Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called "troponin bridges," by analyzing real-time X-ray diffraction "movies" from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin's steric blocking of myosin-actin binding. This enables subsequent force production, with crossbridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.S tretch activation is a striking example of mechanical signal transduction, in which stretching a partially activated muscle yields, after a delay, greater activation. It is observed to varying degrees in all striated muscles, is prominent in vertebrate cardiac muscle where it may underlie the Frank-Starling relationship (1), and is essential to flight in multiple orders of insects, which together comprise ∼75% of insect species (2) and fully half of all animal taxa (3). Since stretch activation was first reported by Pringle (4), a great deal has been learned about how muscular contraction is driven by cyclic myosin-actin interactions (5) which, in vertebrate skeletal muscles, are controlled by calcium's effect on the steric blocking action of troponin-tropomyosin (6). However, even atomic resolution models of myosin-actin (7) and the troponin complex (8, 9) fail to shed any light on the mechanism of activation by stretch. The central question of stretch activation remains: How does mechanical stress convert to myosinactin activation while the requisite [Ca 2þ ] stays constant?Recently we observed cross-bridges between thick (mainly myosin) filaments and thin (mainly actin-troponin-tropomyosin) filaments at the level of the troponin (10). These myosin-troponin connections, referred to here simply as troponin bridges, comprised about 15% of all cross-bridges identified in an insect flight muscle (IFM) quick frozen during an isometric contraction. Troponin bridges represent a newly recognized class of crossbridges, distinct from the "traditional" force-generating myosin cross-bridges that bind to actin target zones halfway between troponins in IFM (10, 11). The direct observation of troponin bridges revived a previously speculative mechanism for stretch activation (12), in which troponin bridges serve as the agent of mechanica...

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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).

Smooth muscle myosin and heavy meromyosin (smHMM) are activated by regulatory light chain (RLC) phosphorylation but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The “free head” can bind to actin, but the actin-binding interface of the “blocked head” is involved in interactions with the free head. We report here a 3-D structure for phosphorylated, active smHMM obtained using electron crystallography of 2-D arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state, but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the “blocked” head of dephosphorylated smHMM. We hypothesize that RLC phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.