These studies have focused on the role of Gi-proteins and their regulators in mitosis and cytokinesis. 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. In collaboration with Zhen Huang at the University of Wisconsin we are initiating studies to examine mice in which Ric-8A has been conditionally deleted from B or T lymphocytes. The non-conditional disruption of Ric-8A causes embryonic lethality. The Ric-8A LoxP mice have been re-derived and crossed to CD19-CRE. Since CD19 and Ric-8A are both located on chromosome 7, mice with only one interrupted allele of Ric-8A in B cells can be characterized. The initial analysis of these mice has revealed a reduction in the numbers of transitional B cells compared to control mice. To examine mice in which both alleles of Ric-8A are subject to deletion in B cells we have obtained MB-1 CRE mice and crossed them with the Ric-8A LoxP mice. We have verified that Ric-8A is deleted in the B cells of these mice. Initial analysis reveals a marked reduction in number of follicular B cells in spleen. We have also begun to cros the Ric-8A LoxP mice with LCK Cre mice to delete Ric-8A in developing T cells. To complement the in vivo study we have began an examination of Ric-8A in the human T cell line Jurkat and the murine B cell line PK-3. During the cell cycle Ric8 protein expression increased during the M phase of the cell cycle in Jurkat cells. An intra-FRET tool to measure changes in Ric-8A conformation during cell cycle was constructed. Analysis of Ric-8A intrafret during the cell cycle indicated that during mitosis and particularly during cytokinesis, Ric-8A is in a closed state. The cytokinesis step of the cell cycle seems to be an important checkpoint for Ric8 function since depletion of Ric-8A increased the length of cytokinesis (photoactivation experiment in live dividing cells). This increase in cytokinesis stage length was correlated with an increase in intracellular-bridge length when Ric-8A expression was downregulated suggesting that Ric-8A acts during the abscission step. An important protein in abscission is Aurora-B. We have shown that Ric-8A and AuroraB colocalize in dividing cells. Co-immunoprecipitation and FRET assays verified that the 2 proteins associate. This interaction was constitutive and not affected by the status of G alphai. The AuroraB localization and mobility (FRAP and siRNA experiments) were also not Ric-8A dependent as assessed by Ric-8A knock-downs. Whether Ric-8A can affect the kinase activity of aurora-B is being tested. Conversely whether Ric-8A is a substrate for Aurora-B kinase activity will also be determined. In C. elegans RGS7 functions in early cell divisions and RGS7 mutants show hyper-asymmetric movementof mitotic spindles. Among the mammalian RGS proteins, RGS3 most closely resembles C. elegans RGS7. We have shown that one isoform of RGS termed PDZ-RGS3 functions to regulate microtubule dynamics and cytokinesis. Two independent mouse lines each with a targeted disruption of Rgs3 have been identified, however, one line is an embryonic lethal while the other is viable. Extensive back-crossing of the two lines onto a C57/Bl6 background has not resolved the differences between the two lines. We are now beginning to immune phenotype the non-lethal Rgs3-/-mice.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2011
Total Cost
$525,658
Indirect Cost
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State
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Harris, James; Lang, Tali; Thomas, Jacinta P W et al. (2017) Autophagy and inflammasomes. Mol Immunol 86:10-15
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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
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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

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