These studies have focused on the role of Gi-proteins and their regulators in mitosis, autophagy, lysosomal function, macrophage 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. 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. Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein are essential in transducing signals from the microenvironment. We use genetically modified mice to investigate the role of Galphai2 in inflammasome activity and macrophage polarization. Galphai2 in murine bone marrow-derived macrophages (BMDMs) regulates inflammasome activity independent of inflammasome activated (NLRP3, AIM2, and NLRC4). This regulation stems from the biased polarity of BMDMs. We determined that BMDMs with excess Galphai2 signaling have a tendency towards classically activated pro-inflammatory (M1) phenotype, Galphai2 deficient are biased towards alternatively activated anti-inflammatory (M2) phenotype. Long-term, but not short-term inhibition of Gi with pertussis toxin recapitulates the knockout phenotype, indicating that the inflammatory changes are built into the macrophage life history. This data indicates that excess Galphi2 signaling promotes an M1 macrophage phenotype, while Galphai2 signaling deficiency promotes an M2 phenotype. Understanding Galphai2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.
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