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 kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or 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 phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation 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 polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.
Post
P L
PL
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
DeBiasio
R L
RL
Taylor
D L
DL
D016475:3T3 Cells; D000818:Animals; D015374:Biosensing Techniques; D002465:Cell Movement; D004735:Energy Transfer; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005895:Gizzard, Avian; D007700:Kinetics; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014422:Turkeys
D005452:Fluoresceins; D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D012235:Rhodamines; D009219:Myosin-Light-Chain Kinase