Proper chromosomal segregation by microtubule dynamics at the mitotic spindle is required for cell division. Aberrations cause severe developmental abnormalities and can contribute to cancer. Genetic studies using model organisms and biochemical studies have demonstrated the existence of a nucleotide cycle for G alpha subunits that is required for proper mitotic spindle function during cell division. This cycle employs highly conserved G?? regulatory proteins including GoLoco motif containing proteins, which are nucleotide dissociation inhibitors (GDI); Regulator of G-protein Signaling (RGS) domain containing proteins, which are GTPase activating proteins (GAP); and Resistance to inhibitors of cholinesterase 8 (Ric-8), a guanine nucleotide exchange factor (GNEF) for G. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. In mammals, the GoLoco motif-containing protein LGN, G??i, and the microtubule-binding nuclear mitotic apparatus protein (NuMA) regulate microtubule pulling forces during cell division. In vitro, Ric-8 dissociates G alpha i-GDP/LGN/NuMA complexes catalytically, releasing G alpha i-GTP, NuMA, from LGN. Our studies have focused on Ric8 and two RGS proteins, RGS14, and RGS3. We have found that Ric-8 is expressed by most human cells and at high levels in lymphocytes as demonstrated by immunoblotting with a Ric-8 antibody. In interphase cells Ric-8 localizes in the cytosol, in centrosomes, and at unknown sites within the nucleus. Expression of a Ric-8-green fluorescent fusion protein (Ric-8GFP) demonstrated a similar expression patter, but in addition showed heightened expression in lamillepodia. During prophase Ric-8 is recruited to the plasma membrane and to the kinetochore, the site of microtubule attachment to the centomeric region of chromosomes. Gi alpha 2 co-localized with Ric-8 at both the plasma membrane and in the kinetochore. Cells expressing the Ric-8GFP underwent mitotic arrest. Reduction of Ric-8 expression by a Ric-8 mRNA targeted shRNA in HeLa cells resulted in cells with multiple spindles and spindle misalignment leading to multi-nucleation. The decreased Ric-8 expression also led to a loss of NuMA from the plasma membrane and reduced Gai2 at the kinetochore. These results suggest that Ric-8 and Ga GDP/GTP exchange are required for proper chromosome segregation during mitosis. Unique among RGS and GDI proteins, RGS14 and RGS12 contain both an RGS domain and a GoLoco motif. Both the RGS domain and the GoLoco motif of RGS14 target members of the G??i subclass. RGS14 also possesses two Raf-like Ras binding domains. RGS14 associates with centrosomes and microtubules, and loss of Rgs14 expression in mice is catastrophic resulting in failure of zygotes to progress to the 2-cell stage. We have co-localized RGS14 with Gi alpha 1 ?nand Gi alpha 2, but not Gi alpha 3, in centrosomes and with all three Gi alpha subunits in the midbody during cytokinesis. Fluorescence resonance energy transfer analysis confirmed a direct interaction between RGS14 and Gi alpha 1 in centrosomes. Supporting the importance of Gi alpha and their regulators at these sites, cells treated with pertussis toxin, which blocks GTP/GDP exchange of Gi alpha, exhibit cytokinesis defects, bi-nucleation, and G1 cell cycle arrest while expression of a GTPase-deficient G??i1, but not a wild type version, causes mitotic failure. These results suggest that the GTP cycle of the Gi?? subunits modulated by their regulators is necessary for normal cell cycle progression and consistent with the above Ric-8 data. To further our studies of Rgs14, mice in which Rgs14 can be conditionally deleted are being developed. Chimeric mice obtained following injection of correctly targeted embryonic stem (ES) cells into blastocytes are being evaluated for germline transmission of the targeted Rgs14 allele. 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. Numerous RGS3 isoforms exist due to extensive alternative mRNA splicing. One isoform of RGS termed PDZ-RGS3 was originally identified as a binding partner of beta-ephrin and implicated in beta-ephrin reverse signaling in neurons. In addition to its C-terminal RGS domain and N-terminal PDZ domain, PDZ-RGS3 also possesses a C2A domain and an unknown domain that bears some resemblance to a Lim domain. PDZ domains bind the C-termini of membrane proteins and have been widely implicated in forming sub-membrane scaffolds to cluster molecules at the cell surface. Recently, we have shown the involvement of PDZ-RGS3 in the regulation of microtubule dynamics and cytokinesis. PDZ-RGS3-GFP localizes predominantly in the cytosol of interphase cells, but is recruited to the midbody in late telophase. Elevating the expression level of PDZ-RGS3 leads to a profound defect in cytokinesis. Using RNA interference to deplete PDZ-RGS3 in cells, a combination of confocal and video time-lapse microscopy revealed disruption of microtubule organization, spindle elongation, and nuclear separation leading to a block in mitotic exit and cytokinesis. Mass spectroscopy analysis of proteins co-precipitating with the PDZ domain of PDZ-RGS3 identified septin proteins. Confirming this interaction PDZ-RGS3 co-immunoprecipitated and co-localized with the septin Nedd5. Reducing PDZ-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 in late anaphase and the midbody in late telophase. Aurora B kinase is a key enzyme involved in the regulation of normal chromosome segregation during mitosis and cytokinesis. The association of PDZ-RGS3 with aurora B kinase was confirmed by co-immunoprecipitation and FRET analysis. The depletion of endogenous PDZ-RGS3 led to a defect in the spatial orientation of aurora B kinase during telophase. To further our studies of Rgs3, mice with targeted deletion of Rgs3 have been obtained. We have two independent mouse lines each with a targeted deletion of Rgs3. The phenotypes are dramatically different with one line failing to produce viable offspring, while no obvious abnormalities have been noted with the other line. We are back-crossing both lines onto a C57/Bl6 background to help sort out 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 |
