Antibodies probe for folded monomeric myosin in relaxed and contracted smooth muscle.
Regulatory light chain phosphorylation is required for assembly of smooth and non-muscle myosins in vitro, but its effect on polymerization within the cell is not understood. Relaxed smooth muscle cells contain dephosphorylated thick filaments, but this does not exclude the presence of a pool of folded myosin monomers which could be recruited to assemble when phosphorylated, thus forming part of smooth muscle's activation pathway. To test this hypothesis, relaxed and contracted avian gizzard cryosections were labeled with a fluorescently conjugated monoclonal antibody specific for the folded monomeric conformation, or with an antibody against the tip of the tail whose epitope is accessible in the monomeric but not the filamentous state. Fluorescence intensity observed in the two physiological states was quantitated by digital imaging microscopy. Only trace amounts of folded monomeric myosin were detected in both the relaxed and contracted states. The amount of monomer also did not increase when alpha-toxin permeabilized gizzard was equilibrated in a solvent that disassembles filaments in vitro. Assembly/disassembly is therefore unlikely to play a major role in regulating the contraction/relaxation cycle in smooth muscle cells.
Horowitz
A
A
Rosentiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
Trybus
K M
KM
Bowman
D S
DS
Fay
F S
FS
D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D005895:Gizzard, Avian; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D009218:Myosins