Dissection of formin pathways coordinating polarity with cell migration downstream of Ras signalling

透明蛋白相关的形成蛋白构成了一个保守的多结构域蛋白家族，它催化线性肌动蛋白细丝的新生成核，并作为Rho GTPase信号通路的下游效应物，控制许多细胞功能，包括细胞形状的建立或细胞迁移。我们对10个Dictyostelium formmins的持续分析意外地揭示，消除受调节的formin dDia1强烈地加速了细胞的运动，而过表达dDia1的细胞的迁移被显著抑制。通过dDia1活性变体的成像评估，甲双胍在质膜周围的薄层中积聚，并且在细胞皮层的囊泡结构中富集最强烈。本项目的目的是通过荧光和相关的电子显微镜来表征这些高度动态的结构，并找出dDia1究竟是如何对细胞迁移产生意想不到的负面影响的。在体内，形成蛋白似乎严格依赖于fh1结构域对profile -actin复合物的招募，以便在FH2结构域进一步组装。因此，我们也打算深入研究Dictyostelium ForC的肌动蛋白聚合，因为它是已知唯一缺乏fh1区域的双胍。由于ForC也缺乏其他典型的g -肌动蛋白结合位点，第二个项目旨在确定和表征这种双胍完成招募肌动蛋白亚基用于随后的丝组装的可能机制。我们期望这两个项目将极大地增加我们对formin介导的肌动蛋白组装的了解。

Diaphanous-related formins constitute a conserved family of ubiquitous multidomain proteins that catalyze the de novo nucleation of linear actin filaments and act as downstream effectors of Rho GTPase signaling pathways to control numerous cellular functions including the establishment of cell shape or cell migration. Our ongoing analysis of the 10 Dictyostelium formins unexpectedly revealed that the elimination of the regulated formin dDia1 strongly accelerated cell motility, whereas migration of dDia1-overexpressing cells was significantly suppressed. As assessed by imaging of an active dDia1 variant, the formin accumulated in a thin layer around the plasma membrane and was most strongly enriched in vesicular structures at the cell cortex. The aim of this project is to characterize these highly dynamic structures by epifluorescence and correlative electron microscopy and find out how exactly dDia1 brings about it unexpected negative effect on cell migration. In vivo formins seem to strictly depend on the recruitment of profilin-actin complexes by the FH1-domain for further assembly by the FH2 2 domain. Therefore, we also intend to thoroughly study actin polymerization by Dictyostelium ForC, because it is the only known formin lacking the FH1-region. As ForC is also devoid of other canonical G-actin binding sites, the second project aims to identify and characterize possible mechanisms by which this formin accomplishes to recruit actin subunits for subsequent filament assembly. We expect that both projects will greatly add to our knowledge of formin-mediated actin assembly.