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

这个项目的重点是如何染色体的真菌，赤壳菌，在有丝分裂过程中分离。以前，有丝分裂的时间进程和超微结构的细节被确定，并证明了在星形细胞中存在拉力和在纺锤体中存在推力，这两者都有助于后期B期间的染色体分离。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研究驱动蛋白样微管相关马达蛋白（KLP）在有丝分裂中的作用。该策略是从N.本发明的目的是尽可能地从红球菌基因组DNA文库中筛选出基因产物，使用免疫荧光显微镜原位定位有丝分裂器中的基因产物，在体外运动测定中确定由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这些基因中的每一个。将通过视频显微镜在体内观察每种突变对有丝分裂进展的影响。对于每种基因，将检验以下假设：（1）消除KLP阻断或抑制有丝分裂，以及（2）KLP过量产生增加有丝分裂运动速率。激光微束实验，然后将被用来作为一个怀疑的作用，KLP在有丝分裂力的产生确认。真核细胞骨架是一个复杂的结构，化学机械（马达）和调节蛋白质的系统，既作为细胞的内部支架，也作为几乎所有细胞和细胞内运动的机械，包括细胞分裂（有丝分裂）时染色体的运动。这项工作代表了一个独特的机会，将特定的马达蛋白与体内证明的已知极性的有丝分裂力（即，导致染色体移动的推力和拉力）。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，并且应该导致关于哪种KLP负责哪种有丝分裂力的明确结论。因此，这项工作有可能成为第一个完全阐明后期B期间星体拉力和纺锤体推力的分子基础。这样做，它将大大有助于我们了解微管介导的有丝分裂过程中的染色体运动的机制。此外，它将大大增加所需的信息库，以利用生物马达作为生物（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.