The ability of cells to sense and respond to physiological forces relies on the actin cytoskeleton, a dynamic structure that can directly convert forces into biochemical signals. Because of the association of muscle actin-binding proteins (ABPs) may affect F-actin and hence cytoskeleton mechanics, we investigated the effects of several ABPs on the mechanical properties of the actin filaments. The structural interactions between ABPs and helical actin filaments can vary between interstrand interactions that bridge azimuthally adjacent actin monomers between filament strands (i.e. by molecular stapling as proposed for caldesmon) or, intrastrand interactions that reinforce axially adjacent actin monomers along strands (i.e. as in the interaction of tropomyosin with actin). Here, we analyzed thermally driven fluctuations in actin's shape to measure the flexural rigidity of actin filaments with different ABPs bound. We show that the binding of phalloidin increases the persistence length of actin by 1.9-fold. Similarly, the intrastrand reinforcement by smooth and skeletal muscle tropomyosins increases the persistence length 1.5- and 2- fold respectively. We also show that the interstrand crosslinking by the C-terminal actin-binding fragment of caldesmon, H32K, increases persistence length by 1.6-fold. While still remaining bound to actin, phosphorylation of H32K by ERK abolishes the molecular staple (Foster et al. 2004. J Biol Chem 279;53387-53394) and reduces filament rigidity to that of actin with no ABPs bound. Lastly, we show that the effect of binding both smooth muscle tropomyosin and H32K is not additive. The combination of structural and mechanical studies on ABP-actin interactions will help provide information about the biophysical mechanism of force transduction in cells.

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.