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细胞分裂中的作用。在秀丽隐杆线虫和果蝇等模式生物中，不依赖受体的异三聚体G蛋白的功能对于有丝分裂纺锤体的定向、微管拉力的产生、aster诱导的细胞分裂以及核-中心体复合物的集中至关重要。这种新模式现在正扩展到哺乳动物细胞。我们和其他人已经证明Gi蛋白及其调控因子如AGS3、LGN和RGS14定位于中心体、有丝分裂细胞皮层和体中部区域。在这些位点上，AGS3、LGN和RGS14可能与Gi α蛋白结合，功能类似于G β / γ亚基。目前尚不清楚G α蛋白是否也在这些位点与G β / γ配对，或者只与LGN和AGS3等蛋白相互作用。为了表明G β / γ可能发生关联，我们已经展示了内源性G β / γ亚基在中心体/纺锤体极、有丝分裂细胞皮层、有丝分裂纺锤体、中央纺锤体和中间体的动态定位。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物靶向这些位点。外源性G β / γ亚基的表达、sirna介导的G β 1的敲低以及G β / γ清除蛋白β - ark -ct的表达都会导致细胞分裂缺陷。我们还关注了一种非gpcr的Gi蛋白激活剂ric8a。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，ric8a定位于细胞质和中心体中。一种绿色荧光融合蛋白（Ric-8A-GFP）也有类似的定位。在前期，Ric-8A被招募到细胞皮层和着丝点，这是微管与染色体的着丝点区域连接的位置。Gi α亚基与Ric-8A在两个位点共定位。在HeLa细胞中，Gi α 1在HeLa细胞有丝分裂梭形极对面的细胞皮层富集。这与NuMA、LGN和p150glue（一种动态蛋白成分）的出现有关。通过靶向Ric-8A mRNA的shRNA或sirna减少Ric-8A的表达，导致细胞分裂纺锤体定向失调和有丝分裂纺锤体运动减少。Ric-8A表达的降低降低了细胞皮层Gi α 1的表达，这可能导致NuMA、LGN和p150glue的显著减少。百日咳毒素阻断由g蛋白偶联受体触发的Gi GTP/ GTP交换，被证明可以阻断由rick - 8a刺激的Gi GTP/GDP交换（G. Tall）。与ric8a敲除表型相似，百日咳毒素处理导致有丝分裂纺锤体定向紊乱，无法正确招募粘在有丝分裂细胞皮层上的Gi α 1、NuMA、LGN和p150。通过靶向Gi α 1、2和3 mRNA表达的sirna减少Gi α的表达，可以反映这些结果。这些研究表明，ric8和G α GDP/GTP交换是有丝分裂纺锤体定向的必要条件。细胞周期分析和成像实验显示，减少ric8a表达可延长有丝分裂的平均持续时间，而分裂细胞中体区表达ric8a对细胞分裂的影响较小。RGS14和RGS12包含一个RGS结构域和一个GoLoco基序。RGS14的RGS结构域和GoLoco基序都以Gi亚类的成员为目标。我们发现RGS14与Gi α亚基共定位于中心体和胞质分裂期间的体中部。为了进一步研究Rgs14，我们开发了Rgs14可以有条件删除的小鼠。尽管有相反的报道，Rgs14的种系缺失并未导致胚胎致死。研究正在对这些小鼠进行表型分析，并检查RGS14在免疫细胞功能中的作用。秀丽隐杆线虫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RGS蛋白中，RGS3与秀丽隐杆线虫RGS7最为相似。我们已经证明RGS的一种异构体PDZ-RGS3具有调节微管动力学和细胞动力学的功能。PDZ-RGS3-GFP在晚期被募集到中体。PDZ-RGS3表达升高导致细胞分裂缺陷。细胞中RGS3表达的缺失破坏了微管组织、正常纺锤体伸长和核分离，导致细胞分裂受损。质谱分析与PDZ- rgs3的PDZ结构域共沉淀的蛋白质鉴定出septin蛋白。PDZ-RGS3与septin Nedd5共免疫沉淀和共定位。RGS3表达减少导致Nedd5不能在中间区/中间体积累。除了septin募集外，PDZ-RGS3还与aurora B激酶在纺锤体中部和中部共定位。极光B激酶是参与有丝分裂和细胞分裂过程中正常染色体分离调控的关键酶。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系，每个系都有Rgs3的靶向破坏，然而，一个系是胚胎致死的，而另一个系是可存活的。这两条线在C57/Bl6背景上的大量反向交叉并没有解决这两条线之间的差异。

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.