Functional proteomics of cellular mechanosensing mechanisms

在过去的10年里，机械生物学领域迅速发展，各种各样的数据加强了这样的假设，即大多数细胞（如果不是全部的话）处理来自环境的机械输入来协调它们的活动。这些机械敏感信号级联的分子细节目前还没有很好地表征。这里提出的项目是我之前关于整合素介导的机械传感机制的工作的延续。我们的初步数据显示，在我的实验室建立了一套高灵敏度的最先进的质谱分析方法，我们将使用它来分析和关联细胞刚度传感过程中的各种分子参数。我们将(1)分析蛋白质亚细胞定位的变化，(2)细胞磷酸化景观的伴随变化，同时细胞探测其ECM环境的刚性。我们将(3)利用CRISPR/Cas9介导的基因组编辑技术生成敲除系以及功能磷位点突变体的损失和获得，并利用粘附微模式上的细胞收缩性试验研究细胞机械传感的变化。敲除系和突变体的磷酸化蛋白质组学分析将揭示机械传感中信号模块的相互依赖性，并揭示细胞中机械输入的基本枢纽和/或阈值调节剂。因此，我们的目标是通过将先进的细胞生物学技术与质谱驱动（磷-）蛋白质组学和CRISPR/Cas9介导的基因组编辑相结合，了解细胞机械传感的基本分子原理。我们将鉴定和功能性询问关键蛋白和磷酸化开关，以了解它们在组织纤维化中成纤维细胞活性控制中的重要性。

In the last 10 years the field of mechanobiology rapidly evolved and a variety of data strengthens the hypothesis that most if not all cells process mechanical inputs from their environment to coordinate their activities. The molecular details of these mechanosensitive signaling cascades are currently not very well characterized. The project proposed here is a continuation of my previous work on integrin mediated mechanosensing mechanisms. Our preliminary data shows that a set of highly sensitive state of the art mass spectrometry assays is established in my laboratory, which we will use to analyze and correlate various molecular parameters during cellular rigidity sensing. We will (1) analyze changes in subcellular localization of proteins, and (2) the accompanying alterations of the cellular phosphorylation landscape, while cells probe the rigidity of their ECM environment. We will (3) generate knock out lines as well as loss and gain of function phosphosite mutants using CRISPR/Cas9 mediated genome editing, and study alterations in cellular mechanosensing using cell contractility assays on adhesive micropatterns. Phosphoproteomic analysis of knock out lines and mutants will reveal the interdependence of signaling modules in mechanosensing and uncover essential hubs and/or threshold modulators for mechanical inputs in cells. Thus, our aim is to understand fundamental molecular principles of cellular mechanosensing by using a combination of advanced cell biology techniques with mass spectrometry driven (phospho-)proteomics and CRISPR/Cas9 mediated genome editing. We will identify and functionally interrogate critical proteins and phosphorylation switches in order to understand their importance in the control of fibroblast activity in tissue fibrosis.