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 crossbridges. 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 force suppression ͉ muscle activation ͉ vanadate ͉ x-ray diffraction M uscle pulls a load during shortening contractions and forcefully resists the pull of the load during lengthening (eccentric) action. Full activation of contraction depends on an adequate supply of ATP, binding of Ca 2ϩ to troponin, and cross-bridge attachment to move tropomyosin (TM) away from the myosin binding site on actin, in accord with the steric blocking model (1-4). The transitions between lifting (shortening), holding steady (isometric), and lowering (eccentric lengthening) a hand weight feel so smooth that intuition suggests a common mechanism for all active force production by muscle myosin. However, a striking asymmetry exists between eccentric vs. shortening or isometric contractions. In lengthening (eccentric) contractions of active skeletal muscle, additional force (up to two times isometric) with very reduced O 2 and ATP consumption (2, 5-7) are observed. During stretches Ͼ2%, overstrain would detach all initially bound myosins (7), so the maintained force increase requires continuing replenishment by new cross-bridges. Attachment-detachment rates in shortening contractions are limited by the relatively slow ATP-hydrolysis cycle (2). The extremely fast rates observed during stretches (3,4,8,9) suggest that there must be novel recruitment mechanisms during active lengthening (2, 4, 7).

Evidence for Unique Structural Change of Thin Filaments upon Calcium Activation of Insect Flight Muscle

“…First, upon calcium activation of IFM fibers, the most intense 6th ALL decreases its intensity and shifts its peak away from the meridian. The 6th ALLs in relaxed and calcium-activated states have actually been compared in previous reports, 19,23 but neither of them described the changes observed here ('No obvious change' 23 or an ∼10% increase in the supplemental material to Ref. 19; the reason for this discrepancy is presently unclear).…”

“…The 6th ALLs in relaxed and calcium-activated states have actually been compared in previous reports, 19,23 but neither of them described the changes observed here ('No obvious change' 23 or an ∼10% increase in the supplemental material to Ref. 19; the reason for this discrepancy is presently unclear). Second, the observed changes of the 6th ALL are not affected by reducing the number of myosin heads attached to actin.…”

“…23 Myosin attachment in this zone negatively interferes with the m1,0,3 while it strengthens the m1,0,6; thus, the intensity ratio of these two reflection spots can be used as a measure of extent of myosin binding to the thin filament. 19,23,28 Upon calcium activation of Lethocerus fibers at 20°C, the m1,0,3 weakened while the m1,0,6 strengthened, increasing the (m1,0,6)/(m1,0,3) ratio by 512%. The 3rd actin (troponin) meridional spot at 1/12.9 nm − 1 weakens by 71%.…”

“…Its tropomyosin molecules move upon activation in the same manner as in vertebrate skeletal muscle, as evidenced by electron-microscopic 16 and X-ray diffraction studies. [17][18][19] However, the regulation of contraction in the asynchronous type of IFM (IFM in which high-frequency wing beats are elicited by low-frequency nerve impulses) is more complex than that in vertebrate skeletal muscle; at least at submaximal calcium levels, asynchronous IFM generates only a small to medium amount of isometric force by calcium activation alone. A fully enhanced force is generated when the muscle is stretched externally (stretch activation 20 ).…”

X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle

“…Proposed mechanisms for the 60-y mystery of stretch activation have included helical matching between thick and thin filaments (34,35), lattice spacing changes (36), stress-induced changes in myosin activity (14,(37)(38)(39), and stress-induced changes in thinfilament activation (12,(40)(41)(42). We favor the last mechanism, and our results, particularly the time-resolved sequence of tropomyosin movement, cross-bridge binding and force production, strongly support the hypothesis that steric blocking-unblocking by tropomyosin regulates stretch activation.…”