Fluidic shear stress alters clathrin dynamics and vesicle formation in endothelial cells.

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs cells. How ECs overcome such unfavorable, from biophysical perspective, conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to show that endocytic dynamics are similar between statically cultured human umbilical vein endothelial cells (HUVECs) and fibroblast-like Cos-7 cells. Here we asked whether biophysical stresses generated by blood flow could favor one mechanism of clathrin-coated vesicle formation to overcome environment present in vasculature. We used our data processing platform - DrSTAR - to examine if clathrin dynamics are altered in HUVECs grown under fluidic sheer stress (FSS). Surprisingly, we found that FSS led to an increase in clathrin dynamics. In HUVECs grown under FSS we observed a 2.3-fold increase in clathrin-coated vesicle formation and a 1.9-fold increase in non-productive flat clathrin lattices compared to cells grown in static conditions. The curvature-positive events had significantly delayed curvature initiation in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local physiological environment and represents an important regulatory mechanism.