Fig. 4. Dynamics of microinjected myosin II analog in (a), locomoting (See Sequence 3 (1 M)). and (b), stationary fragments (See Sequence 4 (1 M)). Overviews of the fragments at time 0 and sequential images of selected regions at times indicated in min:s are shown. Dotted lines fixed with respect to the substratum are drawn to highlight that myosin spots (several representative spots are indicated by arrows) intensify in brightness and remain stationary with respect to the substratum throughout most of the lamellum of the locomoting fragment but move forward in the vicinity of its rear edge. In the stationary fragment, myosin spots both grow and diminish in brightness while moving slowly to the center.
(c) Quantification of dynamics of individual spots in a locomoting fragment. Average brightness of spot above brightness of local background (in arbitrary units) and position of spot along the axis of locomotion in substrate coordinate system (in arbitrary units) are plotted versus time (corresponding images of the spot for each time point are shown on top of the plot). Brightness of the spot increases and reaches a plateau, but then increases again as the rear approaches and the spot starts to move forward. (d) Change in spot brightness in sequential images (in arbitrary units) is plotted versus spot brightness for six individual spots in the same fragment. Only the data points before the onset of retraction are used for this plot to reflect changes in brightness due to assembly-disassembly as opposed to contraction. The plot is fitted by a kinetic equation for the myosin polymer association-dissociation reaction, n m <=> p, k+/k- = p/mn, where dp, change in polymer concentration (change in spot brightness), dt, time interval, k+ , association rate constant, m, concentration of soluble myosin molecules (assumed to be nearly uniform across the fragment), n, order of the reaction, k-, dissociation rate constant, and p, local polymer concentration (spot brightness). Dissociation rate constant estimated from the slope of the plot equals 0.05±0.2 s-1 (mean ± standard error). Bars, 2 µm in (a), (b), 1 µm in (c).
