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

这些研究集中在Gi蛋白及其调节剂在有丝分裂和胞质分裂中的作用。 在模式生物如秀丽隐杆线虫和果蝇中，受体独立的异源三聚体G蛋白功能对于有丝分裂纺锤体的定向、微管拉力的产生、紫菀诱导的胞质分裂和核-中心体复合物的集中是至关重要的。这种新的模式现在正在扩展到哺乳动物细胞。我们和其他人已经表明，Gi蛋白及其调节因子，如AGS 3，LGN和RGS 14定位在中心体，在有丝分裂细胞皮质，并在中间体区域。 在这些位点，AGS 3、LGN和RGS 14可能结合Gi α蛋白，并且功能类似于G β/γ亚基。 G α蛋白是否也在这些位点与G β/γ配对，或者只能与LGN和AGS 3等蛋白质相互作用尚不清楚。 表明G β/γ协会可能会发生，我们已经显示了动态定位的内源性G β/γ亚基的中心体/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。 分裂的YFP标记的G β/γ的双分子荧光互补证实了这些二聚体靶向这些位点。 G β/γ亚基的外源性表达、G β 1的siRNA介导的敲低以及G β/γ清除蛋白Beta-ARK-ct的表达均导致细胞分裂缺陷。 我们还关注了Gi蛋白的非GPCR激活剂，称为Ric-8A。 Ric-8A在大多数人类细胞中表达，在淋巴细胞中表达水平较高。 在间期，Ric-8A定位于细胞质和中心体。Ric-8A-绿色荧光融合蛋白（Ric-8A-GFP）定位相似。 在前期，Ric-8A被募集到细胞皮层和动粒，即微管附着到染色体中心区的位点。 Gi α亚基与Ric-8A在两个位点共定位。在HeLa细胞中，Gi α 1富集在HeLa细胞中有丝分裂纺锤体极对面的细胞皮质。 这与NuMA、LGN和p150 glued（一种dynactin组分）的出现相关。 通过靶向Ric-8A mRNA的shRNA或siRNA减少Ric-8A表达导致细胞具有定向混乱的有丝分裂纺锤体和减少的有丝分裂纺锤体运动。 Ric-8A表达的降低降低了细胞皮层的Gi α 1表达，这可能导致NuMA、LGN和p150 glucose的显著降低。百日咳毒素阻断由G蛋白偶联受体触发的Gi GDP/GTP交换，显示阻断由Ric-8A刺激的Gi GTP/GDP交换（G.高）。与Ric-8A敲低表型相似，百日咳毒素处理导致有丝分裂纺锤体定向错误，并且不能正确募集到Gi α 1、NuMA、LGN和粘附到有丝分裂细胞皮质的p150。 通过靶向Gi α 1、2和3 mRNA表达的siRNA降低Gi α表达模拟了这些结果。 这些研究表明，Ric-8和G α GDP/GTP交换是正确的有丝分裂纺锤体方向所必需的。细胞周期分析和成像实验表明，减少Ric-8A表达延长有丝分裂的平均持续时间，而胞质分裂的影响较小，尽管Ric-8A表达在分裂细胞的中间体区域。 RGS 14和RGS 12含有RGS结构域和GoLoco基序。 RGS 14的RGS结构域和GoLoco基序都靶向Gi亚类的成员。 我们已经共同定位RGS 14与Gi α亚基在中心体和在胞质分裂的中间体。 为了进一步研究Rgs 14，已经开发了Rgs 14可以条件性缺失的小鼠。尽管有相反的报道，生殖细胞缺失Rgs 14并不导致胚胎致死。研究正在进行中，以表型这些小鼠，并检查RGS 14在免疫细胞功能中的作用。 C.线虫RGS 7在早期细胞分裂中起作用，而RGS 7突变体显示有丝分裂纺锤体的超不对称运动。 在哺乳动物RGS蛋白中，RGS 3与C. elegans RGS7.我们已经表明，RGS的一种亚型称为PDZ-RGS 3的功能，以调节微管动力学和胞质分裂。 PDZ-RGS 3-GFP在末期被募集到中间体。 PDZ-RGS 3表达升高导致胞质分裂缺陷。 细胞中RGS 3表达的缺失破坏微管组织、正常纺锤体伸长和核分离，导致胞质分裂受损。 与PDZ-RGS 3的PDZ结构域共沉淀的蛋白质的质谱分析鉴定了septin蛋白。 PDZ-RGS 3与隔蛋白Nedd 5共免疫沉淀和共定位。 降低RGS 3表达导致Nedd 5不能在中间区/中间体中积累。 除了septin募集，PDZ-RGS 3与极光B激酶共定位于纺锤体中间区和中间体。 极光B激酶是参与调节有丝分裂和胞质分裂过程中正常染色体分离的关键酶。 内源性PDZ-RGS 3的缺失导致极光B激酶的空间取向缺陷。 为了进一步研究Rgs 3，获得了Rgs 3靶向缺失的小鼠。 已经鉴定了两个独立的小鼠品系，每个品系具有Rgs 3的靶向破坏，然而，一个品系是胚胎致死的，而另一个品系是存活的。将两个品系广泛回交到C57/B16背景上没有解决两个品系之间的差异。

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.