The mechanisms underlying the GPCR-mediated chemotaxis in D. discoideum

1：cAR1介导的RAS信号的时空动力学。为了揭示抑制机制，我们通过观察荧光探针活性RAS结合结构域与GFP融合(RBD-GFP)对cAMP梯度的响应来监测RAS的时空激活。我们观察到的RAS激活动力学表明，一个负调控因子，可能是RasGAP，逐渐被招募到膜上，促进RAS失活。在哺乳动物细胞中，含有磷脂结合域的RasGAP被证明移位到质膜内表面，使RAS失活，从而抑制PI3K。我们认为，一个类似的磷脂结合的RasGAP很可能是盘状螺旋藻中cAR1介导的化学传感的重要抑制物。为了验证这一假说，我们正在研究RasGAP在盘状芽孢杆菌化学传感中的作用。 2：一种新的GBG效应器ElmoE在趋化过程中将GPCR信号转导到肌动蛋白网络。 G蛋白偶联受体(GPCRs)的激活导致异源三聚体G蛋白解离成GA和GBG亚基，进而调节参与一系列细胞反应的各种效应因子。在趋化过程中，GBG亚单位调节肌动蛋白的组装和迁移，但连接GBG和肌动蛋白细胞骨架的蛋白(S)仍然未知。在这里，我们发现了一种新的GBG效应因子ElmoE，它存在于Dictyostelius中，并且证明它是GPCR介导的趋化作用所必需的。值得注意的是，ElmoE与GBG和Dock样蛋白相互作用，以一种依赖于GPCR的方式激活小GTP酶Rac，并与Arp2/3复合体和F-肌动蛋白结合。因此，ElmoE是趋化因子GPCRs、G蛋白和肌动蛋白细胞骨架之间的第一个直接联系。该途径由GPCR、GBG、Elmo/Dock、Rac和Arp2/3组成，在趋化过程中空间指导真核细胞伪足中树突状肌动蛋白网络的生长(Yan等人Dev.单元格2012)。

1: cAR1-mediated spatiotemporal dynamics of Ras signaling. To reveal the inhibitory mechanism, we monitored spatiotemporal activation of Ras by observing the membrane translocation of a fluorescent probe, active Ras Binding Domain fused to GFP (RBD-GFP) in response to a cAMP gradient. The Ras-activation dynamics we observed indicate that a negative regulator, possibly a RasGAP, is gradually recruited to the membrane and promotes Ras inactivation. In mammalian cells, a RasGAP containing a phospholipid-binding domain has been shown to translocate to the plasma membrane's inner face, deactivate Ras, and thereby inhibit PI3K. We proposed that a similar phospholipid-bound RasGAP is likely to be an important inhibitor in cAR1-mediated chemosensing in D. discoideum. To test this hypothesis, we are studying the roles of RasGAP in chemosensing in D. discoideum. 2: A novel Gbg effector, ElmoE, transduces GPCR signaling to the actin network during chemotaxis. Activation of G-protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Ga and Gbg subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gbg subunits regulate actin assembly and migration, but the protein(s) linking Gbg to the actin cytoskeleton remains unknown. Here, we identified a new Gbg effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE interacts with Gbg and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as the first direct link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gbg, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis (Yan et al Dev. Cell 2012).