Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro.

Abstract

We have reinvestigated the effects of Ca ++ and ATP on brush borders isolated from intestinal epithelial cells. At 37°C, Ca ++ (1/tM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37°C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca ++ (1 /xM), and occurs in both membrane-intact and demembranated brush borders. Ca÷÷-dependent-solation of microvillus cores requires a concentration of Ca ÷+ slightly greater (10/~M) than that required for contraction.Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC2o), is stimulated by Ca ÷+. At 37°C, BBMLC2o phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC2o phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca ++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca +÷-and calmodulin-dependent brush border myosin light chain kinase.Ca ++ has been implicated as a regulator of both motility and cytoplasmic structure in nonmuscle ceils. For example, changes in free Ca ++ concentration may regulate motility in vertebrate nonmuscle and smooth muscle cells by controlling phosphorylation of the regulatory light chain of myosin by a Ca ++-calmodulin-dependent myosin light chain kinase. Phosphorylation of the regulatory light chain of smooth and nonmuscle myosin increases its actin-activated Mg++-ATPase activity, presumably increasing force production and causing motility (for review, see reference 1).Ca ++ may also regulate cytoplasmic structure by disrupting the bundles or isotropic networks of cross-linked actin filaments that have been postulated to contribute to the gel-like properties of cytoplasm (see reference 12, for review). Raising the concentration of free Ca ++ in cytoplasmic extracts disrupts supramolecular actin filament structures. This indicates that Ca ++ may regulate the rapid changes in cytoplasmic viscosity that can occur in vivo. Recently, two major classes of Ca ++-sensitive actin-binding proteins which could modulate the reversible breakdown of actin filament structures have been identified. These include proteins which cross-link actin filaments in the absence, but not in the presence, of Ca ++ (9,50,58,60,79) and proteins which reversibly cut or shorten filaments in the presence of Ca ++ (6,13,26,30,46,53). The mechanism by which changes in structure are coordinated with motility in vivo is still a matter of speculation (32).One of the most extensively ordered arrays of contractile prote...

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.

“…We also observed a reduction in the density of cross-linking filaments associated with the basal ends of the core rootlets. In addition, the circumferential, "contracting" ring (37,35,48,49) of actin filaments associated with the junctional margin of the brush border at the level ofthe zonula adherens was intact in the salt-extracted brush borders, but it appeared somewhat less densely packed than in control preparations (results not shown, but see reference 37) . Numerous crosslinking filaments resembling myosin filaments (Fig.…”

“…For example, TW 260/240 may participate in endocytosis of apical membrane, which is mediated, at least in part, by coated vesicles (53). In addition, apical membrane may be replaced by Goggi-derived vesicles (54) that we have speculated are actively transported to the membrane along core rootlets, perhaps by vesicle-associated myosin (35,37). This speculation was based on the observation that in QFDERR preparations of isolated brush borders there are numerous vesicles attached to the core rootlets.…”

Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

“…However, long-term exposure of endothelial monolayers to low Ca2+ finally caused (beginning about 60 min after addition of EDTA) disintegration of the monolayer and a concomitant rise in permeability. TFP and W7, two compounds known to interfere with Ca2+-calmodulin-dependent mechanisms such as stimulation of actin-activated myosinATPase (Sheterline, 1980;Hidaka, Asano & Tanaka, 1981;Keller & Mooseker, 1982;Sobieszek, 1989), inhibited both the Ca2+/A23187-induced rise in permeability (Fig. 8B) and gap formation (Fig.…”

Commensal bacterial internalization by epithelial cells: An alternative portal for gut leakiness

“…Previous reports indicated that the Ca 2C /calmodulin-dependent phosphorylation of MLC induced TW contraction associated with BB fanning on isolated brush border sheets interconnected by TJs. 51,52 We speculated that ultrastructural changes of the BB were required to allow entry of intestinal bacteria into cells. By using human C2BBe cell cultures (a clone of Caco-2 cells with long BB), we demonstrated that IFNg stimulation caused internalization of nonpathogenic bacteria by epithelial cells through a cholesterol-rich lipid raft and caveolin-1-dependent endocytotic pathways.…”