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.

Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
Zip Code
Harris, James; Lang, Tali; Thomas, Jacinta P W et al. (2017) Autophagy and inflammasomes. Mol Immunol 86:10-15
Robichaux 3rd, William G; Branham-O'Connor, Melissa; Hwang, Il-Young et al. (2017) Regulation of Chemokine Signal Integration by Activator of G-Protein Signaling 4 (AGS4). J Pharmacol Exp Ther 360:424-433
Kehrl, John H (2016) The impact of RGS and other G-protein regulatory proteins on G?i-mediated signaling in immunity. Biochem Pharmacol 114:40-52
Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C et al. (2016) Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection. J Immunol 196:846-56
Boularan, Cedric; Hwang, Il-Young; Kamenyeva, Olena et al. (2015) B Lymphocyte-Specific Loss of Ric-8A Results in a G? Protein Deficit and Severe Humoral Immunodeficiency. J Immunol 195:2090-102
Hwang, Il-Young; Park, Chung; Harrison, Kathleen et al. (2015) An essential role for RGS protein/G?i2 interactions in B lymphocyte-directed cell migration and trafficking. J Immunol 194:2128-39
Boularan, Cedric; Kamenyeva, Olena; Cho, Hyeseon et al. (2014) Resistance to inhibitors of cholinesterase (Ric)-8A and G?i contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity. PLoS One 9:e86680
Boularan, Cédric; Kehrl, John H (2014) Implications of non-canonical G-protein signaling for the immune system. Cell Signal 26:1269-82
Branham-O'Connor, Melissa; Robichaux 3rd, William G; Zhang, Xian-Kui et al. (2014) Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3). J Biol Chem 289:10738-47
Woodard, Geoffrey E; Huang, Ning-Na; Cho, Hyeseon et al. (2010) Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle. Mol Cell Biol 30:3519-30

Showing the most recent 10 out of 11 publications