Roles of Microtubule-Associated Motor Proteins in Mitosis

细胞分裂及其伴随的遗传物质在子细胞间的均匀分布，是所有生命过程的绝对基础。真核生物细胞分裂过程中，染色体形式的遗传物质均匀分布在两个子细胞之间的过程称为有丝分裂。在有丝分裂过程中，染色体凝聚并聚集在纺锤体两极之间，这是一种因其形状而得名的微管阵列。染色体附着在纺锤体内特殊微管的末端。由于由运动蛋白（即动力蛋白）介导的基于微管的化学机械运动，染色体分成两组并向纺锤体极点移动，从每一组纺锤体极点放射出另一组微管，称为aster。有丝分裂过程中向两极迁移的阶段称为后期。这个项目的目的是确定有丝分裂装置及其附着的染色体是如何产生的。之所以选择丝状子囊菌红赤霉病菌（又名茄枯萎菌）作为研究对象，是因为它是细胞学上最容易处理的真菌，也适用于常规和分子遗传技术。它是唯一的生物体，其中星体拉力和主轴推力已被实验证明在体内发生；有丝分裂的时间过程和超微结构细节已被确定。四个微管相关运动蛋白基因已被克隆并测序，细胞质动力蛋白重链基因在有丝分裂中的一个作用已被证实。继续的目标是利用红链球菌优越的细胞学和光学特性，通过直接观察活体有丝分裂进一步确定这些运动蛋白的作用。该策略是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，然后观察每个突变引起的体内有丝分裂表型。对于每一种运动蛋白，消除或过量生产将改变有丝分裂运动速率的假设将得到检验。然后，激光微束实验将用于确认运动蛋白在有丝分裂力产生中的可疑作用。此外，使用绿色荧光蛋白-马达蛋白融合物将每个马达蛋白定位在活体有丝分裂装置中，并使用体外运动测定来确定由马达蛋白相关蛋白产生的力的极性。该项目代表了一个难得的机会，将特定的马达蛋白与体内已知极性和位置的有丝分裂力相关联。通过对突变体进行激光微束实验来测试功能推断的策略是该项目独有的，并且应该导致关于哪种运动蛋白负责哪种有丝分裂力的明确结论。因此，该项目有潜力成为第一个充分阐明星体拉力和主轴推力和反作用力的分子基础。这样做，它将大大有助于我们对有丝分裂机制的理解，这是真核生物所有生长和发育的基础。

9807829 Aist Cell division, with its concomitant uniform distribution of genetic material between daughter cells, is absolutely fundamental to all life processes. The part of the cell division process in eukaryotes whereby the genetic material, in the form of chromosomes, becomes evenly distributed between the two daughter cells is called mitosis. During the mitotic process, the chromosomes become condensed and collect halfway between the poles of the spindle, a microtubular array so named because of its shape. The chromosomes become attached to the ends of specialized microtubules within the spindle. As a result of microtubule-based chemomechanical movements mediated by motor proteins known as dyneins, the chromosomes separate into two groups and move toward the spindle poles, from each of which another set of microtubules, termed asters, radiate. The phase of mitosis during which this migration toward the poles occurs is termed anaphase. The objective of this project is to determine how forces that move the mitotic apparatus and its attached chromosomes are generated. The filamentous ascomycete, Nectria haematococca (a.k.a. Fusarium solani), has been chosen for study because it 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 experimentally to occur in vivo; both of these forces contribute to chromosome separation during anaphase B. The time-course and ultrastructural details of mitosis have been determined. Four microtubule-associated motor protein genes have been cloned and sequenced, and one role of the cytoplasmic dynein heavy chain gene in mitosis has been demonstrated. The continuing goal is to use the superior cytological and optical properties of N. haematococca to further ascertain the roles of these motor proteins by direct observation of mitosis in vivo. The strategy is to destroy t he function of each cloned gene by site-specific mutation in the genome and then to observe the in vivo mitotic phenotypes caused by each mutation. With each motor 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 motor proteins in mitotic force generation. Additionally, each motor protein will be localized in the mitotic apparatus in vivo using green fluorescent protein - motor protein fusions, and the polarity of force produced by the kinesin-related proteins will be determined using in vitro motility assays. The project 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 microbeam experiments on mutants is unique to this project and should lead to unequivocal conclusions as to which motor protein is responsible for which mitotic force. Thus, the project has the potential to be the first to fully elucidate the molecular basis of both the astral pulling force and the spindle pushing and counter forces. In doing so, it would contribute substantially to our understanding of the mechanisms of mitosis, which is fundamental to all growth and development in eukaryotes.