The Role of Kinesin-like Motor Proteins in Astral and Spindle Forces

这个项目的重点是真菌的染色体是如何在有丝分裂过程中分离的。在此之前，有丝分裂的时间过程和超微结构细节已经被确定，并且纺锤体中的拉力和纺锤体中的推力的存在被证明，这两者都有助于染色体在后期b的分离。目前的目标是确定这些有丝分裂力是如何产生的。为此，将结合细胞学和分子生物学的先进技术来检查运动蛋白样微管相关运动蛋白（KLPs）在有丝分裂中的作用。该策略是尽可能多地从红链球菌基因组DNA文库中克隆klp编码基因，使用免疫荧光显微镜在有丝分裂装置中原位定位基因产物，在体外运动测定中确定每个基因产物产生的力的极性，然后通过位点特异性突变破坏基因组中的每个这些基因。每个突变对有丝分裂进程的影响将通过视频显微镜在体内观察。对于每个基因，(1)KLP的消除阻断或抑制有丝分裂，(2)KLP的过量产生增加有丝分裂运动的速度的假设将被检验。激光微束实验将用于确认KLPs在有丝分裂力产生中的可疑作用。真核细胞骨架是一个由结构、化学机械（马达）和调节蛋白质组成的复杂系统，它既是细胞的内部支架，也是几乎所有细胞和细胞内运动的机械，包括细胞分裂时染色体的运动。这项工作代表了一个独特的机会，将特定的运动蛋白与体内已知极性的有丝分裂力（即导致染色体运动的推力和拉力）联系起来。通过对KLP缺陷突变体进行激光微束实验来测试功能推断的策略是独特的，并且应该得出明确的结论，即哪个KLP负责哪个有丝分裂力。因此，这项工作有可能是第一个完全阐明后期b中星体拉力和纺锤体推力的分子基础。这样做将大大有助于我们理解有丝分裂期间微管介导的染色体运动机制。此外，它将大大增加所需的信息库，以便开发生物马达作为生物（纳米）制造和仿生逆向工程的新型生物分子材料。

This project focuses on how the chromosomes of a fungus, Nectria haematococca, are separated during mitosis. Previously, the time- course and ultrastructural details of mitosis were determined and the presence of pulling forces in the asters and pushing forces in the spindle was demonstrated, both of which contribute to chromosome separation during anaphase B. The present objectives are to determine how these mitotic forces are generated. To do this, advanced technologies of cytology and molecular biology will be combined to examine the role of kinesin-like, microtubule- associated motor proteins (KLPs) in mitosis. The strategy is to clone as many KLP-encoding genes from an N. haematococca genomic DNA library as possible, localize the gene products in the mitotic apparatus in situ using immunofluorescence microscopy, determine the polarity of force produced by each gene product in an in vitro motility assay and then disrupt each of these genes in the genome by site-specific mutation. The effect of each mutation on progression through mitosis will be observed in vivo by video microscopy. For each gene, the hypothesis that (1) elimination of the KLP blocks or inhibits mitosis, and (2) overproduction of the KLP increases the rate of mitotic movements will be tested. Laser microbeam experiments will then be used as a confirmation of the suspected roles of the KLPs in mitotic force generation. %%% The eukaryotic cytoskeleton is a complex system of structural, chemomechanical (motor), and regulatory proteins which serve as both an internal scaffolding for cells and as the machinery for virtually all cellular and intracellular movement, including the movement of chromosomes at the time of cell division (mitosis). This work represents a unique opportunity to correlate specific motor proteins with in vivo-demonstrated mitotic forces of known polarity (i.e., the pushing and pulling forces that result in chromosome movements). The strategy of testing functional inferences by performing laser microbeam experiments on KLP- deficient mutants is unique and should lead to unequivocal conclusions as to which KLP is responsible for which mitotic force. Thus, the work has the potential to be the first to fully elucidate the molecular basis of both the astral pulling force and the spindle pushing force during anaphase B. In doing so, it would contribute substantially to our understanding of the mechanisms of microtubule-mediated movement of chromosomes during mitosis. In addition, it will add substantially to the information base needed in order to exploit biological motors as novel biomolecular materials for bio(nano)fabrication and biomimetic reverse engineering.