Isolation of the bile canalicular actin-myosin II motor.
Cytoskeleton-rich canalicular membranes (CCMs) with preserved cytoskeleton and demembranated CCMs, consisting only of cytoskeletal elements, were used to examine the relationship of pericanalicular microfilaments, myosin II phosphorylation, and canalicular contraction. The components of CCMs were visualized by fluorescence microscopy using the filamentous actin probe rhodamine-phalloidin and by electron microscopy, before and after incubation in 1 microM Ca2+/1 mM ATP (contraction solution). Canalicular contraction (luminal closure) was evaluated by morphometric analysis. Myosin II was extracted from CCMs, purified by immunoprecipitation, and analyzed on Western blots. In sequential experiments, autoradiographs of gels from [gamma-32P]-ATP-treated CCMs in the presence or absence of Ca2+ were examined after 0.25, 0.50, 1, 2, 3, 5, and 10 min, and the effects of W7 (a calmodulin antagonist) and ML9 (a myosin light chain kinase inhibitor) were evaluated. The results showed that phosphorylation of the 20-kDa protein was low in controls but enhanced beginning 0.25-0.50 min after addition of contraction solution. Both W7 and ML9 significantly inhibited this reaction and inhibited canalicular contraction. The results indicate that phosphorylation of the regulatory 20-kDa myosin light chain of canaliculus-associated myosin II coincides with or precedes contraction of the canaliculus. We conclude that the canalicular contractile apparatus is composed of actin filaments and a myosin II motor.
Tsukada
N
N
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Azuma
T
T
Phillips
M J
MJ
D000199:Actins / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001648:Bile Canaliculi / Q000378:metabolism / Q000648:ultrastructure*; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003599:Cytoskeleton / Q000378:metabolism*; D008566:Membranes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats
D000199:Actins; D009218:Myosins; D002118:Calcium