These studies have focused on the role of Gi-proteins and their regulators in mitosis, autophagy, lysosomal function, and actin dynamics. 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 Galphai 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 including 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 Galphai1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. To test the role of Ric-8A 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. 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. In B lymphocytes, Ric-8A is essential for the normal Gi and Gq levels, B cell differentiation, trafficking, and antibody responses. In collaboration with J. Blumer's laboratory (MUSC)we have assessed the importance of AGS4 in immune cell function. AGS4 is well expressed in immune cells with its highest expression in neutrophils. Loss of AGS4 resulted in neutropenia and leukocytosis. Dendritic cells, T lymphocytes, and neutrophils from the AGS4 deficient mice respond sub-optimally to chemoattractants. An in vivo peritonitis model revealed an inability of the AGS4-null neutrophils to migrate normally to primary sites of inflammation. These data suggest that AGS4 is required for proper chemokine signal processing in leukocytes. In non-canonical G-protein signaling, Galphai associates with guanine nucleotide dissociation inhibitors (GDI) other than Gbeta/gamma. Wave proteins, which help regulate the actin cytoskeleton have a domain that resembles a GoLoco motif and Wave1 has been shown to bind Galphai. We have observed that Galphai protein can adopt a filamentous-like structure, which coordinates dynamically with actin polymerization during the developments of filopodia and lamellipodia. Galphai partially co-localizes with WAVE1 and Arp2/3 both by confocal microscopy and electon microscopy. FRET studies are consistent with a close physical interaction between actin and the Galphai protein. The GTP-bound form of the protein recruits more WAVE1 and Arp2/3 to cell protrusion regions than does the GDP-bound form. Modeling protein-protein interactions suggests that the GDP-bound form of this G protein would likely competes with G-actin for binding to the WH2 domain (part of VCA domain) of WAVE1 and WAVE2 protein. We have established collaborations with Phillip Cruz (NIAID, NIH) to assist with bioinformatics and molecular modeling of the interactions of Galphai with the WAVE regulatory complex proteins and with Baoyn Chen (Iowa State University) to examine the interactions of Galphai proteins with the WAVE regulatory complex via direct biochemical studies. To better understand the role of actin regulation in vivo we have established a novel four-dimensional imaging platform to precisely determine the profile and dynamics of lymphocyte transmigration in vivo. This 4D imaging system allows for advanced spatial and temporal resolution. By labeling the lymph node vasculature with fluorescently-labeled antibody against PECAM-1 we documented that lymphocytes predominated crossed high endothelia venules (HEVs) by migrating through endothelial cell junctions. Furthermore, we observed real-time HEV pocket formation. To monitor F-actin dynamics we have used LifeAct-GFP bone-marrow reconstituted mice as a source of lymphocytes for adoptive transfer. Since the cells very rapidly access the HEVs following intravenous infusion, we can treat the cells prior to transfer with various inhibitors of actin polymerization. We have found that ARP2/3 and formin inhibitors curtail lymphocyte actin dynamics in the HEVs and largely prevent transmigration. Finally, 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.
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