Background and aims: It is known that smooth muscle possesses substantial
mechanical plasticity in that it is able to adapt to large changes in length without
compromising its ability to generate force. It is believed that structural malleability of
the contractile apparatus underlies this plasticity. There is strong evidence suggesting
that myosin thick filaments of the muscle are relatively labile and their length in vivo
is determined by the equilibrium between monomeric and filamentous myosin. The
equilibrium in turn is governed by the state of phosphorylation of the 20-kD
regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of
the myosin light chain favors formation of the filaments; it is not known how the light
chain phosphorylation affects the lability of the filaments. The major aim of this
thesis was to measure the mechanical integrity of the filaments formed from purified
myosin molecules from bovine airway smooth muscle, and to determine whether the
integrity was influenced by phosphorylation of the myosin light chain.
Methods: Myosin was purified 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 RLC within 20-40 s 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: RLC 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.
Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in
modifying the structural properties of myosin filaments in smooth muscle, where
formation and dissolution of the filaments are required in the cells’ adaptation to
different cell length.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU./4131 |
| Date | 11 1900 |
| Creators | Ip, Kelvin |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | 1611143 bytes, application/pdf |
Page generated in 0.002 seconds