1 Role of N - WASP in Endothelial Monolayer Formation and Integrity

Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of Factin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function. INTRODUCTION Endothelial cells (ECs) line the vasculature, and they regulate the trafficking of fluid, metabolites and cells between the blood and tissue. Endothelial cell-cell junctions are highly dynamic and tightly regulated (1-3); dysfunctional junctions can be associated with human disease (4-7) . Actin filaments are organized as a loose circumferential band near endothelial cell junctions. The actin network and cell junctions are organized in a coordinated manner to regulate a variety of physiological and pathological processes, including transendothelial migration by immune and cancer cells. Endothelial cells are highly dynamic at their junctions, frequently sending out lamellipodial protrusions to establish and maintain the cell-cell contacts that hold the monolayer together (8, 9). Endothelial junctions consist of adherens junctions and tight junctions, each with their associated molecules. The junctions between endothelial cells are not spatially segregated into zones as found in epithelial monolayers (10); the junctional molecules of ECs are dispersed throughout the junctional area (11). A large http://www.jbc.org/cgi/doi/10.1074/jbc.M115.668285 The latest version is at JBC Papers in Press. Published on June 12, 2015 as Manuscript M115.668285 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from