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

这些研究集中于Gi蛋白及其调节剂在有丝分裂、胞质分裂、自噬和溶酶体功能中的作用。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。我们已经显示了Gi蛋白的非GPCR激活剂Ric-8A在人类细胞分裂中的作用。Ric-8A在大多数人类细胞中表达，在淋巴细胞中表达水平较高。我们有证据表明，Ric-8A是重要的招募信号复合物的中期细胞皮质组成的NuMA，LGN，动力蛋白，p150胶合，和Gi α 1。干扰该复合物的定位引起有丝分裂纺锤体方向和正常细胞分裂的缺陷。 Ric-8A触发GDP从与含有GoLoco基序的蛋白质结合的Gi释放，并且在胚胎干细胞系中，它促进新生Gi 1/2和G q亚基与细胞膜的缔合。为了测试其在造血细胞和B淋巴细胞中的作用，我们产生了ric 8 fl/flvav-1和ric 8 fl/flmb 1-cre小鼠。来自这些小鼠的B细胞具有降低水平的Galpha 1 2/3和Galphaq蛋白。虽然骨髓造血细胞发育相对正常，但脾边缘区B细胞发育不正常，总体B细胞数量减少。ric 8 fl/flmb 1-cre B细胞表现出对趋化因子的不良反应、异常运输和不适当的原位定位。这些小鼠具有严重破坏的淋巴结构，对新抗原的反应差，表现出差的B细胞记忆，并且具有低水平的血清免疫球蛋白。Ric-8A缺陷的生发中心B细胞经历较少的不对称细胞分裂。在B淋巴细胞中，Ric-8A对于正常G i和G q水平、B细胞分化、运输和抗体应答是必需的。 由于G i亚基及其调节因子位于胞质分裂前的中间体，并与细胞分裂的最后阶段有关，因此我们研究了Ric-8A在胞质分裂中的作用。我们已经确定了Ric-8A通过控制Vps 34活性影响胞质分裂和胞质分裂的分子机制。我们发现，Ric-8A蛋白的表达是转录后控制在细胞周期中达到其最高水平在有丝分裂。为了通过FLIM（荧光寿命成像显微镜）测量Ric-8A的构象变化而创建的FRET生物传感器显示Ric-8A在有丝分裂期间处于闭合状态，特别是在胞质分裂期间。降低Ric-8A表达延迟分裂细胞的分裂时间，这与细胞间桥长度增加和多核化相关。在胞质分裂过程中，Ric-8A与Vps 34共定位在中间体沿着，与Gi和LGN一起，在那里这些蛋白质起调节Vps 34磷脂酰肌醇3-激酶活性的作用。 增强宿主-病原体界面的宿主免疫应答是对抗耐药细菌感染的有利策略。操纵溶酶体作为内化细菌的最终降解细胞器具有这种干预的潜力。 我们已经发现AGS 3在调节溶酶体功能中的作用，AGS 3是异源三聚体G蛋白信号传导的辅助调节蛋白，具有广泛的功能活性。研究重点是阐明AGS 3相关的信号传导机制，揭示了溶酶体生物合成的上调，这增强了宿主细胞对抗几种细胞内病原体的能力。

These studies have focused on the role of Gi-proteins and their regulators in mitosis, cytokinesis, autophagy, and lysosomal function. 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. Ric-8A triggers the release of GDP from Gαi bound to GoLoco motif containing proteins and in embryonic stem cell lines it promotes the association of nascent Gαi1/2 and Gαq subunits with cellular membranes. To test its role in hematopoietic cells and B lymphocytes specifically, we generated ric8fl/flvav-1 and ric8fl/flmb1-cre mice. B cells from these mice have reduced levels of Galphai2/3 and Galpha q proteins. While bone marrow hematopoietic cell development proceeds relatively normally; splenic marginal zone B cells development does not, and overall B cell numbers are reduced. The ric8fl/flmb1-cre B cells exhibit poor responses to chemokines, abnormal trafficking, and improper in situ positioning. The mice have a severely disrupted lymphoid architecture, respond poorly to neo-antigens, exhibit poor B cell memory, and have low levels of serum immunoglobulins. Ric-8A deficient germinal center B cells undergo fewer asymmetric cell divisions. In B lymphocytes, Ric-8A is essential for the normal Gαi and Gαq levels, B cell differentiation, trafficking, and antibody responses. Since Gαi subunits and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division, we have studied the role of Ric-8A in cytokinesis. We have identified a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its 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 Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity. Enhancing the host immune response at host-pathogen interface is an advantageous strategy to combat drug-resistant bacterial infections. Manipulation of lysosomes acting as the ultimate degradation organelle for internalized bacteria bears the potential for such an intervention. We have found a role for AGS3, an accessory regulator protein of heterotrimeric G-protein signaling with a broad array of functional activities, in modulating lysosomal function. Studies focus on elucidation of AGS3-associated signaling mechanisms revealed an upregulation of lysosomal biogenesis that enhances the ability of host cells to combat several intracellular pathogens.