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，我们已经开始研究有条件地从B或T淋巴细胞中删除Ric-8A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Ric-8A的两个等位基因都在B细胞和其他类型的造血细胞中缺失的小鼠，我们获得了Vav-1CRE小鼠，并将它们与Ric-8A loxP小鼠杂交。我们已经证实Ric-8A在这些小鼠的脾细胞和骨髓来源的巨噬细胞中是缺失的。初步结果表明，这些小鼠患有严重的免疫缺陷。 我们已经证明，Ric-8A蛋白的表达、磷酸化和泛素化在细胞周期中变化，在有丝分裂时达到最大水平。FRET生物传感器通过FLIM(荧光寿命成像显微镜)测量Ric-8A的构象变化，发现Ric-8A在有丝分裂期间处于关闭状态，特别是在细胞质分裂期间。Ric-8A的表达降低延迟了分裂细胞的脱落时间，这与细胞间桥长度的增加和多核有关。在细胞质分裂过程中，Ric-8A与磷脂酰肌醇3-激酶(PI3-K)Vps34以及G-alphai和LGN共同定位在中体，这些蛋白调节PtdIns(3)P的产生，PtdIns(3)P是正常细胞质分裂所需的磷脂。因为细胞质缺陷通常伴随着人类癌症，所以我们对人类原发肿瘤进行了Ric-8A表达水平的筛查。我们发现，与正常组织相比，肿瘤样本中的它们发生了显着变化。 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RGS的一种亚型PDZ-RGS3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 have begun 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 and other hematopoietic cell types we have obtained Vav-1 CRE mice and crossed them with the Ric-8A LoxP mice. We have verified that Ric-8A is deleted in the spleen cells of these mice and in bone marrow derived macrophages. Prelimary results indicate that these mice suffer from a severe immunodeficiency. We have shown that Ric-8A protein expression, phosphorylation and ubiquitination vary during the cell cycle, reaching their maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis Ric-8A co-localized with the phosphatidylinositol 3-kinase (PI3-K) Vps34 at the midbody along with G alphai and LGN, where these proteins functioned to regulate the production of PtdIns(3)P, a phospholipid needed for proper cytokinesis. Because cytokinesis defects often accompany human cancers we screened primary human tumors for Ric-8A expression levels. We found that they were significantly altered in the tumor samples compared to normal tissues. 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 termedPDZ-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.