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、细胞分裂、自噬和溶酶体功能中的作用。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。我们已经证明了Gi蛋白的非gpcr激活剂ric8a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对于向中期细胞皮层募集由NuMA、LGN、dynein、p150 glue和Gi alpha1组成的信号复合体很重要。对该复合体定位的干扰导致有丝分裂纺锤体取向和正常细胞分裂的缺陷。

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.