The overall objective of my laboratory is to understand the molecular mechanisms by which smooth muscle cells modulate the interaction of the contractile proteins actin and myosin to regulate smooth muscle contraction. We are currently investigating regulatory proteins associated with the actin filament. The focus of this proposal is to determine how the actin-binding proteins tropomyosin and calponin modulate the activation of smooth muscle actomyosin. The present proposal will test the following hypothesis: The """"""""turned-on"""""""" state of actin.Tm activates dephosphorylated cross bridges by increasing the on- rate. Actin.Tm filaments can be """"""""turned-on"""""""" by the high-affinity (i.e. force producing) state of phosphorylated cross bridges. Calponin (CaP) prolongs the high-affinity state of phosphorylated cross bridges by reducing the off-rate, and thereby, enhances the turning-on of actin.Tm and the subsequent activation of dephosphorylated crossbridges. These studies will advance our understanding of the complex regulatory events that are associated with contraction of smooth muscle. These studies are based entirely on the use of an in vitro motility assay. This assay combines the major biochemical advantages of in vitro assays using purified soluble contractile proteins with the mechanical insights that can be derived from intact muscle studies, while avoiding many of the disadvantages of these other methods. Several recent innovations allow us for the first time to routinely measure isometric force changes on single actin filaments. Both unloaded filament velocity and isometric force production Bill be measured for single reconstituted, regulated actin filaments. Native and chemically-modified myosin subfragments will be used to probe the activation mechanisms of regulated actin. These studies will directly address the question of how smooth muscles regulate dephosphorylated cross bridges during the """"""""latch-state"""""""" - a highly economical contractile state of smooth muscle.
| Haeberle, J R (1999) Thin-filament linked regulation of smooth muscle myosin. J Muscle Res Cell Motil 20:363-70 |
