Abstract

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Highlights

  • Activation in response to contractile stimuli [2]

  • We previously found that paxillin phosphorylation at Tyr-31 and Tyr-118 regulates activation of the actin-nucleation initiator protein neuronal Wiskott-Aldrich Syndrome protein (N-WASp) in tracheal smooth muscle and that both paxillin tyrosine phosphorylation and N-WASp activation are required for the initiation of actin polymerization by the Arp2/3 complex in response to a contractile stimulus [8]

  • Our results provide the first demonstration that the contractile stimulation regulates the interaction of vinculin with talin and actin at adhesion junctions in smooth muscle and that the regulation of vinculin activity plays an important role in the development of active tension in this tissue

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Summary

Introduction

Activation in response to contractile stimuli [2]. there is growing evidence that tension development in smooth muscle depends on dynamic cytoskeletal processes outside of the actomyosin interaction [3, 4]. Contractile stimulation initiates an integrated array of cytoskeletal events that are orchestrated by macromolecular protein complexes at adhesion junctions where cytoskeletal proteins link actin filaments to the extracellular matrix. The head domain of vinculin (Vh) can bind to talin and ␣-actinin, and the vinculin tail domain (Vt) can bind to actin filaments, supporting linkages between the actin cytoskeleton and integrin adhesion junctions [13, 17, 29, 31]. We hypothesized that the formation of connections between integrin adhesion junctions and actin filaments by vinculin during the contractile stimulation of SMCs may be critical for the regulation of tension transmission from the contractile apparatus to the extracellular matrix. We evaluated changes in vinculin phosphorylation at Tyr-1065 during the contractile stimulation of tracheal muscle tissues

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