Functional proteomics of cellular mechanosensing mechanisms
Final Report Abstract
Cells actively sense their environment by translating mechanical properties of the extracellular matrix, such as stiffness or shear stress, into biochemical signals. This cellular mechanosensing is crucial for development, tissue homeostasis and the outcome of many diseases. Mechanosensitive signaling pathways are important in the pathophysiology of fibrotic diseases, where the composition of the extracellular matrix (ECM) and its mechanical properties are altered in disease progression. The activity and survival of activated fibroblasts in fibrotic diseases may be largely controlled by mechanical signals. In this process, the structure and organization of the cytoskeleton is modified, which also induces long term gene expression changes. The precise molecular nature of many elements in these feedback connections is currently unknown. In this work, we explored proteome-wide molecular changes in protein phosphorylation during mechanosensing and cell spreading in human lung fibroblasts using mass spectrometry driven phosphoproteomics. We used a label-free quantification of phosphopeptides to generate time-resolved phosphoproteomes during fibroblast mechanosensing. The analysis of differential phosphorylation on varying substrates stiffnesses allowed us to identify rigidity- dependent regulated phosphosites. The pro-fibrogenic phenotype of lung fibroblasts on stiff substrates was accompanied by a massive remodelling of the phosphoproteome, including induction of the Yap signaling pathway, increased cell contractility and cytoskeletal rearrangements. We identified stiffness dependent regulation of novel transcriptional regulators and components of the mTOR pathway that are currently investigated functionally using CRISPR mediated point mutations on the identified target sites for phosphorylation. On completion, this work has the potential to reveal novel therapeutic targets for modulation of (myo-)fibroblast identity in fibrotic diseases.
Publications
- (2017). Functional proteomics of cellular mechanosensing mechanisms. Semin. Cell Dev. Biol. 71, 118–128
Wasik, A.A., and Schiller, H.B.
(See online at https://doi.org/10.1016/j.semcdb.2017.06.019)
