The Role of Motor Proteins in Astral and Spindle Forces

9408249 Aist本项目的目的是确定如何产生移动有丝分裂器的力。选择的生物体，丝状子囊菌Nectria haematococca，是细胞学上最易处理的真菌，也适用于常规和分子遗传技术。它是唯一一种在体内同时存在星体拉力和纺锤体推力的生物体;这两种力都有助于后期B的染色体分离。以前的研究也确定了N.红球菌属本研究的分子靶标是驱动蛋白样蛋白（KLP），其是在纺锤体中产生推力的候选者，以及动力蛋白样蛋白（DLP），其被认为在紫菀中产生拉力。已知KLP对于其他三种真菌（酿酒酵母、粟酒裂殖酵母和构巢曲霉）中的正常有丝分裂进程是必需的，并且最近已显示DLP对于S.啤酒。N.具有优良的上级细胞学和光学特性。红球菌将被用于观察这些蛋白在体内有丝分裂中的作用。其策略是从N.从编码KLP和DLPs的红球菌中克隆基因，通过基因组中的位点特异性突变破坏每个克隆基因的功能，并通过视频显微镜观察由每个突变引起的细胞学表型（如果有的话）。对于每种蛋白质，将检验消除或过量产生将改变有丝分裂运动速率的假设。激光微束实验，然后将被用来作为有丝分裂力产生的蛋白质的怀疑作用的确认。此外，将使用免疫荧光显微术将每种蛋白质原位定位在有丝分裂器中，并使用体外运动测定法确定每种蛋白质产生的力的极性。一个KLP编码基因Klp 1已经被克隆，基因组拷贝已经被破坏，KLP 1缺失的表型效应已经被证明是有丝分裂纺锤体的异常行为。将研究KLP 1在有丝分裂中的作用，并将分析扩展到生物体中的其他KLP和DLP。拟议的工作代表了一个难得的机会，在体内证明有丝分裂力已知的极性和位置相关的特定马达蛋白。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，并且如果不同的马达蛋白参与有丝分裂的不同方面，则结果可以清楚地证明哪个马达蛋白负责哪个有丝分裂力。这将大大有助于我们了解有丝分裂的机制，这是一个过程，是所有真核生物的生长和发育的基础。有丝分裂，细胞分裂的过程，是所有真核生物的基础。该过程确保细胞的遗传物质在两个子细胞之间均匀分布，通过在细胞物理分裂成两个之前仔细调节成对的姐妹染色体向细胞的相对端的运动。这对子细胞的生存能力至关重要。这一过程是通过一系列复杂的、高度调节的和可预测的微管介导的染色体运动来完成的。虽然有证据表明两种基于微管的运动蛋白（化学机械转换器），驱动蛋白和动力蛋白，参与这些运动及其调节的生化机制尚未得到很好的理解。在先前NSF的支持下，该实验室使用最先进的激光显微镜技术来研究在有丝分裂的各个阶段由微管装置（称为“纺锤体”）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。我们越了解这个过程，我们就越能更好地操纵它，要么破坏不需要的有丝分裂事件（例如，农业中的害虫或杂草控制、癌症的新治疗等）或者理解并干预不想要的、病理性的破坏。此外，由于这些过程发生在非常小的尺寸尺度上（微管直径在纳米范围内），因此更好地理解这些小而精确的运动的机制和调节可以作为有用的范例，甚至作为纳米纤维工程中的新材料。***

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. ***