Of central importance to the process of cell division is the accurate transmission of a complete set of chromosomes to each daughter cell, which in eukaryotes is achieved through the function of the mitotic spindle. The goal of this research proposal is to elucidate the role of the highly conserved GTPase Ran in cell division. The current model suggests that RanGTP functions as a spatial marker that signals the position of the genome in eukaryotic cells. During mitosis, RanGTP is thought to promote spindle assembly by stimulating microtubule polymerization and organization in the vicinity of chromosomes. This """"""""local effect"""""""" results from the chromosomal localization of RCC1, the guanine nucleotide exchange factor that generates RanGTP, which binds to transport factors causing them to release cargoes required for spindle assembly. However, the nature of the cargoes, their mitotic function and the molecular mechanisms underlying the RCC1-Ran-microtubule signaling cascade are still poorly understood. A major experimental approach described in this proposal takes advantage of complex cellular extracts prepared from eggs of the African frog Xenopus laevis that can be studied using biochemical and functional assays. This provides an excellent model system to characterize the components and function of mitotic cargoes, to characterize mitotic gradients, and to develop novel reconstitution assays.
Our aims are: (1) To elucidate the function of a Ran-regulated cargo, Rae1 that exists in a complex requiring RNA for its activity, and investigate spindle-associated RNAs. (2) To use fluorescent sensors to visualize and characterize RCC1-induced gradients throughout the cell cycle in extracts and in a variety of cell types. (3) To reconstitute RanGTP gradients and spindle assembly using RCC1-coated beads. (4) To use a newly developed small molecule inhibitor of the Ran pathway to further dissect the functions of RanGTP during cell division. In all eukaryotes, assembly and function of the spindle apparatus is essential to accurately distribute the genetic information during cell division, and errors in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is not only fundamental to our understanding of mitotic and meiotic spindle assembly, but may also provide important new insight into our understanding of human diseases.
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