A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.
Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.
Post
P L
PL
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Trybus
K M
KM
Taylor
D L
DL
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002645:Chickens; D005456:Fluorescent Dyes; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D055095:Optics and Photonics; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D014422:Turkeys
D005456:Fluorescent Dyes; C038518:acrylodan; D015081:2-Naphthylamine; D009218:Myosins