Bert Poedts

Endothelial cells lining blood vessels constantly interact with their extracellular environment. While the chemical interactions have been extensively studied, there is a whole range of physical signals underexplored (e.g. stiffness of the ECM and shear stress from the blood flow) which elicit biological reactions. Unsurprisingly, these mechanical signals are altered in vascular diseases such as cerebral cavernous malformations (CCM), leading us to increasingly apply techniques to expand our understanding of endothelial cell behavior in response to physical forces. However, there is a limited comprehension of force propagation in sub-cellular structures. Here, we describe the use of FRET-based molecular tension sensors to determine forces across particular proteins in endothelial cells. The critical element in molecular force sensors is the stress sensitive module, placed between the FRET donor and acceptor molecules. These sensing modules will deform and extend in length upon application of a physiological force and can be placed within relevant structural proteins, targeting the sensor to specific intracellular structures. To calculate the distance between donor and acceptor, and therefore the applied force, we perform fluorescence lifetime FRET (FRET-FLIM) where the fluorescence lifetime of the donor is recorded. The resulting data will be analyzed using phasor-FLIM, fit-free analysis is facilitated leading to more accurate and unbiased results to extract FRET efficiency. Using this, FRET-based molecular tension sensors linked to structural proteins in cell-cell and cell-ECM interactions, exposed to a range of extrinsic mechanical and biochemical factors, further paves the way to provide a molecular force connectivity map of the vascular mechanotransduction pathways. This in turn will help us to not only better understand all factors involved in vascular diseases, but also lead to more comprehensive treatments.