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Isolation of the bile canalicular actin-myosin II motor
N Tsukada1, T Azuma, M J Phillips
1Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract:
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.
Insights
The study reveals that myosin II phosphorylation drives canalicular contraction. This process involves actin filaments and myosin II, crucial for canalicular contractile apparatus function.
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- The canalicular contractile apparatus regulates bile flow.
- Understanding the molecular mechanisms of canalicular contraction is essential.
Purpose of the Study:
- To investigate the relationship between pericanalicular microfilaments, myosin II phosphorylation, and canalicular contraction.
- To identify the molecular components responsible for canalicular contraction.
Main Methods:
- Utilized cytoskeleton-rich canalicular membranes (CCMs) and demembranated CCMs.
- Employed fluorescence and electron microscopy for visualization.
- Analyzed myosin II phosphorylation using Western blots and [gamma-32P]-ATP labeling.
- Assessed canalicular contraction via morphometric analysis and evaluated the effects of W7 and ML9.
Main Results:
- Myosin II phosphorylation increased significantly within 0.25-0.50 minutes of adding the contraction solution.
- Both W7 (calmodulin antagonist) and ML9 (myosin light chain kinase inhibitor) suppressed phosphorylation and contraction.
- Phosphorylation of the 20-kDa myosin light chain preceded or coincided with canalicular contraction.
Conclusions:
- Phosphorylation of the 20-kDa myosin light chain is a key event in canalicular contraction.
- The canalicular contractile apparatus comprises actin filaments and a myosin II motor.
- Myosin II motor activity, regulated by phosphorylation, is essential for canalicular function.