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。我们已经证明了一种名为Ric-8A的Gi蛋白的非GPCR激活剂在人类细胞分裂中的作用。RIC-8A在大多数人类细胞中表达，在淋巴细胞中高水平表达。我们有证据表明，Ric-8A对于向中期细胞皮质招募由NUMA、LGN、Dynein、P150胶体和Giα1组成的信号复合体是重要的。干扰这种复合体的定位会导致有丝分裂纺锤体取向和正常细胞分裂的缺陷。 与威斯康星大学的黄震合作，我们正在开始研究Ric-8A已有条件地从B或T淋巴细胞中删除的小鼠。无条件中断Ric-8A会导致胚胎死亡。Ric-8A loxP小鼠已重新衍生，并与CD19-CRE杂交。由于CD19和Ric-8A都位于7号染色体上，因此在B细胞中只有一个Ric-8A突变等位基因的小鼠是典型的。对这些小鼠的初步分析显示，与对照组小鼠相比，过渡性B细胞的数量有所减少。为了检测B细胞中Ric-8A的两个等位基因都有缺失的小鼠，我们获得了MB-1Cre小鼠，并将它们与Ric-8A loxP小鼠杂交。我们已经证实Ric-8A在这些小鼠的B细胞中是缺失的。初步分析显示，脾中滤泡B细胞的数量显著减少。我们还开始将Ric-8A loxP小鼠与LCK Cre小鼠杂交，以删除发育中的T细胞中的Ric-8A。 为了补充体内研究，我们已经开始在人类T细胞系Jurkat和小鼠B细胞系PK-3中检测Ric-8A。在细胞周期中，Jurkat细胞中Ric8蛋白的表达在细胞周期的M期增加。构建了一种FRET内工具来测量细胞周期中Ric-8A构象的变化。对Ric-8A在细胞周期内的分析表明，在有丝分裂期间，特别是在胞质分裂期间，Ric-8A处于关闭状态。细胞周期的胞质分裂步骤似乎是Ric8功能的重要检查点，因为Ric-8A的耗尽增加了胞质分裂的长度(活分裂细胞的光激活实验)。当Ric-8A表达下调时，胞质分裂阶段长度的增加与细胞内桥长度的增加相关，这表明Ric-8A在脱落步骤中起作用。脱落过程中的一种重要蛋白质是极光B。我们已经证明Ric-8A和AuroraB在分裂细胞中共存。免疫共沉淀和FRET检测证实这两种蛋白相互关联。这种相互作用是结构性的，不受G-Alphai状态的影响。根据Ric-8A敲除的评估，AuroraB的定位和迁移率(FRAP和siRNA实验)也不依赖于Ric-8A。Ric-8A是否可以影响极光-B的激酶活性正在测试中。相反，Ric-8A是否是Aurora-B激酶活性的底物也将被确定。 在线虫中，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。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，一个品系是胚胎致死的，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。我们现在开始免疫非致死性RGS3-/-小鼠的表型。

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. We have shown a role for a non-GPCR activator of Gi protein termed Ric-8A in human cell division. Ric-8A expression occurs in most human cells and at high levels in lymphocytes. We have evidence that Ric-8A is important for recruiting a signaling complex to the metaphase cell cortex consisting of NuMA, LGN, dynein, p150 glued, and Gi alpha1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. In collaboration with Zhen Huang at the University of Wisconsin we are initiating studies to examine mice in which Ric-8A has been conditionally deleted from B or T lymphocytes. The non-conditional disruption of Ric-8A causes embryonic lethality. The Ric-8A LoxP mice have been re-derived and crossed to CD19-CRE. Since CD19 and Ric-8A are both located on chromosome 7, mice with only one interrupted allele of Ric-8A in B cells can be characterized. The initial analysis of these mice has revealed a reduction in the numbers of transitional B cells compared to control mice. To examine mice in which both alleles of Ric-8A are subject to deletion in B cells we have obtained MB-1 CRE mice and crossed them with the Ric-8A LoxP mice. We have verified that Ric-8A is deleted in the B cells of these mice. Initial analysis reveals a marked reduction in number of follicular B cells in spleen. We have also begun to cros the Ric-8A LoxP mice with LCK Cre mice to delete Ric-8A in developing T cells. To complement the in vivo study we have began an examination of Ric-8A in the human T cell line Jurkat and the murine B cell line PK-3. During the cell cycle Ric8 protein expression increased during the M phase of the cell cycle in Jurkat cells. An intra-FRET tool to measure changes in Ric-8A conformation during cell cycle was constructed. Analysis of Ric-8A intrafret during the cell cycle indicated that during mitosis and particularly during cytokinesis, Ric-8A is in a closed state. The cytokinesis step of the cell cycle seems to be an important checkpoint for Ric8 function since depletion of Ric-8A increased the length of cytokinesis (photoactivation experiment in live dividing cells). This increase in cytokinesis stage length was correlated with an increase in intracellular-bridge length when Ric-8A expression was downregulated suggesting that Ric-8A acts during the abscission step. An important protein in abscission is Aurora-B. We have shown that Ric-8A and AuroraB colocalize in dividing cells. Co-immunoprecipitation and FRET assays verified that the 2 proteins associate. This interaction was constitutive and not affected by the status of G alphai. The AuroraB localization and mobility (FRAP and siRNA experiments) were also not Ric-8A dependent as assessed by Ric-8A knock-downs. Whether Ric-8A can affect the kinase activity of aurora-B is being tested. Conversely whether Ric-8A is a substrate for Aurora-B kinase activity will also be determined. In C. elegans RGS7 functions in early cell divisions and RGS7 mutants show hyper-asymmetric movementof 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. Two independent mouse lines each with a targeted disruption of Rgs3 have been identified, 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. We are now beginning to immune phenotype the non-lethal Rgs3-/-mice.