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Functional proteomics of cellular mechanosensing mechanisms

细胞机械传感机制的功能蛋白质组学

基本信息

批准号：
324864813
负责人：
Dr. Herbert Schiller
金额：
--
依托单位：
Institute of Lung Health and Immunity (LHI)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/324864813?language=en
关键词：
Functional proteomics cellular mechanosensing mechanisms

项目摘要

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.

在过去的10年里，机械生物学领域迅速发展，各种数据支持了这样一个假设，即大多数细胞(如果不是所有细胞)都会处理来自环境的机械输入，以协调它们的活动。这些机械敏感信号级联的分子细节目前还没有得到很好的描述。这里提出的这个项目是我之前在整合素介导的机械传感机制方面工作的继续。我们的初步数据显示，我的实验室建立了一套高度灵敏的质谱分析方法，我们将使用它们来分析和关联细胞刚性检测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，以及(2)当细胞探测其ECM环境的刚性时，伴随的细胞磷酸化格局的变化。我们将(3)使用CRISPR/Cas9介导的基因组编辑来产生功能亚磷酸盐突变体的敲除线以及丢失和获得，并使用粘附性微图案上的细胞收缩分析来研究细胞机械感觉的变化。对基因敲除系和突变体的磷酸蛋白质组学分析将揭示机械感知中信号模块的相互依赖关系，并揭示细胞机械输入的必要中枢和/或阈值调节器。因此，我们的目标是通过使用先进的细胞生物学技术与质谱学驱动(磷酸)蛋白质组学和CRISPR/Cas9介导的基因组编辑相结合来了解细胞机械传感的基本分子原理。我们将识别和功能询问关键蛋白质和磷酸化开关，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。