MLCK Induces Actin Filament Bundling and Moves on Bundled Actin and Stress Fibers of Smooth Muscle Cells

Abstract

We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. We have been studying the interactions between MLCK, actin, SMM and smooth muscle stress fibers at a single molecule level using total internal reflectance fluorescence microscopy (TIRF). We observed that MLCK induces actin filament bundling. The number and length of the bundles depends upon the MLCK concentration and incubation time. MLCK also induced actin-tropomyosin complex (actin-Tm) bundling, but with longer bundle length. The N-terminal 1-75 peptide of MLCK (GST-N1-75 MLCK), which has three DFRxxL actin binding motifs does not cause actin or actin-Tm bundling, suggesting that other actin binding domains on MLCK are required for bundling. These findings may suggest that MLCK plays a role in stabilizing stress fibers in smooth muscle cells.With quantum dots labeled MLCK (QD-MLCK), we have observed that MLCK not only causes actin bundling, but moves along the actin bundles and actin-Tm bundles. In a more physiological system, skinned human airway smooth muscle cells, direct observations of single QD-MLCK molecules show clearly that MLCK co-localizes with and can move along the actin- and myosin-containing stress fibers, at high ionic strength, or at physiological ionic strength with CaM-Ca2+ and ATP. GST-N1-75 MLCK also moves along smooth muscle cell stress fibers. The diffusion coefficient, calculated from mean-squared-displacement (MSD) data from MLCK-QDs' trajectories, indicates that the mechanism by which one MLCK phosphorylates multiple SMMs may involve MLCK movement along thin and/or thick filaments on a time scale measured in seconds.