Regulation of small GTPase signalling networks and chaperone-assisted selective autophagy through mechanical forces in leukocytes and cardiomyocytes

机械应力作用于免疫细胞，这些免疫细胞从血管系统中出来，在血管腔的内皮层上爬行，或者在组织界面或间隙中遇到机械障碍。我们的实验室专注于蛋白质功能和动力学的生物物理传感器的构建，以及控制白细胞粘附和迁移的力转导的细胞内通信途径的生化和功能阐明。通过对小鼠进行基因消融，我们鉴定了adp -核糖基化因子(Arf)- gtpase的鸟嘌呤核苷酸交换因子（GEFs）细胞素家族，作为整合素介导的白细胞细胞粘附的调节因子。免疫系统和心肌细胞中细胞胞素-2的组织特异性基因消融分别导致所谓树突状细胞粘附信号和迁移的改变，以及小鼠心脏纤维化和过早死亡。此外，我们早些时候与Höhfeld实验室合作表明，BAG3伴侣蛋白机制不仅在肌肉细胞中参与机械转导，而且在免疫细胞中参与机械转导。最近的证据表明，在免疫细胞和心肌细胞中，细胞分裂素依赖的功能和整合素激活的基本调节因子Rap1-GTPase信号传导集中在RhoA和BAG3上。因此，在本项目中，我们将阐明Arf-GEF/Arf-GTPase依赖的信号机制在控制机械应力保护中的作用。我们将利用遗传学，生化方法和局部亚细胞GTPase信号的动态可视化，使用最先进的生物传感器技术来阐明通过细胞分裂素/Rap1/RhoA/BAG3轴的应激信号。我们的工作将建立白细胞和心肌细胞机械应力保护所需的力依赖信号网络。

Mechanical stress acts on immune cells, which exit from the vasculature, which crawl on endothelial layers in the vascular lumen, or which are confronted with mechanical barriers in at tissue interfaces or in interstitial spaces. Our labs specialize in the construction of biophysical sensors of protein functions and dynamics, as well as the biochemical and functional elucidation of intracellular communication pathways, which control force transduction in leukocyte adhesion and migration. Using genetic ablation in the mouse, we identified the cytohesin family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor (Arf)-GTPases, as regulators of integrin-mediated cell adhesion in leukocytes. Tissue specific genetic ablations of cytohesin-2 in the immune system and in cardiomyocytes led to altered adhesion signalling and migration in so-called dendritic cells, and to heart fibrosis and premature death in mice, respectively. Furthermore, we showed earlier in collaboration with the Höhfeld lab that the BAG3 chaperone machinery contributes to mechanotransduction not only in muscle cells, but also in immune cells. Recent evidence suggests that cytohesin-dependent functions and Rap1-GTPase signalling, a fundamental regulator of integrin activation, converge on RhoA and BAG3 in immune cells and in heart muscle cells. In this project, we will therefore elucidate the role of Arf-GEF/Arf-GTPase dependent signalling mechanism in the control of mechanical stress protection. We will utilize genetics, biochemical methods and dynamic visualization of local subcellular GTPase signalling, using state-of-the-art biosensor technologies to elucidate stress signalling via the cytohesin/Rap1/RhoA/BAG3 axes. Our work will establish force-dependent signalling networks required for mechanical stress protection in leukocytes and cardiomyocytes.