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

这些研究集中在GI蛋白及其调节因子在有丝分裂、自噬、溶酶体功能、巨噬细胞功能和肌动蛋白动力学中的作用。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Galphai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都有表达，在淋巴细胞中也有高水平的表达。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 Gled和Galphai1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。 在非典型的G蛋白信号中，Galphai与鸟嘌呤核苷酸解离抑制物(GDI)有关，而不是Gbeta/Gamma。帮助调节肌动蛋白细胞骨架的WAVE蛋白有一个类似于GoLoco基序的结构域，并且Wave1已被证明与Galphai结合。我们观察到Galphai蛋白可以采用丝状结构，在丝足和片脂发育过程中与肌动蛋白聚合动态协调。通过共聚焦显微镜和电子显微镜，Galphai与WAVE1和Arp2/3部分共定位。FRET研究与肌动蛋白和Galphai蛋白之间的密切物理相互作用是一致的。GTP结合形式的蛋白质比GDP结合形式的蛋白质在细胞突起区域招募更多的WAVE1和Arp2/3。对蛋白质-蛋白质相互作用的模拟表明，该G蛋白的GDP结合形式可能与G-肌动蛋白竞争结合到WAVE1和WAVE2蛋白的WH2结构域(VCA结构域的一部分)。我们已经与Phillip Cruz(NIAID，NIH)建立了合作关系，以协助Galphai与波调节复合体蛋白质的相互作用的生物信息学和分子建模，并与爱荷华州立大学的Paulyn Chen通过直接的生化研究来研究Galphai蛋白与波调节复合体的相互作用。 为了更好地了解肌动蛋白在体内的调节作用，我们建立了一个新的四维成像平台来精确地确定体内淋巴细胞移行的轮廓和动力学。这种4D成像系统可以实现更高的空间和时间分辨率。通过用PECAM-1荧光标记抗体标记淋巴结血管，我们证明淋巴细胞主要通过内皮细胞连接游走穿过高内皮微静脉(HEV)。此外，我们还观察到了HEV气囊的实时形成。为了监测F-肌动蛋白的动态变化，我们使用LifeAct-GFP重组小鼠的骨髓作为过继转移的淋巴细胞来源。由于细胞在静脉输注后非常迅速地进入HEV，我们可以在转移之前使用各种肌动蛋白聚合抑制剂来处理细胞。我们发现，Arp2/3和福尔明抑制剂抑制HEV中淋巴细胞肌动蛋白的动态，并在很大程度上防止移行。 巨噬细胞以先天免疫亚群的形式存在，表现出表型异质性和功能可塑性。它们的表型由组织微环境的输入决定。G蛋白在从微环境中传递信号方面是必不可少的。我们使用转基因小鼠来研究Galphai2在炎症体活性和巨噬细胞极化中的作用。小鼠骨髓源性巨噬细胞(BMDM)中的Galphai2调节炎症小体活动，而不依赖于激活的炎症小体(NLRP3、AIM2和NLRC4)。这一规定源于BMDM的偏向极性。我们确定Galphai2信号过多的BMDM倾向于经典激活的促炎(M1)表型，而Galphai2缺乏的BMDM倾向于交替激活的抗炎(M2)表型。长期，但不是短期百日咳毒素抑制胃肠道，重现了敲除表型，表明炎症变化是建立在巨噬细胞的生活史中的。这一数据表明，Galphi2信号过度促进M1巨噬细胞表型，而Galphai2信号缺乏促进M2表型。了解Galphai2介导的巨噬细胞极化效应可能有助于了解疾病的发病机制和巨噬细胞的重新编程，从而开发新的治疗方法。

These studies have focused on the role of Gi-proteins and their regulators in mitosis, autophagy, lysosomal function, macrophage function, and actin dynamics. 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 Galphai 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 including 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 Galphai1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. In non-canonical G-protein signaling, Galphai associates with guanine nucleotide dissociation inhibitors (GDI) other than Gbeta/gamma. Wave proteins, which help regulate the actin cytoskeleton have a domain that resembles a GoLoco motif and Wave1 has been shown to bind Galphai. We have observed that Galphai protein can adopt a filamentous-like structure, which coordinates dynamically with actin polymerization during the developments of filopodia and lamellipodia. Galphai partially co-localizes with WAVE1 and Arp2/3 both by confocal microscopy and electon microscopy. FRET studies are consistent with a close physical interaction between actin and the Galphai protein. The GTP-bound form of the protein recruits more WAVE1 and Arp2/3 to cell protrusion regions than does the GDP-bound form. Modeling protein-protein interactions suggests that the GDP-bound form of this G protein would likely competes with G-actin for binding to the WH2 domain (part of VCA domain) of WAVE1 and WAVE2 protein. We have established collaborations with Phillip Cruz (NIAID, NIH) to assist with bioinformatics and molecular modeling of the interactions of Galphai with the WAVE regulatory complex proteins and with Baoyn Chen (Iowa State University) to examine the interactions of Galphai proteins with the WAVE regulatory complex via direct biochemical studies. To better understand the role of actin regulation in vivo we have established a novel four-dimensional imaging platform to precisely determine the profile and dynamics of lymphocyte transmigration in vivo. This 4D imaging system allows for advanced spatial and temporal resolution. By labeling the lymph node vasculature with fluorescently-labeled antibody against PECAM-1 we documented that lymphocytes predominated crossed high endothelia venules (HEVs) by migrating through endothelial cell junctions. Furthermore, we observed real-time HEV pocket formation. To monitor F-actin dynamics we have used LifeAct-GFP bone-marrow reconstituted mice as a source of lymphocytes for adoptive transfer. Since the cells very rapidly access the HEVs following intravenous infusion, we can treat the cells prior to transfer with various inhibitors of actin polymerization. We have found that ARP2/3 and formin inhibitors curtail lymphocyte actin dynamics in the HEVs and largely prevent transmigration. Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein are essential in transducing signals from the microenvironment. We use genetically modified mice to investigate the role of Galphai2 in inflammasome activity and macrophage polarization. Galphai2 in murine bone marrow-derived macrophages (BMDMs) regulates inflammasome activity independent of inflammasome activated (NLRP3, AIM2, and NLRC4). This regulation stems from the biased polarity of BMDMs. We determined that BMDMs with excess Galphai2 signaling have a tendency towards classically activated pro-inflammatory (M1) phenotype, Galphai2 deficient are biased towards alternatively activated anti-inflammatory (M2) phenotype. Long-term, but not short-term inhibition of Gi with pertussis toxin recapitulates the knockout phenotype, indicating that the inflammatory changes are built into the macrophage life history. This data indicates that excess Galphi2 signaling promotes an M1 macrophage phenotype, while Galphai2 signaling deficiency promotes an M2 phenotype. Understanding Galphai2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.