Abstract
Localized Ca2+ influx via TRPV4 on the surface of endothelial cells greatly influences endothelial adaptation to blood flow, but how mechanical stress from blood flow controls TRPV4 integration into this physiological function is not fully understood. Here, we studied the spatial organization of TRPV4 and its relationship to the adherens junction component β-catenin using single- and dual-color direct stochastic optical reconstruction microscopy (dSTORM). In non-stimulated endothelial cells, TRPV4 is clustered in small protein islands, as is β-catenin. Using dual-color imaging, we found that TRPV4 and β-catenin reside in similar islands and can be found at both the basolateral and basal membranes. Following shear stress stimulation, TRPV4 molecules formed smaller clusters, with the majority residing outside of clusters. Further shear stress stimulation changed the molecular distribution of TRPV4 molecules, limiting them to the basal membrane. This redistribution and the smaller clusters resulted in the segregation of TRPV4 from β-catenin. Furthermore, TRPV4 trafficking was controlled by focal adhesion kinase and activation of the α5ß1 integrin. These highly differentiated spatial redistributions suggest that mechanotransduction of blood flow is controlled via a more complex hierarchy than previously thought.
Highlights
We have previously shown that in HEK293 cell stably expressing TRPV4 (TRPV4-HEK293) shear stress activates TRPV4 and leads to increase in [Ca2+]i level in a does dependent manner[12,13,14]
Endothelial cells and human umbilical cord endothelial cells (HUVECs), we have shown that shear stress sensitizes the response of TRPV4 to its selective agonist[12,15,16] and in Human umbilical vein endothelial cells (HUVECs), shear stress increases the exocytosis of functional TRPV4 channels to the cell membrane[16]
We labeled TRPV4 channels expressed in HUVECs with anti-TRPV4 antibodies directly conjugated to a photoactivatable fluorescent dye, Alexa 647
Summary
We have previously shown that in HEK293 cell stably expressing TRPV4 (TRPV4-HEK293) shear stress activates TRPV4 and leads to increase in [Ca2+]i level in a does dependent manner[12,13,14]. TRPV4 interacts with ß-catenin at adherens junctions, linking them to the actin cytoskeleton[17]. An increase in [Ca2+]i disrupts the adherens junction via activation of myosin light-chain kinase and the RhoA-Rho kinase pathway and induces actin stress fiber formation[18,19]. TRPV4 channels formed smaller clusters, with the majority of them relocated from the basolateral membrane to basal membrane, and TRPV4 lost its interaction with β-catenin. The shear-induced translocation of TRPV4 channels was controlled by focal adhesion kinase and α4ß1/α5ß1 integrin
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