An integrated experimental and theoretical approach to investigate endothelial cell alignment.

Endothelial cells respond to both internal and external mechanical stimuli. Forces within the cytoskeleton and cell-substrate interactions regulate their elongation. This study examines the role of these forces in cellular alignment, combining in vitro experiments with mathematical modelling. Our in vitro approach employs a sparse endothelial cell colony, distinctly tagged for myosin-II, F-actin, and focal adhesion proteins. We modulate myosin-II pharmacologically and through live imaging, determine actin-myosin dynamics and resulting cell shape changes. The empirical results show that by increasing myosin-II levels, contractility and alignment are enhanced, evidenced by the prominence of stress fibres. This alignment is more pronounced on stiffer substrates due to increased cell-substrate interactions from a higher concentration of focal adhesions. Using this data, we formulated a coarse-grained active nematic model accounting for the viscoelastic properties of the actin network, active forces from bundling proteins, actin turnover, interactions between the actin network and the extracellular matrix via focal adhesion complexes, and contrasts between isotropic and bundled nematic actin networks. The numerical solution of the model reveals the internal mechanism involved in the progression of cell elongation. Initially, within a circular cell configuration, stress fibres emerge due to a symmetry-breaking phenomenon. Subsequently, a distinct differential increase in contractility is observed in a particular direction, promoting the strengthening of stress fibres in that orientation. Simultaneously, focal adhesions bind to contractile stress fibres and further enhance the alignment of stress fibres through dynamic actin remodelling. The improved alignment of stress fibres allows the bundling protein to exert anisotropic active forces, leading to a pronounced alignment in the overall cellular morphology.