Comparison of Unitary Displacements and Forces Between 2 Cardiac Myosin Isoforms by the Optical Trap Technique by Seiryo Sugiura, Naoshi Kobayakawa, Hideo Fujita, Hiroshi Yamashita, Shin-ichi Momomura, Shigeru Chaen, Masao Omata, and Haruo Sugi Circulation Research Volume 82(10):1029-1034 June 1, 1998 Copyright © American Heart Association, Inc. All rights reserved.

Schematic diagram of the laser optical trap system. Schematic diagram of the laser optical trap system. A fluorescently labeled actin filament (AF) with a polystyrene bead (Pb) attached to its barbed end is made to interact with a single or a few myosin molecules sparsely bound to a glass surface, while the bead is captured in the optical trap in the flow cell (F), mounted on the mechanical stage (S) of a microscope. Direction of ATP-dependent actin-myosin sliding is indicated by an arrow. Bright-field illumination with a xenon lamp (Xe) is used to project the bead image onto a quadrant photodiode detector (QD). Two piezoelectric mirrors (PM) rapidly deflect the beam of Nd-YAG laser (thick solid line) before it enters into the back aperture of the objective lens (O) to change the trap position. Epifluorescence illumination with a mercury lamp (Hg) is used to observe the fluorescent actin filament with an silicone-intensified camera (SIT). Other optics in the system are a condenser (C), 2 mirrors (M), and 3 dichroic filters (D). The output from the quadrant detector is fed to the feedback circuit (FC) driving the 2 PMs. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Responses of the optical trap system to triangular movements of the microscope stage position, on which the flow cell is mounted, with the feedback loop opened (left) and with the feedback loop closed (right). Responses of the optical trap system to triangular movements of the microscope stage position, on which the flow cell is mounted, with the feedback loop opened (left) and with the feedback loop closed (right). Left, The bead position shows square-shaped changes due to alternating constant viscous drag forces produced by the stage movement. Right, The trap position shows square-shaped changes, while the bead position remains stationary. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Examples of displacement records obtained from V1 and V3 isoforms under a low trap stiffness. Examples of displacement records obtained from V1 and V3 isoforms under a low trap stiffness. Vertical deflections in the records indicate movements of the trapped bead in the direction parallel to the actin filament long axis. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Frequency histograms showing distribution of the amplitude of the displacement in V1 and V3 isoforms. Frequency histograms showing distribution of the amplitude of the displacement in V1 and V3 isoforms. Displacements with amplitudes ≤5 nm were not scored. Dashed curves represent distributions of gaussian components; solid curves show reconstructed distributions of the gaussian components. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Distributions of the duration of the displacement events in V1 and V3 isoforms. Distributions of the duration of the displacement events in V1 and V3 isoforms. Solid curves are single exponential fits to the distributions of durations. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Examples of force transient records obtained for V1 and V3 isoforms under a high trap stiffness with feedback. Examples of force transient records obtained for V1 and V3 isoforms under a high trap stiffness with feedback. Vertical deflections in the record indicate movements of the trap position, representing force transients. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Frequency histograms showing distribution of the amplitude of the force transients in V1 and V3 isoforms. Frequency histograms showing distribution of the amplitude of the force transients in V1 and V3 isoforms. Force transients ≤0.5 pN were not scored. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Force transient record obtained from V3 isoform, in which the force transients exhibited 2 distinct amplitudes with a ratio of ≈2 (high force, ≈4 pN; low force, ≈2 pN). Force transient record obtained from V3 isoform, in which the force transients exhibited 2 distinct amplitudes with a ratio of ≈2 (high force, ≈4 pN; low force, ≈2 pN). Bar=5 pN. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.

Distribution of the duration of the force transients in V1 and V3 isoforms. Distribution of the duration of the force transients in V1 and V3 isoforms. Solid curves are single exponential fits to the distribution of durations. Seiryo Sugiura et al. Circ Res. 1998;82:1029-1034 Copyright © American Heart Association, Inc. All rights reserved.