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. 2013 Jul 15;305(2):C215-27.
doi: 10.1152/ajpcell.00103.2013. Epub 2013 May 22.

Vasoconstrictor-induced Endocytic Recycling Regulates Focal Adhesion Protein Localization and Function in Vascular Smooth Muscle

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Free PMC article

Vasoconstrictor-induced Endocytic Recycling Regulates Focal Adhesion Protein Localization and Function in Vascular Smooth Muscle

Ransom H Poythress et al. Am J Physiol Cell Physiol. .
Free PMC article

Abstract

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, 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 α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. 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 dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.

Keywords: Src; endosomes; microtubules; zyxin.

Figures

Fig. 1.
Fig. 1.
Differential centrifugation of differentiated vascular smooth muscle (dVSM) tissue distinguishes known cytoskeletal proteins from membrane and cytosolic proteins. A: typical Coomassie blue-stained blot of proteins in cytosolic (C), membrane (M), cytoskeletal (SK) fractions. The majority of overall protein is localized in the cytoskeletal fraction. B: densitometry from A (n = 3). C: Western blots of unstimulated tissues of 3 different proteins typically associated with the 3 subcellular compartments of interest. D: quantitative densitometric analysis of Western blots of total GAPDH, Rab5, and actin (n = 3–15), proteins characteristically associated with cytosol, membrane, and cytoskeleton, respectively. Brightness has been uniformly altered for visual display.
Fig. 2.
Fig. 2.
The α-agonist phenylephrine (PE) triggers redistribution of zyxin, vasodilator-stimulated phosphoprotein (VASP), paxillin, and metavinculin/vinculin to the membrane fraction from the cytosolic fraction. A: typical blots illustrating the time course of stimulus-induced changes in protein subcellular distribution. unstim, Unstimulated (resting). B–E: densitometric analysis of Western blots of focal adhesion (FA) proteins in cytosolic, membrane, and cytoskeletal fractions normalized as a percentage of total protein levels (left) or as a percentage of unstimulated levels (right) from unstimulated or PE-stimulated (10 μmol/l) tissues (n = 4–17). Data for each experiment are from a single gel. Blank space is a spliced-out empty lane. Brightness has been uniformly altered for visual display. *P < 0.05 vs. unstim (2-tailed, paired t-test).
Fig. 3.
Fig. 3.
Crk-associated substrate (CAS) and Src redistribute to the membrane fraction from the cytoskeletal fraction in the presence of PE. Time course of stimulus-induced changes in protein subcellular distribution. A: typical blots. B and C: densitometric analysis of CAS and Src in cytosolic, membrane, and cytoskeletal fractions normalized as a percentage of total protein levels (left) or as a percentage of unstimulated levels (right) from unstimulated or PE-stimulated (10 μmol/l) tissues (n = 6–18). Data for each experiment are from a single gel. Blank space is a spliced out empty lane. Brightness has been uniformly altered for visual display. *P < 0.05 vs. unstim (2-tailed, paired t-test).
Fig. 4.
Fig. 4.
FA kinase (FAK), β-integrin, and α-actinin show no detectable vasoconstrictor-induced redistribution. A: typical blots. B–D: densitometric analysis of FAK, β-integrin, and α-actinin in cytosolic, membrane, and cytoskeletal fractions normalized as a percentage of total protein levels from unstimulated or PE-stimulated (10 μmol/l for 10 min) tissues (n = 4–12). Data for each experiment are from a single gel. Blank space is a spliced-out empty lane. Brightness has been uniformly altered for visual display. *P < 0.05 vs. unstim (2-tailed, paired t-test).
Fig. 5.
Fig. 5.
The FA marker protein zyxin shows increased colocalization with endosome markers in the presence of PE. A: immunofluorescent colocalization of Rab5 and zyxin before (left) and after (right) 10 min of stimulation with PE (10 μmol/l). Arrows indicate colocalization; arrowheads show lack of colocalization and zyxin localization at FA-like structures. B: colocalization of zyxin and early endosomal antigen 1 (EEA1) before (left) and after (right) 10 min of PE (10 μmol/l) stimulation. Arrows indicate colocalized points. Scale bars, 5 μm. C: percentage of the first protein that colocalizes with the second protein. DAPI, 4′,6-diamidino-2-phenylindole. Values are means ± SE; n = 10–14 cells. *P < 0.05 vs. unstim (2-tailed, paired t-test).
Fig. 6.
Fig. 6.
Amount of zyxin and EEA1 in close proximity increases after PE stimulation. Confirmation by proximity ligation assay of close association that increases after 10 min of PE stimulation. A: freshly dissociated dVSM cells were fixed and incubated with zyxin and EEA1 antibodies or zyxin alone as a negative control. Representative images of each condition are shown. Scale bars, 10 μm. Images have been inverted to gray scale for better visualization. Cell outline is based on visible light capture. B: number of signal dots, as a measure for proximity of an antigen pair, was analyzed on the basis of 17–41 cells per experimental condition in 3–5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control, unless otherwise indicated (2-tailed t-test).
Fig. 7.
Fig. 7.
Primaquine (PQ) blocks vasoconstrictor-induced membrane redistribution and reduces contractility. A–D: primaquine reduces PE-induced protein redistribution in response to PE stimulation (n = 4–18) +P < 0.05 vs. PE. *P < 0.05 vs. untreated (Untreat). E: effect of primaquine on the time to 50% steady-state contraction amplitude (n = 6). *P < 0.05. F: primaquine reduces maximal steady-state contractile force in response to PE stimulation (n = 6). *P < 0.05.
Fig. 8.
Fig. 8.
Actin inhibition with cytochalasin D (CytoD) and latrunculin B (LatB) blocks vasoconstrictor-induced redistribution to the membrane fraction. A–D: membrane fraction analysis after differential centrifugation of untreated, PE-stimulated, or cytochalasin D-pretreated and PE-stimulated tissue samples (n = 3–17). +P < 0.05 vs. PE. *P < 0.05 vs. untreated (−). E and F: densitometric analysis of Western blots stained for VASP and zyxin after differential centrifugation of latrunculin B-treated tissue (n = 3–19). +P < 0.05 vs. PE. *P < 0.05 vs. untreated. G: pretreatment with latrunculin B (0.1 μmol/l) for 1 h significantly inhibits maximal tissue contraction strength (n = 3). *P < 0.05.
Fig. 9.
Fig. 9.
Microtubule inhibition with colchicine (Colch) blocks vasoconstrictor-induced redistribution to the membrane fraction. A and B: densitometric membrane fraction analysis after differential centrifugation of untreated, PE-stimulated, or colchicine-treated (15 μmol/l) and PE-stimulated tissue samples (n = 3–17). +P < 0.05 vs. PE. *P < 0.05 vs. untreated. C: colchicine pretreatment for 1 h did not affect maximal tissue contraction to PE. D: colchicine pretreatment significantly increased time to 50% maximal contraction in response to PE (n = 3). *P < 0.05.
Fig. 10.
Fig. 10.
Expected proximity to the plasma membrane suggests degree of redistribution to the membrane fraction. A: hypothesized role of endosomes in FA protein redistribution. B: potential FA architecture based on the model of Kanchanawong et al. (29). C: percent increase in the membrane fraction after 10 min of PE stimulation compared with rest. Proteins are arranged by expected distance from the plasma membrane based on the model of Kanchanawong et al.

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