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

点击翻译按钮获取中文摘要

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.