Shear stress induces concentration gradient distributions of membrane proteins in live cells

Cells sense and respond to fluid shear stress. Cell surfaces are exposed to flow, yet the influence of shear stress on the behavior of plasma membrane proteins remains unclear. Here, we show that extracellular flow induces the gradient distribution of cell membrane proteins with increasing concentration toward the downstream direction of the flow. Shear stress of 10-30 dynes/cm² caused the formation of concentration gradients of both glycosylphosphatidylinositol (GPI)-anchored proteins and transmembrane proteins, including integrin β1, E-cadherin, and the insulin receptor in Xenopus XTC cells. Using single-molecule live-cell imaging, we found that GPI-anchored T-cadherin molecules are dragged along the direction of flow under shear stress. The extent of gradient formation varied among membrane proteins. Vascular endothelial (VE)-cadherin showed minimal gradient formation, and its response was unaffected by disruption of the actin cytoskeleton or cholesterol depletion. We also observed cell line-dependent differences in the response of GPI-anchored proteins. In Xenopus A6 kidney epithelial cells, gradient formation of GPI-anchored EGFP (EGFP-GPI) was less pronounced than in XTC cells. Actin disruption modestly enhanced EGFP-GPI gradient formation, suggesting a partial role for the cortical actin network in modulating cell line-dependent responses. In Cos-7 cells and human umbilical vein endothelial cells (HUVECs), similar gradients of GPI-anchored proteins were observed but required higher shear stress. Our findings suggest that external flow directly transports membrane proteins, establishing concentration gradients that may contribute to the cellular flow-sensing mechanism.