title: Kinetic characterization of nonmuscle myosin IIb at the single molecule level.
abstract: Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s(-1)) of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s(-1)). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s(-1)). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.
authors.affiliation: Laboratory of Molecular Physiology, HLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Attila
authors.initials: A
authors.lastname: Nagy
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.forename: Sara A
authors.initials: SA
authors.lastname: Sun
authors.forename: Davin K T
authors.initials: DK
authors.lastname: Hong
authors.forename: Earl
authors.initials: E
authors.lastname: Homsher
authors.forename: Aibing
authors.initials: A
authors.lastname: Wang
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D001703:Biophysics / Q000379:methods; D002417:Cattle; D002465:Cell Movement; D002645:Chickens; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron / Q000379:methods; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000378:metabolism; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB / Q000378:metabolism*; D052898:Optical Tweezers; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D024501:Nonmuscle Myosin Type IIB