Focal adhesion protein dynamics and the role of endosomes in contractile, fully differentiated, vascular smooth muscle
Poythress, Ransom Harold
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Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle cells (VSMCs). However, it is often assumed that FAs in non-migratory, differentiated vascular smooth muscle cells (dVSMCs) embedded in the wall of healthy blood vessels are static structures. Recent work from our lab has demonstrated agonist-induced actin polymerization and Src-dependent focal adhesion phosphorylation in dVSMCs, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the alpha-agonist phenylephrine. VASP and Zyxin displayed the largest redistributions while beta-integrin and FAK showed undetectable redistribution. Vinculin, metavinculin, Src, CAS, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and EEA1. Endosomal disruption with the small molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B and colchicine, indicating that the redistribution is actin and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus, these studies are consistent with the concept that FAs in dVSMCs, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.