Role of RAB8 and Caveolin-1 in cell mechanotransduction and vascular biology

Résumé

Mechanical forces are constantly generated during organ function and within tissues during cellular activities. Eukaryotic cells have evolved mechanotransduction pathways that allow them to sense physical stimuli and trigger adaptive responses, resulting in their functional reprogramming and the remodeling of the architecture of their extracellular environment. Recent research supports that these mechanoadaptive pathways, beyond protecting cells from physical damage, can profoundly impact cell behavior and affect the progression of complex diseases such as atherosclerosis. The thesis research reported here focuses on two proteins involved in cell mechanoadaption, specifically in the regulation of intracellular trafficking and plasma membrane (PM) organization: RAB8 and caveolin-1 (CAV1). A combination of different biophysical techniques has demonstrated that RAB8 localization is modulated by changes in PM tension or integrity, suggesting that specific RAB8 traffic pathways are activated in response to mechanical stimuli. In order to characterize the physiological role of RAB8 traffic in the vascular system, CRISPr-Cas9-edited endothelial cell lines were developed and tested in microfluidics systems. The data obtained indicates that RAB8 is required for the activation of flow shear stress mechanotransduction pathways, and for the regulation of CAV1 phosphorylation dynamics in response to flow. These results suggest that RAB8 may play a relevant role in endothelial homeostasis, and in the physiopathology of cardiovascular disorders such as atherosclerosis. Since RAB8 and CAV1 are both involved in lipid homeostasis and mechanoadaption, we hypothesized that intervening their expression would affect LDL transport in the endothelium in response to different mechanical conditions. To test that, in vitro assays were developed ad hoc to study the effect of substrate architecture and stiffness on LDL trafficking and retention. While our observations were not conclusive regarding the specific involvement of RAB8 or CAV1 in the coupling of substrate sensing and LDL management, these optimized, quantitative in vitro assays offer a versatile opportunity for the detailed study of the different phases of LDL transport that occurs in blood vessels during atherosclerosis progression