Mechanisms of FHOD1 regulated stress fiber formation

肌动蛋白细胞骨架的调控在细胞迁移和细胞分裂等许多过程中起着至关重要的作用。在Rho GTP酶的下游，Forin蛋白家族的多个成员已被确定为体外和体内肌动蛋白成核的关键调节因子。特别是，Forins mDia1和FHOD1被认为调节基于肌动蛋白的应激纤维和细胞收缩能力。MDia1本身并不主要与收缩结构(如应力纤维)结合，但与它们的调节有关。相反，FHOD1与应力纤维本身有很强的相关性，我们的初步结果也发现了这种位于局部粘连的肌动蛋白核因子，这形成了应力纤维的锚点。在目前的研究阶段，这些福尔马林的许多生化特性已经被阐明，这就相当准确地描绘了它们是如何作为单独的分子机器工作的。然而，它们与细胞机制的相互作用仍然知之甚少。特别是，尚不清楚这些Form in的招募和时空激活动力学如何控制局部的肌动蛋白聚合，以建立动态的细胞结构，如应力纤维。Rho GTP酶已被证明在Forins的激活机制中起着至关重要的作用。有趣的是，尽管对于mDia1和FHOD1来说，生化激活机制--通过打开头到尾的分子内抑制--是相似的，但在细胞背景下调节Rho GTP酶下游的特异性仍然知之甚少--特别是对于研究较少的Forin FHOD1。在这里，我们建议使用多方面的方法，包括RNA干扰，荧光活性生物传感器和TIRF散斑显微镜来研究Forin肌动蛋白核苷如何控制Rho信号通路下游的细胞收缩。首先，我们将研究福尔马林和上游的全球环境基金-H1/RhoA/ROCK通路是如何调节细胞收缩的。其次，我们将评估mDia1和FHOD1的定位动力学对Rho GTP酶RhoA、rac1和CDc42的依赖性。第三，我们将把Forin动力学与Rho GTP酶的时空活性模式联系起来。最后，我们将使用光诱导技术来操纵Rho GTP酶活性的亚细胞分布，并研究这种外部操纵如何影响Forin功能。

Regulation of the actin cytoskeleton is critical for many processes such as cell migration and cell division. Downstream of Rho GTPases multiple members of the formin protein family have been identified as critical regulators of actin nucleation in vitro and in vivo. In particular, the formins mDia1 and FHOD1 are thought to regulate actin based stress fibers and cell contractility. mDia1 itself is not predominantly bound to contractile structures, such as stress fibers, but has been implicated in their regulation. In contrast, FHOD1 is strongly associated with stress fibers themselves and our preliminary results also identify this actin nucleator at focal adhesions, which form anchor points for stress fibers. At the current stage of research, many biochemical properties of these formins have been elucidated, which draw a fairly precise picture, how they work as individual molecular machines. However, their interplay with the cellular machinery is still poorly understood. In particular, in is not clear, how the recruitment and spatio-temporal activation dynamics of these formins controls localized actin polymerization to build dynamic cellular structures, such as stress fibers. Rho GTPases have been shown to play crucial role in the activation mechanism of formins. Interestingly, although for both mDia1 and FHOD1, the biochemical activation mechanism – via opening of a head-to-tail intramolecular inhibition - is similar, specificity of regulation downstream of Rho GTPases in the cellular context is still poorly understood – especially for the less studied formin FHOD1. Here, we propose to use a multi-facetted approach including RNA interference, fluorescence activity biosensors and TIRF speckle microscopy to study how formin actin nucleators control cell contractility downstream of Rho signaling pathways. First, we will study how cell contractility is regulated by formins and the upstream GEF-H1/RhoA/ROCK pathway. Second, we will assess the dependence of mDia1 and FHOD1 localization dynamics on the Rho GTPases RhoA, Rac1 and Cdc42. Third, we will correlate formin dynamics with spatio-temporal Rho GTPase activity patterns. Finally, we will use a light inducible technique to manipulate the subcellular distribution of Rho GTPase activity and study, how such external manipulations affect formin function.