The sensing of force by cells is determinant for processes like morphogenesis, wound healing, or tumour formation, and can control functions such as proliferation, differentiation, or gene expression. However, the specific mechanisms by which cells sense mechanical forces and respond accordingly remain largely unknown. The proposed project aims to characterize cell mechanotransduction through the study of the Src family kinase substrate p130Cas (Cas, Crk associated substrate), a key protein involved in many cellular processes which is known to be activated (phosphorylated) by force. In the first part of the project, carried out at the outgoing institution, optical and magnetic tweezers and Total Internal Reflection Microscopy (TIRF) will be employed to examine how the phosphorylation of single Cas molecules is regulated by force. Individual Cas molecules will be stretched, and TIRF will be used to determine the force required for phosphorylation, if increased forces lead to increased phosphorylation (providing thus for a mechanism for the sensing of force magnitude), and if discontinuing the application of force leads to dephosphorylation (providing for a mechanism for the sensing of force interruption). In the second part of the project, which will take place mostly at the return institution, magnetic tweezers will be used to study force-dependent Cas phosphorylation in living single cells. The sensing of mechanical forces by cells will be analyzed by studying how these stimuli phosphorylate Cas locally at the site of force application, and globally through cytoskeletal force transmission. The role of the cytoskeleton in integrating local mechanical stimuli will be examined by modifying and measuring its mechanical state through micropatterning technologies. The interest of the project covers disciplines such as biophysics, cell biology nanotechnology, and tissue engineering.
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