Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle

这些研究主要集中在GI蛋白及其调节因子在有丝分裂和胞质分裂中的作用。在秀丽线虫和果蝇等模式生物中，不依赖受体的异三聚体G蛋白功能对有丝分裂纺锤体的定位、微管拉力的产生、Aster诱导的胞质分裂和核-中心体复合体的集中至关重要。这一新的范例现在正在扩展到哺乳动物细胞。我们和其他人已经证明GI蛋白及其调节因子如AGS3、LGN和RGS14定位于中心体、有丝分裂细胞皮质和中体区域。在这些位点，AGS3、LGN和RGS14可能与Giα蛋白结合，其功能类似于Gβ/γ亚基。Gα蛋白是否也与Gβ/伽马在这些位点配对，或者只与LGN和AGS3等蛋白质相互作用尚不清楚。内源性Gβ/γ亚基在中心体/纺锤体极、有丝分裂细胞皮质、有丝分裂纺锤体、中央纺锤体和中体的动态定位表明可能存在Gβ/伽马结合。分裂的YFP标记的Gβ/伽马的双分子荧光互补证实了这些二聚体针对这些位置的靶向。外源Gβ/γ亚基的表达，siRNA介导的Gβ1的下调，以及Gβ/γ清除蛋白Beta-Ark-ct的表达，都导致了细胞分裂缺陷。我们还重点研究了一种名为Ric-8A的GI蛋白的非GPCR激活剂。RIC-8A在大多数人类细胞中表达，在淋巴细胞中高水平表达。在间期，Ric-8A定位于胞浆和中心体。Ric-8A-绿色荧光融合蛋白(Ric-8A-GFP)定位类似。在前期，Ric-8A被招募到细胞皮质和着丝粒，即微管附着到染色体着丝粒区域的位置。Giα亚基与Ric-8A在两个位点共定位。在HeLa细胞中，Giα1在HeLa细胞中与有丝分裂纺锤体极相对的皮质中表达丰富。这与NUMA、LGN和动力蛋白成分p150gled的出现有关。通过靶向Ric-8A mRNA的shRNA或siRNAs降低Ric-8A的表达，会导致细胞出现错位的微纺锤体，并减少有丝分裂纺锤体的运动。Ric-8A表达的降低降低了Giα1在细胞皮质的表达，这可能导致了NUMA、LGN和p150gled的显著减少。百日咳毒素可阻断G蛋白偶联受体触发的GI GTP/GTP交换，阻断Ric-8A(G.Tall)刺激的GI GTP/GDP交换。与Ric-8A击倒表型百日咳毒素类似，百日咳毒素治疗导致有丝分裂纺锤体迷失方向，未能正确招募到粘着有丝分裂细胞皮质的GIα1、NUMA、LGN和p150。通过靶向Giα1、2和3mRNA表达的siRNAs减少Giα的表达可以复制这些结果。这些研究表明，Ric-8和GαGDP/GTP交换是有丝分裂纺锤体定位所必需的。细胞周期分析和成像实验表明，尽管Ric-8A在分裂细胞的中体区表达，但减少Ric-8A的表达延长了有丝分裂的平均持续时间，而对细胞质分裂的影响较小。RGS14和RGS12含有一个RGS结构域和一个GoLoco基序。RGS14的RGS结构域和GoLoco基序都针对Gi亚类成员。在胞质分裂过程中，我们已经将RGS14与Giα亚基共同定位于中心体和中体。为了进一步我们对RGS14的研究，已经发展了可以有条件地缺失RGS14的小鼠。尽管有相反的报道，RGS14的生殖系缺失并不会导致胚胎死亡。研究正在进行中，以表型这些小鼠，并检查RGS14在免疫细胞功能中的作用。线虫RGS7在早期细胞分裂中起作用，RGS7突变体表现出有丝分裂纺锤体的超不对称运动。在哺乳动物的RGS蛋白中，RGS3与线虫RGS7最为相似。我们已经证明了一种称为PDZ-RGS3的RGS亚型具有调节微管动力学和胞质分裂的功能。PDZ-RGS3-GFP在末期晚期被募集到中体。上调PDZ-RGS3的表达会导致胞质分裂缺陷。RGS3在细胞中的表达缺失破坏了微管组织，正常的纺锤体延长和核分离导致胞质分裂受损。与PDZ-RGS3的PDZ结构域共沉淀的蛋白质的质谱分析鉴定了Septin蛋白质。PDZ-RGS3与Septin Nedd5免疫共沉淀和共定位。RGS3表达降低导致Nedd5不能在中间带/中体积聚。除了Septin募集，PDZ-RGS3与极光B激酶共同定位在纺锤体中区和中体。极光B激酶是有丝分裂和胞质分裂过程中调节正常染色体分离的关键酶。内源性PDZ-RGS3的缺失导致极光B激酶的空间取向缺陷。为了进一步研究RGs3，我们获得了靶向缺失RGs3的小鼠。已经确定了两个独立的小鼠品系，每个品系都有RGs3的靶向干扰，然而，一个品系是胚胎致命的，而另一个品系是活的。两条线路在C57/BL6背景上的广泛回交并没有解决两条线路之间的差异。

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.