Abstract
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.
Highlights
Myosin I1 is an important motor inthe contraction of vitro, light chain phosphorylation transforms smooth muscle smooth and striatedmuscle as well as in a variety of non- and mammaliannonmuscle myosin I1 from a folded 10s to an extended 6s conformation, contractions during migration of fibroblasts, and cap- induces the assemblyof bipolar filaments, increases the actinping of receptors
The acrylodan-labeledlight chain was A new class of fluorescent analog, termed“protein-based opexchanged into the A-bands of chicken pectoralis myo- tical biosensors,” combines the molecular specificity of a pepfibrilsin Sift4 to demonstratethe localizationand activity tide or protein with the sensing capoafbpilhiytysiological fluoof the biosensor in a highlyordered contractile system. rescentindicators (15)
Labelelidght chains were development of a myosin regulatory light chain phosphorylaalso incorporated into stress fibers of living fibroblasts and smooth muscle cellsT. his general approach of combining molecular biologyand fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tooflosr investigationswith model systemsand solution studies and yieldimportant information about temporal-spatial chemical and motion biosensor that responds spectroscopically to phosphorylation by MLCK
Summary
The activity and conformation of both smooth muscle and mammalian nonmuscle myosinI1 is regulatedby phosphorylation of serine 19of the 20-kDa regulatory light chaibny myosin light chain kinase(MLCK).’ Under physiological conditions in so t h a t an optimally constructed, environmentally sensitive, fluorescent probe can be placed in specific regions of the molecule should guide the creation of new protein-based optical biosensors. Under physiological conditions in so t h a t an optimally constructed, environmentally sensitive, fluorescent probe can be placed in specific regions of the molecule should guide the creation of new protein-based optical biosensors This will elucidate the temporal-spatial dynamics of the regulatory andeffector events during a wide. Light chain; &LC,,,gizzard-purified20-kDalight chain; C~S’~.LC,,, Cys” mutant 20-kDa lightchain;MOPS, 4-morpholinepropanesulfonic acid; Dl”, dithiothreitol; ATPyS,adenosine 5’-O-(thiotriphosphate); IAEDANS, 5-((~~2-iodoacetyl)amino)ethyl)amino)naphthalene-l-sulfonic acid; IANBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; BODIPY FLIA, N-(4,4-difluoro-5,7-dimethyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)-N’-iodoacetylethylenediamine; BODIPY 530/550, N-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4adiaza-s-indacene-3-propionyl~-N”iodoacetylethylenediaminBe;rBODIPY 493/503, 8-bromomethyl-4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a.4a-diaza-3-indacene.
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