Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation

Abstract

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca2+-free PSS or regular PSS or were activated in 10 muM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane.

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells

“…In a previous study (16) we reported that AJ-associated proteins remain localized near the SMC periphery in tissues incubated in Ca 2ϩ -depleted PSS or PSS and with CCh activation. However, this work was done on untethered tissues, and thus it was not possible to confirm the contractile status of the tissue.…”

“…In contrast, we (16) have reported that the AJ-associated proteins vinculin, talin, and fibronectin are stably localized near the membrane of SMCs in gastrointestinal organs under relaxed and activated conditions, suggesting that these proteins are very stable at the cell periphery in intact organs. However, many laboratories (including our own) use enzymatic treatment of SM tissue to isolate SMCs for a wide range of in vitro and cell culture experiments.…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…Vinculin binds directly to actin filaments and anchors them to transmembrane integrin proteins indirectly through its binding to talin and ␣-actinin, which are integrin-binding proteins that can also bind directly to filamentous actin (14 -19). Although vinculin has traditionally been viewed as a stable constituent of adhesion junctions in smooth muscle tissues (20,21), it is widely recognized to play a dynamic role in the growth and maturation of adhesion junctions during cell migration and traction in substrate adherent cells (22). Our studies have shown that vinculin plays an active role in tension generation in airway smooth muscle tissues during active contraction (10,23).…”

Altered hemodynamics, endothelial function, and protein expression occur with aortic coarctation and persist after repair

“…Talin is a cytoskeletal focal adhesion protein on the cytosolic side of the membrane, and fibronectin is an extracellular glycoprotein (20,21). When considered together, expression of these contractile and focal adhesion proteins provides an additional indicator of SM differentiation beyond previous studies, which observed only SM ␣-actin or SM myosin.…”