Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain.
OBJECTIVE
Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity.


METHODS
We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy.


RESULTS
MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated.


CONCLUSIONS
Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.
Ip
Kelvin
K
James Hogg iCAPTURE Centre, St. Paul's Hospital, Vancouver, Canada.
Sobieszek
Apolinary
A
Solomon
Dennis
D
Jiao
Yuekan
Y
Paré
Peter D
PD
Seow
Chun Y
CY
D000818:Animals; D002417:Cattle; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D024745:Smooth Muscle Myosins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure
D018994:Myosin Light Chains; D024745:Smooth Muscle Myosins