A FLUORESCENT PROTEIN BIOSENSOR OF MYOSIN-II REGULATORY LIGHT-CHAIN PHOSPHORYLATION REPORTS A GRADIENT OF PHOSPHORYLATED MYOSIN-II IN MIGRATING CELLS

Phosphorylation of the regulatory light chain by myosin light chain ki nase (MLCK) regulates the motor activity of smooth muscle and nonmuscl e myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin I I regulatory light chain phosphorylation ((F)RLC-(R)myosin II) is desc ribed here. The biosensor depends upon energy transfer from fluorescei n-labeled regulatory light chains to rhodamine-labeled essential and/o r heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphory lation by MLCK (versus phosphorylation by protein kinase C). Folding/u nfolding, filament assembly, and actin binding do not have a large eff ect on the energy transfer ratio. (F)RLC-(R)myosin II has been microin jected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing (F)RLC-(R)myos in II with the kinase inhibitor staurosporine produced a lower ratio o f rhodamine/fluorescein emission, which corresponds to a lower level o f myosin II regulatory light chain phosphorylation. Locomoting fibrobl asts containing (F)RLC-(R)myosin II showed a gradient of myosin II pho sphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their p olarized shape, retract the tail as the cell moves forward, and delive r disassembled subunits to the leading edge for incorporation into new fibers.