Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells.
Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells.
Kolega
J
J
Department of Anatomy and Cell Biology, State University of New York at Buffalo School of Medicine and Biomedical Sciences, 14214, USA. jkolega@ubmedb.buffalo.edu
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D002232:Carbocyanines; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D009130:Muscle, Smooth / Q000737:chemistry; D009218:Myosins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D012235:Rhodamines
D002232:Carbocyanines; D005456:Fluorescent Dyes; D012235:Rhodamines; C085321:cyanine dye 5; D009218:Myosins