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. Whether G alpha proteins also pair with G beta/gamma at these sites or can only interact with proteins such as LGN and AGS3 is not known. Indicating that G beta/gamma association may occur, we have shown the dynamic localization of endogenous G beta/gamma subunits to centrosomes/spindle poles, the mitotic cell cortex, the mitotic spindle, the central spindle, and the midbody. Bimolecular fluorescence complementation of a split YFP-tagged G beta/gamma confirmed targeting of these dimmers to these sites. Exogenous expression of G beta/gamma subunits, siRNA-mediated knock-down of G beta1, and expression of a G beta/gamma scavenging protein, Beta-ARK-ct, all caused cell division defects. We have also focused on a non-GPCR activator of Gi protein termed Ric-8A. Ric-8A expression occurs in most human cells and at high levels in lymphocytes. At interphase Ric-8A localizes to the cytosol and in centrosomes. A Ric-8A-green fluorescent fusion protein (Ric-8A-GFP) localizes similarly. During prophase Ric-8A is recruited to the cell cortex and to the kinetochore, the site of microtubule attachment to the centomeric region of chromosomes. Gi alpha subunits co-localized with Ric-8A at both sites. In HeLa cells, Gi alpha 1 was enriched at the cell cortex opposite the mitotic spindle poles in HeLa cells. This correlated with the appearance of NuMA, LGN, and p150glued, a dynactin component. Reducing Ric-8A expression by a shRNA or siRNAs that target Ric-8A mRNA resulted in cells with disoriented miotitc spindles and reduced mitotic spindle movements. The decreased Ric-8A expression reduced Gi alpha 1 expression at the cell cortex, which likely led to the noted reductions of NuMA, LGN, and p150glued. Pertussis toxin, which blocks Gi GDP/GTP exchange triggered by G-protein coupled receptors, was shown to block Gi GTP/GDP exchange stimulated by Ric-8A (G. Tall). Similar to the Ric-8A knock-down phenotype pertussis toxin treatment caused disorieinted mitotic spindles and a failure to properly recruit to Gi alpha 1, NuMA, LGN, and p150glued to the mitotic cell cortex. Reducing Gi alpha expression by siRNAs that target Gi alpha 1,2, and 3 mRNA expression phenocopies these results. These studies indicate that Ric-8 and G alpha GDP/GTP exchange are required for proper mitotic spindle orientation. Cell cycle analysis and imaging experiments revealed that reducing Ric-8A expression prolonged the average duration of mitosis, while cytokinesis was less affected despite Ric-8A expression at the midbody region of dividing cells. RGS14 and RGS12 contain an RGS domain and a GoLoco motif. Both the RGS domain and the GoLoco motif of RGS14 target members of the Gi subclass. We have co-localized RGS14 with Gi alpha subunits in centrosomes and in the midbody during cytokinesis. To further our studies of Rgs14, mice in which Rgs14 can be conditionally deleted have been developed. Despite a report to the contrary, germline deletion of Rgs14 did not cause embryonic lethality. Studies are in progress to phenotype these mice and to examine the role of RGS14 in immune cell function. C. elegans RGS7 functions in early cell divisions and RGS7 mutants show hyper-asymmetric movement of 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. PDZ-RGS3-GFP is recruited to the midbody in late telophase. Elevating PDZ-RGS3 expression causes defective cytokinesis. Depletion of RGS3 expression in cells disrupted microtubule organization, normal spindle elongation, and nuclear separation resulting in impaired cytokinesis. Mass spectroscopy analysis of proteins co-precipitating with the PDZ domain of PDZ-RGS3 identified septin proteins. PDZ-RGS3 co-immunoprecipitated and co-localized with the septin Nedd5. Reducing RGS3 expression led to a failure of Nedd5 to accumulate in the middle zone/midbody. In addition to septin recruitment, PDZ-RGS3 co-localized with the aurora B kinase at the spindle midzone and midbody. Aurora B kinase is a key enzyme involved in the regulation of normal chromosome segregation during mitosis and cytokinesis. The depletion of endogenous PDZ-RGS3 led to a defect in the spatial orientation of aurora B kinase. To further our studies of Rgs3, mice with targeted deletion of Rgs3 have been obtained. Two independent mouse lines each with a targeted disruption of Rgs3 have been identifed, 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.
| Cho, Hyeseon; Kehrl, John H (2007) Localization of Gi alpha proteins in the centrosomes and at the midbody: implication for their role in cell division. J Cell Biol 178:245-55 |
