We have developed a new microscopic technique to measure the force generated on a single actin filament (FA) in the A-band in which the intact lattice structure composed of myosin thick filaments is maintained; we call this newly developed system "Bionanomuscle (or an A-band motility assay system)". The A-bands were prepared by selective removal of thin filaments from rabbit skeletal glycerinated myofibrils under optical microscope with the use of gelsolin (a severing and barbed (B)-end capping protein of FA) that was prepared from bovine serum. A polystyrene bead of 1 μm in diameter attached to the B-end of FA (through a gelsolin molecule attached to the surface of the bead) was trapped and manipulated with optical tweezers. The displacement of the bead up to 200 nm (corresponding to the force of ∼ 40 pN) was determined by phase-contrast image analysis. At the initial stage of this study, the overlapping length of an FA with the A-band was determined from the fluorescence image of FA labeled with rhodamine-phalloidin (Rh-Ph) and the phase-contrast image, but we later improved the method of determination by moving the sample stage stepwise using the piezo actuator. The average force per overlap was subsequently estimated and the histogram was fitted with two Gaussian distributions. Each peak is supposed to correspond to the force developed by FA interacting outside or inside the A-band, and the peak value of the latter was estimated to be 140 pN/μm. From this value, the average force developed per each cross-bridge (CB; a two-headed myosin molecule) was determined to be 1.3 pN.
|Number of pages
|Advances in Experimental Medicine and Biology
|Published - 2004
ASJC Scopus subject areas
- General Biochemistry,Genetics and Molecular Biology