In vivo FRET imaging in freely moving C. elegans demonstrated that the sarcomeric kinase domain of twitchin (TwcK) undergoes periodic, stretch-linked conformational changes during muscle contraction cycles, supporting the hypothesis that mechanically induced unfolding of its autoinhibitory tails activates this cytoskeletal kinase in living muscle. The mechanism that connects this conformational activation to muscle physiology, however, remains unresolved: the physiological substrate and downstream pathway of TwcK (or a rigorous demonstration that it acts through a non-canonical, moonlighting output) are unknown, it is undetermined which of the two autoinhibitory tails (NL or CRD) unfolds in the working sarcomere and at what mechanical force threshold, and the homologous human titin kinase has never been tested for stretch-unfolding in vivo. A 2026 preprint from the same consortium reports that the myosin regulatory light chain MLC-4, the leading substrate candidate, segregates from TwcK in vivo and is not a functional substrate, proposing instead that TwcK is a catalytically-primed moonlighting kinase — a non-peer-reviewed reframing that sharpens rather than closes the mechanism question, which stands in unresolved tension with the hypercontractile phenotype of catalytically-inactive TwcK mutants.

Background

Striated muscle contracts through the sliding of thick (myosin-based) and thin (actin-based) filaments within the sarcomere, the muscle contractile unit. Twitchin (encoded by unc-22 in C. elegans) is a giant protein of the thick filament, homologous to vertebrate titin, whose C-terminal region contains a protein kinase domain termed twitchin kinase (TwcK). In the crystal structure, the kinase catalytic core is enveloped by two autoinhibitory tail extensions — the NL tail (about 45 residues N-terminal to the kinase domain) and the CRD tail (about 60 residues C-terminal, also called the C-terminal regulatory domain) — which bind against the kinase hinge and active site and shut off phosphotransfer activity (von Castelmur et al. 2012). Single-molecule atomic-force-microscopy pulling experiments and molecular-dynamics simulations had shown that these tails can be mechanically unravelled in vitro, leading to the proposal that the pulling forces a kinase molecule experiences inside the working sarcomere unfold the tails and thereby activate the kinase — making TwcK and its human homologue titin kinase (TK) candidate mechanosensory switches of the myofibril (Greene et al. 2008; Puchner et al. 2008 for TK). FRET (Förster resonance energy transfer, a spectroscopic ruler in which energy transfer between two fluorescent proteins falls off steeply with their separation, here about 4.8 nm) was used by Porto et al. (2021) to test this in vivo: transgenic and CRISPR-edited C. elegans expressed twitchin fragments carrying two fluorescent proteins flanking the NL-kinase-CRD assembly, and computer-vision tracking of freely moving worms extracted FRET ratios phase-locked to the muscle contraction cycle. FRET fell during relaxation and rose during contraction, statistically significant versus an inextensible control construct — the first in vivo evidence that a defined sarcomeric protein locus undergoes stretch-linked conformational extension during physiological muscle activity. What the measurement cannot tell is which tail moved (a single FRET pair reports the combined NL-plus-CRD extension), and body curvature was used as a proxy for stretch, so the actual force needed to trigger unfolding in the sarcomere is unmeasured. The functional side is equally open. Biochemically, TwcK phosphorylates a model peptide derived from myosin light chain in vitro, but its physiological substrate is unknown. Genetics shows the kinase matters: CRISPR mutants in which TwcK was rendered catalytically inactive move faster and hypercontract under optogenetic (light-driven) stimulation of muscle, implying that TwcK phosphotransfer normally inhibits muscle activity, most plausibly by phosphorylating a substrate that reduces contraction strength or duration (Matsunaga et al. 2017). An upstream activator is known — MAK-1, the C. elegans homologue of MAPKAP kinase 2 (MAP-kinase-activated protein kinase 2, an enzyme activated downstream of the p38 stress-signalling cascade), binds the CRD and can phosphorylate and activate TwcK in vitro (Matsunaga et al. 2015) — but no downstream target has ever been identified. Whether the mechanically driven conformational change primarily gates phosphotransfer, or instead serves as a scaffold for recruiting other proteins, is undetermined; in the human homologue the situation is inverted, since TK is considered an inactive pseudokinase (a kinase-fold domain without detectable phosphotransfer activity) whose role is scaffolding of MuRF (muscle RING-finger) E3-ubiquitin-ligase and Nbr1/p62 autophagy-receptor recruitment, with mechanical unfolding proposed to regulate that scaffolding (Bogomolovas et al. 2014; Bogomolovas et al. 2021).

Problem Statement

For twitchin kinase (TwcK) in living C. elegans body-wall muscle, whose stretch-linked conformational extension during contraction cycles was demonstrated by in-vivo FRET imaging, determine the mechanotransduction mechanism end to end, namely: (1) the molecular output through which mechanically regulated TwcK modulates muscle contraction — the identity of its physiological substrate(s) and downstream signalling pathway, or a rigorous demonstration that TwcK acts through a non-canonical (moonlighting) output, that is, an output other than phosphorylation of a physiological substrate; whichever branch is established must identify TwcK's molecular output and account for the faster movement and greater contraction of catalytically-inactive TwcK mutants; and (2) the structural-mechanical parameter of activation in the working sarcomere — which autoinhibitory tail (NL, CRD, or both) unfolds during muscle activity. These two components jointly close the chain from mechanical stretch, to tail-specific conformational activation, to an identified molecular and physiological effect, and the problem is complete when a domain expert can state how TwcK converts muscle mechanical activity into altered contractile behaviour. The mechanical force at which unfolding is triggered in the sarcomere remains unmeasured, because the in-vivo FRET signal was referenced to a body-curvature proxy rather than to molecular load; characterizing that force is not part of this task's completion boundary. Whether stretch-unfolding of the homologous human titin kinase (TK) is coupled to muscle inactivity has not been tested in vivo in living vertebrate muscle and is not part of this task's completion boundary.

References

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