Analysis of the Functional Roles of a Novel G-alpha Nucl

细胞分裂需要在有丝分裂纺锤体上通过微管动力学进行适当的染色体分离。异常会导致严重的发育异常，并可能导致癌症。利用模式生物的遗传研究和生物化学研究表明，在细胞分裂过程中，G α亚基的核苷酸循环是有丝分裂纺锤体正常功能所必需的。这个循环使用高度守恒的G?？调节蛋白包括含有GoLoco基序的蛋白，它们是核苷酸解离抑制剂（GDI）；g蛋白信号（RGS）结构域的调节因子，该结构域是GTPase激活蛋白（GAP）；以及对胆碱酯酶8（一种鸟嘌呤核苷酸交换因子）抑制剂（g的GNEF）的抗性。这些蛋白相互作用调节染色体有丝分裂运动过程中的微管牵引力。在哺乳动物中，含有GoLoco基序的蛋白LGN、G？i，微管结合核有丝分裂器蛋白（NuMA）在细胞分裂过程中调节微管拉力。在体外，ric8催化解离G α i-GDP/LGN/NuMA复合物，从LGN释放G α i-GTP， NuMA。我们的研究主要集中在Ric8和两个RGS蛋白，RGS14和RGS3。我们发现Ric-8在大多数人类细胞中表达，并在淋巴细胞中高水平表达，这是用ric8抗体进行免疫印迹的结果。在间期细胞中，ric8定位于细胞质、中心体和细胞核内的未知位置。ric -8绿色荧光融合蛋白（Ric-8GFP）的表达表现出类似的表达模式，但在层足中表达增加。在前期，ric8被招募到质膜和着丝点，即微管附着于染色体的着丝点区域。Gi α 2与ric8在质膜和着丝点上共定位。表达Ric-8GFP的细胞发生有丝分裂阻滞。在HeLa细胞中，通过靶向ric8 mRNA的shRNA减少ric8的表达，导致细胞出现多纺锤体和纺锤体错位导致多核。Ric-8表达的减少还导致质膜上NuMA的缺失和着丝点上Gai2的减少。这些结果表明，在有丝分裂过程中，ric8和Ga GDP/GTP交换是染色体正确分离所必需的。在RGS和GDI蛋白中，RGS14和RGS12具有独特的RGS结构域和GoLoco基序。RGS14的RGS结构域和GoLoco基序都以G?？我的子类。RGS14还具有两个类似raf的Ras结合域。RGS14与中心体和微管相关，RGS14在小鼠中的表达缺失是灾难性的，导致受精卵无法进入2细胞阶段。我们有共定位的RGS14和Gi α 1 ？nand和Gi α 2，但不包括Gi α 3，在中心体和所有三个Gi α亚基在胞质分裂期间在中间体。荧光共振能量转移分析证实RGS14与Gi α 1在中心体中直接相互作用。支持Gi α及其在这些位点的调节因子的重要性，百日咳毒素处理的细胞，阻断Gi α的GTP/GDP交换，表现出细胞分裂缺陷，双核和G1细胞周期阻滞，同时表达gtpase缺陷的G？I1，但不是野生型，导致有丝分裂失败。这些结果表明，Gi?？由它们的调节因子调节的亚基是正常细胞周期进程所必需的，这与上面的Ric-8数据一致。为了进一步研究Rgs14，我们正在开发Rgs14可以有条件删除的小鼠。将正确靶向的胚胎干细胞注入母细胞后获得的嵌合小鼠正在评估靶向Rgs14等位基因的种系传播。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。由于广泛的替代mRNA剪接，存在许多RGS3亚型。RGS的一种异构体PDZ-RGS3最初被确定为β -ephrin的结合伙伴，并参与神经元中β -ephrin的反向信号传导。除了c端RGS结构域和n端PDZ结构域外，PDZ- rgs3还具有一个C2A结构域和一个类似于Lim结构域的未知结构域。PDZ结构域结合膜蛋白的c端，并广泛参与形成亚膜支架以在细胞表面聚集分子。最近，我们已经证明PDZ-RGS3参与了微管动力学和细胞动力学的调节。PDZ-RGS3-GFP主要定位于间期细胞的细胞质中，但在末期后期被募集到中间体。PDZ-RGS3的表达水平升高会导致细胞分裂的严重缺陷。利用RNA干扰来消耗细胞中的PDZ-RGS3，共聚焦和视频延时显微镜结合发现微管组织破坏，纺锤体伸长和核分离导致有丝分裂退出和细胞分裂受阻。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。证实PDZ-RGS3与septin Nedd5共免疫沉淀和共定位的相互作用。PDZ-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激酶共定位于后期纺锤体中部和晚期体中部。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。通过共免疫沉淀和FRET分析证实了PDZ-RGS3与极光B激酶的关联。内源性PDZ-RGS3的缺失导致极光B激酶在终末期的空间取向缺陷。

Proper chromosomal segregation by microtubule dynamics at the mitotic spindle is required for cell division. Aberrations cause severe developmental abnormalities and can contribute to cancer. Genetic studies using model organisms and biochemical studies have demonstrated the existence of a nucleotide cycle for G alpha subunits that is required for proper mitotic spindle function during cell division. This cycle employs highly conserved G?? regulatory proteins including GoLoco motif containing proteins, which are nucleotide dissociation inhibitors (GDI); Regulator of G-protein Signaling (RGS) domain containing proteins, which are GTPase activating proteins (GAP); and Resistance to inhibitors of cholinesterase 8 (Ric-8), a guanine nucleotide exchange factor (GNEF) for G. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. In mammals, the GoLoco motif-containing protein LGN, G??i, and the microtubule-binding nuclear mitotic apparatus protein (NuMA) regulate microtubule pulling forces during cell division. In vitro, Ric-8 dissociates G alpha i-GDP/LGN/NuMA complexes catalytically, releasing G alpha i-GTP, NuMA, from LGN. Our studies have focused on Ric8 and two RGS proteins, RGS14, and RGS3. We have found that Ric-8 is expressed by most human cells and at high levels in lymphocytes as demonstrated by immunoblotting with a Ric-8 antibody. In interphase cells Ric-8 localizes in the cytosol, in centrosomes, and at unknown sites within the nucleus. Expression of a Ric-8-green fluorescent fusion protein (Ric-8GFP) demonstrated a similar expression patter, but in addition showed heightened expression in lamillepodia. During prophase Ric-8 is recruited to the plasma membrane and to the kinetochore, the site of microtubule attachment to the centomeric region of chromosomes. Gi alpha 2 co-localized with Ric-8 at both the plasma membrane and in the kinetochore. Cells expressing the Ric-8GFP underwent mitotic arrest. Reduction of Ric-8 expression by a Ric-8 mRNA targeted shRNA in HeLa cells resulted in cells with multiple spindles and