Functional proteomics of cellular mechanosensing mechanisms

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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.