Myosin Monomer Density and Exchange in Synthetic Thick Filaments Investigated Using Fluorescence Microscopy with Single Molecule Sensitivity

Overview

Journal Proc Biol Sci

Specialty Biology

Date 2000 Mar 18

PMID 10722224

Citations 4

Authors

P B Conibear

C R Bagshaw

Affiliations

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Abstract

The number of myosin molecules in synthetic thick filaments, prepared by dialysis at 0.12 M NaCl and pH 7.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into filaments at a nominal ratio of 1:1000. At this dilution, single fluorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a one- or two-step profile but, in around 30% of the cases, fluctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between filaments in suspension on a time-scale of several hours at 4 degrees C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the fluctuations in fluorescence intensity in the single molecule assays.

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References

Ishijima A, Kojima H, Funatsu T, Tokunaga M, Higuchi H, Tanaka H . Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell. 1998; 92(2):161-71. DOI: 10.1016/s0092-8674(00)80911-3. View

Margossian S, LOWEY S . Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982; 85 Pt B:55-71. DOI: 10.1016/0076-6879(82)85009-x. View

Pierce D, Vale R . Single-molecule fluorescence detection of green fluorescence protein and application to single-protein dynamics. Methods Cell Biol. 1999; 58:49-73. DOI: 10.1016/s0091-679x(08)61948-2. View

Moerner W, Orrit M . Illuminating single molecules in condensed matter. Science. 1999; 283(5408):1670-6. DOI: 10.1126/science.283.5408.1670. View

Ishijima A, Kojima H, Higuchi H, Harada Y, Funatsu T, Yanagida T . Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces. Biophys J. 1996; 70(1):383-400. PMC: 1224937. DOI: 10.1016/S0006-3495(96)79582-6. View

Conibear P, Jeffreys D, Seehra C, Eaton R, Bagshaw C . Kinetic and spectroscopic characterization of fluorescent ribose-modified ATP analogs upon interaction with skeletal muscle myosin subfragment 1. Biochemistry. 1996; 35(7):2299-308. DOI: 10.1021/bi951824+. View

Funatsu T, Harada Y, Tokunaga M, Saito K, Yanagida T . Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature. 1995; 374(6522):555-9. DOI: 10.1038/374555a0. View

Davis J . Myosin thick filaments and subunit exchange: a stochastic simulation based on the kinetics of assembly. Biochemistry. 1993; 32(15):4035-42. DOI: 10.1021/bi00066a026. View

Yamada A, Wakabayashi T . Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction. Biophys J. 1993; 64(2):565-9. PMC: 1262362. DOI: 10.1016/S0006-3495(93)81388-2. View

10.

Saad A, Dennis J, Tan I, Fischman D . Visualization of myosin exchange between synthetic thick filaments. J Muscle Res Cell Motil. 1991; 12(3):225-34. DOI: 10.1007/BF01745111. View

11.

Uyeda T, Kron S, Spudich J . Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin. J Mol Biol. 1990; 214(3):699-710. DOI: 10.1016/0022-2836(90)90287-V. View

12.

Davis J . Assembly processes in vertebrate skeletal thick filament formation. Annu Rev Biophys Biophys Chem. 1988; 17:217-39. DOI: 10.1146/annurev.bb.17.060188.001245. View

13.

Wells C, Bagshaw C . The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins. J Muscle Res Cell Motil. 1984; 5(1):97-112. DOI: 10.1007/BF00713154. View

14.

Bagshaw C, Conibear P . Single molecule enzyme kinetics: application to myosin ATPases. Biochem Soc Trans. 1999; 27(2):33-7. DOI: 10.1042/bst0270033. View

15.

Emes C, ROWE A . Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle. Biochim Biophys Acta. 1978; 537(1):125-44. DOI: 10.1016/0005-2795(78)90608-6. View

16.

KAMINER B, Bell A . Myosin filamentogenesis: effects of pH and ionic concentration. J Mol Biol. 1966; 20(2):391-401. DOI: 10.1016/0022-2836(66)90070-2. View

17.

Conibear P, Kuhlman P, Bagshaw C . Measurement of ATPase activities of myosin at the level of tracks and single molecules. Adv Exp Med Biol. 1999; 453:15-26; discussion 26-7. DOI: 10.1007/978-1-4684-6039-1_3. View