spindle misalignment leading to multi-nucleation. The decreased Ric-8 expression also led to a loss of NuMA from the plasma membrane and reduced Gai2 at the kinetochore. These results suggest that Ric-8 and Ga GDP/GTP exchange are required for proper chromosome segregation during mitosis. Unique among RGS and GDI proteins, RGS14 and RGS12 contain both an RGS domain and a GoLoco motif. Both the RGS domain and the GoLoco motif of RGS14 target members of the G??i subclass. RGS14 also possesses two Raf-like Ras binding domains. RGS14 associates with centrosomes and microtubules, and loss of Rgs14 expression in mice is catastrophic resulting in failure of zygotes to progress to the 2-cell stage. We have co-localized RGS14 with Gi alpha 1 ?nand Gi alpha 2, but not Gi alpha 3, in centrosomes and with all three Gi alpha subunits in the midbody during cytokinesis. Fluorescence resonance energy transfer analysis confirmed a direct interaction between RGS14 and Gi alpha 1 in centrosomes. Supporting the importance of Gi alpha and their regulators at these sites, cells treated with pertussis toxin, which blocks GTP/GDP exchange of Gi alpha, exhibit cytokinesis defects, bi-nucleation, and G1 cell cycle arrest while expression of a GTPase-deficient G??i1, but not a wild type version, causes mitotic failure. These results suggest that the GTP cycle of the Gi?? subunits modulated by their regulators is necessary for normal cell cycle progression and consistent with the above Ric-8 data. To further our studies of Rgs14, mice in which Rgs14 can be conditionally deleted are being developed. Chimeric mice obtained following injection of correctly targeted embryonic stem (ES) cells into blastocytes are being evaluated for germline transmission of the targeted Rgs14 allele. 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. Numerous RGS3 isoforms exist due to extensive alternative mRNA splicing. One isoform of RGS termed PDZ-RGS3 was originally identified as a binding partner of beta-ephrin and implicated in beta-ephrin reverse signaling in neurons. In addition to its C-terminal RGS domain and N-terminal PDZ domain, PDZ-RGS3 also possesses a C2A domain and an unknown domain that bears some resemblance to a Lim domain. PDZ domains bind the C-termini of membrane proteins and have been widely implicated in forming sub-membrane scaffolds to cluster molecules at the cell surface. Recently, we have shown the involvement of PDZ-RGS3 in the regulation of microtubule dynamics and cytokinesis. PDZ-RGS3-GFP localizes predominantly in the cytosol of interphase cells, but is recruited to the midbody in late telophase. Elevating the expression level of PDZ-RGS3 leads to a profound defect in cytokinesis. Using RNA interference to deplete PDZ-RGS3 in cells, a combination of confocal and video time-lapse microscopy revealed disruption of microtubule organization, spindle elongation, and nuclear separation leading to a block in mitotic exit and cytokinesis. Mass spectroscopy analysis of proteins co-precipitating with the PDZ domain of PDZ-RGS3 identified septin proteins. Confirming this interaction PDZ-RGS3 co-immunoprecipitated and co-localized with the septin Nedd5. Reducing PDZ-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 in late anaphase and the midbody in late telophase. Aurora B kinase is a key enzyme involved in the regulation of normal chromosome segregation during mitosis and cytokinesis. The association of PDZ-RGS3 with aurora B kinase was confirmed by co-immunoprecipitation and FRET analysis. The depletion of endogenous PDZ-RGS3 led to a defect in the spatial orientation of aurora B kinase during telophase. To further our studies of Rgs3, mice with targeted deletion of Rgs3 have been obtained. We have two independent mouse lines each with a targeted deletion of Rgs3. The phenotypes are dramatically different with one line failing to produce viable offspring, while no obvious abnormalities have been noted with the other line. We are back-crossing both lines onto a C57/Bl6 background to help sort out the differences between the two lines.