Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle

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These studies have focused on the role of Gi-proteins and their regulators in mitosis, cytokinesis, autophagy, and lysosomal function. In model organisms such as Caenorhabditis elegans and Drosophila receptor-independent heterotrimeric G protein function is vital for the orientation of mitotic spindle, generation of microtubule pulling force, aster-induced cytokinesis, and centration of the nucleus-centrosome complex. This new paradigm is now being extended to mammalian cells. We and others have shown that Gi proteins and their regulators such as AGS3, LGN, and RGS14 localize in centrosomes, at the mitotic cell cortex, and at the midbody region. At these sites AGS3, LGN, and RGS14 likely bind Gi alpha proteins and function similar to G beta/gamma subunits. We have shown a role for a non-GPCR activator of Gi protein termed Ric-8A in human cell division. Ric-8A expression occurs in most human cells and at high levels in lymphocytes. We have evidence that Ric-8A is important for recruiting a signaling complex to the metaphase cell cortex consisting of NuMA, LGN, dynein, p150 glued, and Gi alpha1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. Ric-8A triggers the release of GDP from Gαi bound to GoLoco motif containing proteins and in embryonic stem cell lines it promotes the association of nascent Gαi1/2 and Gαq subunits with cellular membranes. To test its role in hematopoietic cells and B lymphocytes specifically, we generated ric8fl/flvav-1 and ric8fl/flmb1-cre mice. B cells from these mice have reduced levels of Galphai2/3 and Galpha q proteins. While bone marrow hematopoietic cell development proceeds relatively normally; splenic marginal zone B cells development does not, and overall B cell numbers are reduced. The ric8fl/flmb1-cre B cells exhibit poor responses to chemokines, abnormal trafficking, and improper in situ positioning. The mice have a severely disrupted lymphoid architecture, respond poorly to neo-antigens, exhibit poor B cell memory, and have low levels of serum immunoglobulins. Ric-8A deficient germinal center B cells undergo fewer asymmetric cell divisions. In B lymphocytes, Ric-8A is essential for the normal Gαi and Gαq levels, B cell differentiation, trafficking, and antibody responses. Since Gαi subunits and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division, we have studied the role of Ric-8A in cytokinesis. We have identified a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity. Enhancing the host immune response at host-pathogen interface is an advantageous strategy to combat drug-resistant bacterial infections. Manipulation of lysosomes acting as the ultimate degradation organelle for internalized bacteria bears the potential for such an intervention. We have found a role for AGS3, an accessory regulator protein of heterotrimeric G-protein signaling with a broad array of functional activities, in modulating lysosomal function. Studies focus on elucidation of AGS3-associated signaling mechanisms revealed an upregulation of lysosomal biogenesis that enhances the ability of host cells to combat several intracellular pathogens.