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 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 have begun 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 and other hematopoietic cell types we have obtained Vav-1 CRE mice and crossed them with the Ric-8A LoxP mice. We have verified that Ric-8A is deleted in the spleen cells of these mice and in bone marrow derived macrophages. Prelimary results indicate that these mice suffer from a severe immunodeficiency. We have shown that Ric-8A protein expression, phosphorylation and ubiquitination vary during the cell cycle, reaching their 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 the phosphatidylinositol 3-kinase (PI3-K) Vps34 at the midbody along with G alphai and LGN, where these proteins functioned to regulate the production of PtdIns(3)P, a phospholipid needed for proper cytokinesis. Because cytokinesis defects often accompany human cancers we screened primary human tumors for Ric-8A expression levels. We found that they were significantly altered in the tumor samples compared to normal tissues. 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 termedPDZ-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.