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The actin cytoskeleton is involved in many cell functions including locomotion, cytokinesis, cell adhesion, signaling complexes, organelle movements, establishment and maintenance of cell morphology and cell contraction. Actin regulatory proteins orchestrate the multiplicity of actin related functions by crosslinking, severing, and capping. Importantly, actin filaments serve as a molecular track for the myosin motor proteins. The ubiquitous myosin II contains one pair of regulatory light chains (RLC) which control the interaction of the myosin head with actin and hence the movement of myosin thick filaments relative to actin filaments. A key step in myosin activation is phosphorylation by Ca2 /calmodulin-dependent myosin light chain kinase (MLCK).
Molecular mechanisms involved in hormone-receptor mediated events, resulting in generation of second messengers and effector processes targeted by intracellular signaling pathways are key subjects of investigations in cell regulation. Smooth muscle represents a particularly interesting model system in which to study such mechanisms owing to the fact that the primary effector process (actomyosin MgATPase) involves a transduction of chemical to mechanical energy. An understanding of the basic processes involved in this transduction and its regulation is pivotal to investigations of neural and hormonal modulation of smooth muscle contractility, and in part to non-muscle motility.
The research in our laboratory addresses two general aspects of regulation: acute and chronic adaptive. First, in acute studies we have concentrated on relating mechanical events manifested during the course of contraction and relaxation to the generation of biochemical intermediates that regulate contractility. Measurements of stress-strain relationships, shortening velocities, and stiffness are used to provide a primary description of physiological function whose molecular basis is explained with the assistance of structural and biochemical studies. Kinetic studies are used to relate rates and sites of phosphorylation of contractile proteins to mechanical activation. The importance of different components of the regulatory pathway (kinases, phosphatases, contractile proteins, etc.) is tested by antisense knockdowns, protein transduction domains, and by permeabilized muscle fibers.
Biosensor MLCKs containing fluorescent indicator proteins at strategic positions measure Ca2 /calmodulin binding and kinase activation in cells. Biosensor MLCKs thus provide a molecular tool for investigating the temporal and spatial properties for the Ca2 -dependence of cell movement processes. They are expressed in specific physiological kinds of muscle cells in transgenic mice for investigations involving second messenger regulation of Ca2 -dependent myosin phosphorylation and contractions. This new experimental approach is thus used to explore biochemical mechanisms that modulate the Ca2 -dependency of cell motility and muscle contractions.
The cellular mechanism by which MLCK phosphorylates myosin RLC is somewhat a mystery since the kinase appears to be tightly bound to actin, not myosin containing filaments. We have recently identified a unique actin-binding motif located at the N-terminal tail of the kinase with the catalytic core in the C-terminus. One possibility is the kinase extends from the actin filament to the myosin filament to affect RLC phosphorylation. Another possibility is that the kinase is released from the actin filament to phosphorylate RLC. Thus MLCK binding to actin filaments may be regulated. Biosensor MLCKs expressed in transgenic mice will be used to determine the dynamic distribution of MLCK binding to functionally different pools of actin filaments in cells.
Additional investigations will combine the Cre-lox conditional knock out with knock-ins in mice to evaluate the physiological importance of key elements of the Ca2 -calmodulin-MLCK cascade in addition to the modulatory pathways. By expanding to mice as a basis of study, a variety of cell types and cell motility processes will be explored.
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