In vitro motility analysis of smooth muscle caldesmon control of actin-tropomyosin filament movement.
We have used the in vitro motility assay to investigate the effect of caldesmon on the movement of actin-tropomyosin filaments over thiophosphorylated smooth muscle myosin and skeletal muscle heavy meromyosin. Using either motor, incorporation of up to 8 nM caldesmon inhibited filament movement by decreasing the proportion of filaments motile from > 85% to < 30%. There was a minimal effect on filament attachment and a modest decrease in motile filament velocity in this concentration range. The reduction in the proportion of filaments motile could be completely reversed by incorporation of an excess of calmodulin at pCa 4.5. The expressed C-terminal fragment, 606C, which retains caldesmon's inhibitory capacity but does not bind to myosin, decreased the proportion of filaments motile but had no effect on velocity. We conclude that the velocity reduction by whole caldesmon is due to actin-myosin cross-linking. A significant decrease in filament attachment was observed when caldesmon was added to an excess over actin (> 10 nM). In the absence of tropomyosin, addition of an excess of caldesmon caused a similar decrease in the filament density, but there was no effect on the proportion of filaments that were motile. Our results demonstrate that caldesmon can switch actin-tropomyosin from motile to non-motile states without controlling velocity of movement or weak binding affinity and show the inhibitory action of caldesmon in the motility assay to be functionally indistinguishable from that reported for troponin.
Fraser
I D
ID
Department of Cardiac Medicine, National Heart and Lung Institute, London, United Kingdom.
Marston
S B
SB
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D002645:Chickens; D066298:In Vitro Techniques; D009068:Movement / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; D009218:Myosins