Reverse actin sliding triggers strong myosin binding that moves tropomyosin

Abstract

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

Fig. 1.

Mechanical force responses of IFM skinned fibers coordinated with timing of length and solution changes and x-ray exposures. Force baseline = ≈30 μN per fiber was imposed by ≈1% stretch required to orient relaxed IFM. (A) Isometric contraction force response in high [Ca²⁺] is abolished upon adding 3.0 mM NaVi, dropping pCa ∼ 4.5 force to relaxed level seen at pCa ∼ 9.0. (B) Active force plateaus slowly at intermediate pCa ∼ 5.7 (rise elided for brevity) to poise IFM for stretch activation, triggered by step stretch (here 2%/5 msec that elicits a delayed rise in active force). Total force peak is the sum of three components, separated by arrowheads. ISOM, initial low isometric force plateau at pCa ∼ 5.7; ELAST, passive elastic response to stretch, which elicits delayed St-ACT rise to peak. Excluding ELAST gives the total active force, ISOM + St-ACT, a sum approximately equal to the maximum isometric force of Ca-activation in A. (C) Ramp-stretch and hold in low Ca²⁺ (pCa ∼ 9.0), ±3 mM Vi, shows the same high passive stiffness (Fig. S4) and relaxed-state structure (Figs. S5 and S6). In high Ca²⁺ (pCa ∼ 4.5) after Vi-trapping in 3 mM Vi, the stretch and hold protocol shows a large increase in stretch-elicited force, concurrent with a major change from relaxed-state x-ray diffraction (Figs. 2 and 3) to active-state signs of cross-bridge attachment to actin, and from partial (Ca²⁺-induced) to maximum (cross-bridge-induced) TM displacement. (Labels 2A–2F and ← → timings match typical x-ray patterns in Fig. 2.)

Fig. 2.

Background-subtracted x-ray diffraction patterns from glycerinated Lethocerus IFM. Fiber axis is vertical. Gain and contrast of center panel was adjusted independently from the rest of each pattern to show the 19.3- and 38.7-nm first row line reflections circled [also magnified (Inset)] in right upper quadrants. Backbent arrows locate the diffuse horizontal TM reflections, axially spaced 19.3 nm from equator. (A–C) IFM, no Vi treatment. (D–F) IFM with 3.0 mM Vi trapped = IFM(Vi). 38.7-nm reflection >19.3-nm reflection in low force states (A, D, and E), whereas 19.3 nm > 38.7 nm in high-force states (B, C, and F). (A) IFM in relaxing solution (pCa ∼ 9.0). (B) Isometric Ca²⁺ activation (pCa ∼ 4.5), during plateau of maximum force. (C) Stretch activation, peak of delayed active force, at 100–200 msec after 2%/5-msec stretch at pCa ∼ 5.7. (D) IFM(Vi) in relaxing solution (pCa ∼ 9.0). (E) IFM(Vi) in high Ca²⁺ (pCa ∼ 4.5), not stretched. (F) IFM(Vi) in high Ca²⁺ (pCa ∼ 4.5), x-ray exposure begun immediately after completing 4%/200-msec stretch (held for 2 sec). Exposure times, 100 msec. (Enlarged views and comparisons of these six plus rigor and relaxed-stretch patterns are available in the difference patterns in Fig. S3 and in large-format blink-comparator galleries provided by Figs. S5 and S6.)

Fig. 3.

State-dependent diffraction intensities. (A) Histograms of integrated intensities of the TM reflections in control and Vi-treated fibers. Intensities are normalized with respect to the 2,0 equatorial reflection intensity (10) in same pattern. Higher values indicate larger displacement of TM from normal relaxed position. The mean and SEM are from n = 5–6 experiments. (B) Intensity ratios of the first row line reflections on 19.3- and 38.7-nm layer lines (LL). For I_19.3/I_38.7 ratios >1, first row line intensity (see Fig. S6) at 19.3-nm LL is higher than at 38.7-nm LL. In the presence of MgATP, higher ratios = increasing attachment of cross-bridges to actin target zones. In rigor, heavier target-zone labeling by double-headed bridges increases the 38.7-nm reflection without affecting the 19.3-nm reflection to reduce this ratio. Note that “stretch” at pCa ∼ 5.7 refers to the Fig. 1B protocol and at pCa ∼ 4.5 to the Fig. 1C protocol. §, group of mean values that are significantly different from each other. ●, group of mean values that are not significantly different (P < 0.05, Student's t test) from each other in pairwise comparisons.