The earliest molecular response to stretch of insect flight muscle as revealed by fast X-ray diffraction recording

Figures

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  Figure 1. Diffraction movie frames recorded from stretch-activated bumblebee flight muscle fibers at a saturating level of calcium (pH = 4.0) and time courses of changes of force and reflection intensities (time resolution, 0.5 ms). (A) and (B) Two frames of diffraction movie, taken 0.25 ms before (A) and 16.75 ms (B) after step stretch. The boxes indicate the reflections of interest, and their magnified views are shown in the upper area of the frame. Cyan boxes, troponin reflections. 1Tn, 2Tn and 3Tn represent the 1st-order off-meridional (101), 2nd-order off-meridional (102) and 3rd-order on-meridional (003) troponin reflections, respectively. Dark green box, 2nd actin layer line (tropomyosin reflection, TM). Light green box, 111 and 201 reflections. This box is further magnified in the four panels below (A) and (B). (C) Force (red) and length (blue), summed for all specimens used. The length step is 1% of fiber length. The force is in arbitrary unit. (D) Time course of intensity changes of troponin reflections. Blue, 1st (101); red, 2nd (102); green, 3rd (003). (E) 2nd actin layer line (tropomyosin) reflection. The curves were fitted to single exponential association function. The data…

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  Figure 2. Responses of the 111 and 201 reflections to a step stretch under various conditions. (A) pCa = 4.0; (B) pCa = 5.75; (C) pCa = 6.25; (D) relaxed; (E) pCa = 4.0, in the presence of 100 μM blebbistatin; (F) Igase-treated fibers, pCa = 4.0, in the presence of 100 μM blebbistatin. Black, 111; gray, 201. The inset in (A) is the fitting of the fast decay of 111 to a single exponential decay function. The numbers of fiber sets used: (A) 33; (B) 27; (C) 19; (D) 27; (E) 19; (F) 10.

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  Figure 3. Responses of reflections to a step release of bumblebee flight muscle fibers activated at pCa = 4.0. (A) The responses of the 1st (black) and 2nd (gray) troponin reflections (101 and 102). (B) The response of the 2nd actin layer line (tropomyosin) reflection. (C) The responses of the 111 (black) and 201 (gray) reflections. (D) The responses of the 111 (black) and 201 (gray) reflections but in the presence of 100 uM blebbistatin. The curves in (A) and (B) were fitted to a single exponential decay or association function. The number of fiber sets used: 19.

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  Figure 4. Schematic drawings of the ideas underlying the model calculations. These drawings summarize structures that can move upon stretch relative to the troponin complexes, causing the change in X-ray interference and resulting in the intensity change of reflections. (A) In the relaxed state. The 4-start helical array of myosin heads (drawn as continuous helical strands) is the only structure that can move with respect to the troponin complexes (located midway between the neighboring actin target zones, not drawn in the figure). (B) Calcium-activated state. The myosin heads close to the target zone leave the helical array (arrows), creating defects in the helical strands. The periodicity of the defects is the same as that of the target zones (or troponin complexes, 38.7 nm) so that these can be sources of interference. (C) Movement of actin-attached myosin heads upon stretch in the weakly binding state. Because of the limited interaction in the actin-myosin interface, a large movement of the center of mass of the myosin head is expected (dots and arrows). (D) Movement of actin-attached myosin heads upon stretch in the strongly binding state. Because of the extensive interaction in the…

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  Figure 5. Model calculations of intensity changes of the 111 and 201 reflections upon stretch of IFM fibers. (A and B) Effect of sliding of the myosin filament (helical object in Fig. 4A) past the actin filament (thinner rod below the myosin filament in Fig. 4A). This corresponds to the stretch of a relaxed IFM fiber. Red curve, 111; blue curve, 201. In (A) The rotational angles of the myosin filaments are in register. In (B) The myosin filaments axially staggered by 38.7/3 nm with respect to the neighboring filaments. (C and D) Calculations simulating the calcium-activated state in which strongly bound state of myosin has not been formed. In (C) The myosin heads nearest to the target zones are attracted to the actin filaments, but they are so disordered that they are invisible by X-ray. Because of this, the myosin helix has defects (see Fig. 4B). The stagger of myosin filaments is as in (B). The graph in (D) is the combination of (C) and (E) In which myosin filaments with defects slide past actin filament by 38.7 nm, while the weakly bound myosin heads travel between the hot spot and the cool spot in (E). (E) Schematic 3-dimensional map for the relative intensities of the 111 and 201…