Mechanisms of FHOD1 regulated stress fiber formation

肌动蛋白细胞骨架的调节对于许多过程如细胞迁移和细胞分裂是至关重要的。Rho GTPases下游的多个Rho蛋白家族成员已被鉴定为体外和体内肌动蛋白成核的关键调节因子。特别是，mDia 1和FHOD 1被认为是调节肌动蛋白的应力纤维和细胞收缩性。mDia 1本身并不主要与收缩性结构（如应力纤维）结合，但与它们的调节有关。相比之下，FHOD 1与应力纤维本身密切相关，我们的初步结果也确定了这种肌动蛋白成核剂在粘着斑，形成应力纤维的锚点。 在目前的研究阶段，这些formins的许多生化特性已经被阐明，这描绘了一个相当精确的画面，它们如何作为单独的分子机器工作。然而，它们与细胞机制的相互作用仍然知之甚少。特别是，在不清楚，如何招聘和时空激活动力学的这些formins控制本地化肌动蛋白聚合，以建立动态的细胞结构，如应力纤维。 Rho GTPases在Formin的活化机制中起着重要的作用。有趣的是，虽然mDia 1和FHOD 1的生化激活机制-通过打开头到尾的分子内抑制-是相似的，但在细胞环境中Rho GTP酶下游调控的特异性仍然知之甚少-特别是对于研究较少的Rho FHOD 1。 在这里，我们建议使用一个多方面的方法，包括RNA干扰，荧光活性生物传感器和TIRF斑点显微镜，研究hepactin成核剂如何控制细胞收缩下游的Rho信号通路。首先，我们将研究细胞收缩性是如何调节formins和上游GEF-H1/RhoA/ROCK通路。其次，我们将评估依赖于mDia 1和FHOD 1的本地化动力学的Rho GTP酶RhoA，Rac 1和Cdc 42。第三，我们将与时空的Rho GTdR活动模式的相关性。最后，我们将使用光诱导技术来操纵Rho GTdR活性的亚细胞分布，并研究这种外部操纵如何影响GTdR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.