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/aevis that can be studied using biochemical and functional assays. This provides an excellent model system for fractionation and reconstitution experiments to identify and characterize downstream factors. In addition, we propose to examine conservation of the mitotic Ran pathway in vivo using somatic cells.
Our aims are: (1) To identify and characterize intermediates in the RanGTP-microtubule cascade. (2) To use fluorescent sensors of the Ran nucleotide state to visualize and characterize the RanGTP gradient in extracts and in molecularly-defined reactions. (3) To determine the existence and role of RanGTP/cargo gradients in vivo using cultured mammalian cell lines combined with microinjection and RNA interference techniques. Proper spindle assembly and function is required to maintain genome stability, and defects in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is fundamental to our understanding of mitotic and meiotic spindle assembly in eukaryotic cells, and supports our long term goal of reconstituting the complex morphogenetic events of cell division using purified components.
