Mechanistic studies of a novel G-alpha nucleotide cycle

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DESCRIPTION (provided by applicant): Proper chromosomal segregation by microtubule (MT) dynamics at the mitotic spindle is an essential component of cell division in all organisms; aberration of this process can lead to severe developmental abnormalities. Recent disparate genetic and biochemical evidence suggests the existence of a nucleotide cycle for G-protein alpha subunits required for proper mitotic spindle function during cell division. This cycle employs G-alpha regulatory proteins conserved across metazoa, including GoLoco motif nucleotide dissociation inhibitors, RGS-domain-containing GTPase-accelerating proteins, and the guanine nucleotide exchange factor RIC-8. Our long-term objective is to define the molecular determinants and spatiotemporal dynamics that underlie this novel G-alpha regulatory cycle and its effects on MT dynamics. Aim 1 is to delineate the functional interplay between the novel G-alpha regulatory factors that impinge on G-alpha subunit activity in cell division via a series of protein biochemical studies. Aim 2 details development and use of novel peptide biosensors for G-alpha nucleotide state to determine the active species that modulates MT dynamics and the spatial and temporal dynamics of this process in live cells. A novel GoLoco-insensitivity mutation to G-alpha will be employed to address the necessity of the G-alpha/GoLoco interaction to these processes. Aim 3 is to map the functional determinants of RGS14 and G-alpha-i1 proteins that directly modulate MT dynamics using in vitro biochemistry and cell viability, MT network, and cell cycle transit studies, thereby testing our initial hypothesis that the coordinated action of G-alpha and G-alpha regulators directly on tubulin and MTs might represent the force generator in mitotic spindle function during mitosis. This research program will lead to a new understanding of the diversity of G-protein action and its impact on microtubule dynamics, and further the ultimate goal of defining the precise molecular mechanisms of microtubule force generation at mitosis. This should facilitate new drug discovery for anticancer therapeutics, as many current anti-proliferative agents target microtubule dynamics. These studies will also afford insight into the regulation of asymmetric cell division and, consequently, mechanisms of cell polarity and cell-fate determination that have specific relevance to neural specification, developmental defects, and the potential future uses of neuroprogenitor stem cells as treatment for neurodegenerative disorders.