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

透明相关的Forins组成了一个普遍存在的保守的多结构域蛋白家族，它催化线性肌动蛋白细丝的从头成核，并作为Rho GTPase信号通路的下游效应器来控制许多细胞功能，包括建立细胞形状或细胞迁移。我们正在进行的对10个DictyostelialForins的分析出人意料地显示，受调控的Forin dDia1的消除强烈地加速了细胞的运动，而dDia1过表达的细胞的迁移显著受到抑制。通过活性dDia1变异体的成像评估，福尔马林聚集在质膜周围的一层薄层中，并在细胞皮质的囊泡结构中最强烈地浓缩。本项目的目的是通过荧光和相关电子显微镜来表征这些高度动态的结构，并找出dDia1到底是如何对细胞迁移产生意想不到的负面影响的。在体内，福尔马林似乎严格依赖于FH1-结构域对Profilin-Actin复合体的招募，以便由FH2-2结构域进一步组装。因此，我们还打算深入研究Dictyostelialforc的肌动蛋白聚合，因为它是唯一已知的缺乏FH1-区的福尔马林。由于Forc也缺乏其他典型的G-肌动蛋白结合位点，第二个项目的目的是识别和表征这种Formin完成招募肌动蛋白亚单位进行后续微丝组装的可能机制。我们预计这两个项目将极大地增加我们对福尔明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.