Shear stress shifts clathrin-coated vesicle formation to favor the flat-to-curved model in endothelial cells.

Abstract

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 conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to resolve how clathrin-coated vesicles (CCVs) from in fibroblast-like cells (Cos-7) and human umbilical vein endothelial cells (HUVECs). This revealed multiple productive ways of CCV formation, supporting the flexible model of CME. We next asked whether biophysical stresses generated by blood flow could favor one mechanism of CCV formation to overcome unfavorable environment present in vasculature. Addressing this question required development of an automated MATLAB-based accelerated STAR data processing pipeline (DrSTAR) to enable the processing of multiple experimental conditions and biological replicates in a robust and reproducible environment. Moreover, DrSTAR employs a dynamic local referencing algorithm, which resolves the curvature of long-lasting CCSs. We used this new platform to examine if clathrin dynamics are altered in HUVECs grown under sheer stress. We found sheer stress led to an increase in clathrin dynamics. Surprisingly, we found this was due to an increase of flat clathrin accumulations in flow-stimulated cells, while the number of curved events remained consistent between groups. 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. Moreover, CME was more resistant to increased osmotic pressure in HUVECs grown under shear stress. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local environment and represents an important regulatory mechanism.