Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils.
To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.
Applegate
D
D
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York 10029.
Pardee
J D
JD
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism* / Q000648:ultrastructure; D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002645:Chickens; D005656:Fungal Proteins / Q000494:pharmacology; D007700:Kinetics; D008840:Microfilament Proteins / Q000494:pharmacology; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D015800:Protozoan Proteins
D000199:Actins; D005656:Fungal Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C036571:severin protein, Dictyostelium; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000205:Actomyosin; D009218:Myosins