FLUORESCENT ANALOGS 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 m yosin II dynamics in non-muscle cells. In order to provide more specif ic tools for tracking non-muscle myosin II in living cytoplasm, fluore scent analogues of non-muscle myosin IIA and IIB were prepared and cha racterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may b e 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 tetrameth ylrhodamine-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 myos in light chain kinase, (4) increased MgATPase upon light-chain phospho rylation, (5) interconversion between 6S and 10S conformations, and (6 ) distribution into endogenous myosin Ii-containing structures when mi croinjected into cultured cells. These fluorescent analogues can be us ed to visualize isoform-specific dynamics of myosin II in living cells . (C) 1998 Wiley-Liss, Inc.