Fluid Flow as a Strategy for Sorting and Localization of Membrane Proteins

Abstract

Many cells, such as leukocytes, endothelial cells, and osteoblasts, exhibit dramatic biochemical and biophysical responses to shear flow. However, the molecular-scale mechanisms of flow mechanotransduction are complex and details remain obscure [1]. It has been observed that large GPI-anchored proteins are reorganized following application of shear flow to living cells [2], but whether this is the result of advection or of active intracellular transport has not yet been determined. Here, we investigate whether physiological levels of fluid flow applied to living cells can redistribute surface proteins. We subject a confluent monolayer of human cells to controlled shear flow. These cells are modified to express a construct comprising extracellular GFP attached to the outer leaflet of the cell plasma membrane through a GPI anchor. We track protein movement across the cells with the goal of estimating the hydrodynamic forces felt by the extracellular domain and the frictional force experienced by the membrane anchor. We relate our results to experiments on supported bilayers. We also investigate the contributions of the cytoskeleton and plasma membrane lipid composition to protein mobility.[1] Conway and Schwarz. Flow-dependent cellular mechanotransduction in atherosclerosis. Journal of Cell Science, 126, 5101 (2013).[2] Zeng, Waters, Honarmandi, Ebong, Rizzo, and Tarbell. Fluid Shear Stress induces the clustering of heparan sulfate via mobility of glypican-1 in lipid rafts. American Journal of Physiology. 305(6) (2013) and also Zeng and Tarbell, Adaptive Remodeling of the Endothelial Glycocalyx in Response to Fluid Shear Stress. PLOS ONE 9 (1) e86249 (2014).