这是一个对所有真核生物的生长和发育至关重要的过程。有丝分裂是细胞分裂的过程，是所有真核生物生命的基础。这一过程确保了细胞的遗传物质在两个子细胞之间均匀分布，方法是在细胞物理分裂为两个之前，仔细调节成对的姐妹染色体到细胞两端的运动。这对子代细胞的活性至关重要。这一过程是通过一系列复杂的、高度调控和可预测的微管介导的染色体运动来完成的。参与这些运动的生化机制及其调控尚不清楚，尽管有证据表明两种基于微管的运动蛋白(化学机械转导)--动蛋白和动力蛋白--参与其中。在NSF先前的支持下，该实验室使用最先进的激光显微技术研究了微管装置(称为纺锤体)在有丝分裂的不同阶段对染色体施加的力。该实验室现在正计划应用最先进的分子遗传学方法来确定有丝分裂过程每个阶段的生化基础。这项工作具有广泛的意义。我们对这个过程了解得越好，我们就越能更好地操纵它，要么扰乱不必要的有丝分裂事件(例如，农业中的害虫或杂草控制，癌症的新疗法，等等)。或者理解或者干预不想要的病理性干扰。此外，由于这些过程发生在非常小的尺度上(微管直径在纳米范围内)，更好地理解这些微小但精确的运动的机制和调节可能在纳米制造工程中作为有用的范例，甚至作为新的材料。***

9408249 Aist The objective of this project is to determine how forces that move the mitotic apparatus are generated. The organism of choice, the filamentous ascomycete Nectria haematococca, is the most cytologically tractable fungus which is also amenable to both conventional and molecular genetic technologies. It is the only organism in which both astral pulling forces and spindle pushing forces have been demonstrated to occur in vivo; both of these forces contribute to chromosome separation during anaphase B. Previous studies have also determined the time-course and ultrastructural details of mitosis in N. haematococca. The molecular targets of this investigation are kinesin-like proteins (KLPs), which are candidates for generating pushing force in the spindle, and dynein-like proteins (DLPs), which are thought to produce pulling force in the aster. KLPs are known to be necessary for normal mitotic progression in three other fungi (Saccharamyces cerevisiae, Schizosaccharomyces pombe, and Aspergillus nidulans), and a DLP has recently been shown to be necessary for mitosis to progress normally in S. cerevisiae. The superior cytological and optical properties of N. haematococca will be used to observe the roles of these proteins in mitosis in vivo. The strategy is to clone genes from N. haematococca encoding KLPs and DLPs, destroy the function of each cloned gene by site-specific mutation in the genome, and observe by video microscopy the cytological phenotype (if any) caused by each mutation. For each protein, the hypothesis that elimination or overproduction will alter the rate of mitotic movements will be tested. Laser microbeam experiments will then be used as a confirmation of the suspected roles of the proteins in mitotic force generation. Additionally, each protein will be localized in the mitotic apparatus in situ using immunofluorescence microscopy, and the polarity of force produced by each protein will be determined using in vitro motility assays. One KLP-encoding gene, Klp1, has already been cloned, the genomic copy has been disrupted, and the phenotypic effect of the loss of KLP1 has been shown to be abnormal behavior of the mitotic spindle. The role of KLP1 in mitosis will be investigated, and the analysis will be extended to additional KLPs and DLPs in the organism. The proposed work represents a rare opportunity to correlate specific motor proteins with in vivo demonstrated mitotic forces of known polarity and location. The strategy of testing functional inferences by performing laser microbean experiments on the mutants is entirely unique and, if different motor proteins are involved in different aspects of mitosis, the results may provide clear demonstrations as to which motor protein is responsible for which mitotic force. This would contribute substantially to our understanding of the mechanisms of mitosis, a process that is fundamental to growth and development of all eukaryotes. %%% Mitosis, the process of cell division, is fundamental to all eukaryotic life. The process ensures that the genetic material of the cell is equally distributed between the two daughter cells, by carefully regulating the movement of paired sister chromosomes to opposite ends of the cell prior to physical division of the cell into two. This is critical to the viability of the daughter cells. The process is accomplished by a complex, highly regulated and predictable series of microtubule-mediated movements of the chromosomes. The biochemical mechanisms involved in these movements and their regulation are not yet well understood, although there is evidence that two kinds of microtubule-based motor proteins (chemomechanical transducers), kinesin and dynein, are involved. With prior NSF support, this laboratory has used state-of-the-art laser microscopic techniques to study the forces exerted by the microtubular apparatus (known as the "spindle") on the chromosomes during the various stages of mitosis. Th e laboratory is now planning to apply state- of-the-art molecular genetic approaches to determine the biochemical basis for each stage of the mitotic process. The work has broad significance. The better we understand the process, the better we can manipulate it, either to disrupt unwanted mitotic events (e.g., pest or weed control in agriculture, novel treatments for cancer, etc.) or to understand and perhaps intervene in unwanted, pathological disruptions. Also, since these processes take place on very small size scales (microtubule diameters are in the nanometer range), a better understanding of the mechanism and regulation of these small but precise movements may serve as useful paradigms, or even as novel materials, in nanofabrication engineering. ***