Kuan Wang

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline‐rich peptides derived from the 28‐mer PEVK modules of titin and the Z‐line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (∼31 and 3.4 μM, respectively), whereas the other four peptides bind weakly (>100 μM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z‐line during sarcomere assembly.

Single molecule measurements of titin elasticity

Titin, with a massive single chain of 3--4MDa and multiple modular motifs, spans the half-sarcomere of skeletal and cardiac muscles and serves important, multifaceted functions. In recent years, titin has become a favored subject of single molecule observations by atomic force microscopy (AFM) and laser optical trap (LOT). Here we review these single titin molecule extension studies with an emphasis on understanding their relevance to titin elasticity in muscle function. Some fundamental aspects of the methods for single titin molecule investigations, including the application of dynamic force, the elasticity models for filamentous titin motifs, the technical foundations and calibrations of AFM and LOT, and titin sample preparations are provided. A chronological review of major publications on recent single titin extension observations is presented. This is followed by summary evaluations of titin domain folding/unfolding results and of elastic properties of filamentous titin motifs. Implications of these single titin measurements for muscle physiology/pathology are discussed and forthcoming advances in single titin studies are anticipated.

Titin as a giant scaffold for integrating stress and Src homology domain 3-mediated signaling pathways: the clustering of novel overlap ligand motifs in the elastic PEVK segment.

The richness of proline sequences in titins qualifies these giant proteins as the largest source of intrinsically disordered structures in nature. An extensive search and analysis for Src homology domain 3 (SH3) ligand motifs revealed a myriad of broadly distributed SH3 ligand motifs, with the highest density in the PEVK segments of human titin. Besides the canonical class I and II motifs with opposite orientations, novel overlapping motifs consisting of one or more of each canonical motif are abundant. Experimentally, the binding affinity and critical residues of these putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle and non-muscle cell extracts were validated with peptide array technology and by the sarcomere distribution of SH3-containing proteins. A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration. Molecular dynamics and molecular docking studies indicated that the overlapping motif can bind in opposite orientations with comparable energy and contact areas and predicts correctly orientation-specific contacts in NMR data. We propose that the overlap ligand motifs are a new class of ligands with innate ability to dictate SH3 domain orientation and to facilitate the rate, strength, and stereospecificity of receptor interactions. Proline-rich sequences of titins are candidates as major hubs of SH3-dependent signaling pathways. The interplay of elasticity and dense clustering of mixed receptor orientations in titin PEVK segment have important implications for the mechanical sensing, force sensitivity, and inter-adapter interactions in signaling pathways.

Surface initiated actin polymerization from top-down manufactured nanopatterns

Protocols to fabricate high aspect-ratio biologically-based nanostructures using a top-down fabricated polymer platform and surface-initiated actin polymerization were developed.

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (KI) were 70-80 micro M for ATPase and 20-30 micro M for tension, nearly >200-fold lower than BDM (20 mM and 5-8 mM, respectively). When W7 (50 microM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.

What the buzz was all about: superfast song muscles rattle the tymbals of male periodical cicadas

Male cicadas produce mating calls by oscillating a pair of superfast tymbal muscles in their anterior abdominal cavity that pull on and buckle stiff‐ribbed cuticular tymbal membranes located beneath the folded wings. The functional anatomy and rattling of the tymbal organ in 17 yr periodical cicada, Magicicada cassini (Brood X), were revealed by high‐resolution microcomputed tomography, magnetic resonance imaging, electron microscopy, and laser vibrometry to understand the mechanism of sound production in these insects. Each 50 Hz muscle contraction yielded five to six stages of rib buckling in the tymbal, and a small release of muscle tension resulted in a rapid recovery due to the spring‐loaded nature of the stiff ribs in the resilin‐rich tymbal. The tymbal muscle sarcomeres have thick and thin filaments that are 30% shorter than those in flight muscles, with Z‐bands that were thicker and configured into novel perforated hexagonal lattices. Caffeine‐treated fibers supercontracted by allowing thick filaments to traverse the Z‐band through its open lattice. This superfast sonic muscle illustrates design features, especially the matching hexagonal symmetry of the myofilaments and the perforated Z‐band that contribute to high‐speed contractions, long endurance, and potentially supercontraction needed for producing enduring mating songs and choruses. —Nahirney, P. C., Forbes, J. G., Morris, H. D., Chock, S. C., Wang, K. What the buzz was all about: Superfast song muscles rattle the tymbals of male periodical cicadas. FASEB J. 20, 2017–2026 (2006)

S100A1 modulates skeletal muscle contraction by desensitizing calcium activation of isometric tension, stiffness and ATPase

S100, a subfamily of the EF‐hand type calcium sensing proteins, is implicated in many cellular functions including muscle contractility. Two isoforms, S100A1 and S100B, at 2–10 μM significantly inhibit active tension, stiffness and ATPase of skinned single rabbit psoas muscle fibers at sub‐maximal (pCa ∼6.1‐5.6), but not at maximal levels of activation (pCa 4.0). S100A1 is a more potent inhibitor than S100B. Hill analysis of the ATPase–pCa and tension–pCa curves indicates that these proteins reduce calcium sensitivity and enhance the cooperativity toward calcium. We propose S100A1, and perhaps S100B, are viable candidates as physiological modulators of muscle contraction.

Extensive and modular intrinsically disordered segments in C. elegans TTN-1 and implications in filament binding, elasticity and oblique striation.

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1s sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an approximately 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats--39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep--were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to alpha-helix (PEVT, CEEEI, DispRep) and beta-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable alpha-helix-like structures coils in aqueous solution and negligible heptad-based, alpha-helical coiled-coils. The alpha-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable alpha-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.

Simultaneous dynamic stiffness and extension profiles of single titin molecules: Nanomechanical evidence for unfolding intermediates

Striated muscle is the primary source of biomechanical force in organisms from worms to man, and is organized as a composite material from nanometer to meter scales. At nanometer scale, an array of cytoskeletal proteins are required to regulate the size, assembly and function of the sarcomere, as well as transmit force and provide elasticity for restoring the structure. One such protein is the giant protein titin (Mr 3–4×106), which spans half of the muscle sarcomere length. The passive elasticity of muscle at a physiological range of stretch arises primarily from the extension of titin. We have measured both the extension force and the dynamic stiffness of native titin with the atomic force microscope. We found that the stiffness of a single molecule of titin varied between 1 and 4 pN/nm over an extension of >1 μm. Regions showing globular domains unfolding in the sawtooth pattern of force versus extension curve exhibit periodic spikes in the stiffness. The results of this study show that simultaneous st...

Nanomechanics of Full-Length Nebulin: An Elastic Strain Gauge in the Skeletal Muscle Sarcomere

Nebulin, a family of giant modular proteins (MW 700-800 kDa), acts as a F-actin thin filament ruler and calcium-linked regulator of actomyosin interaction. The nanomechanics of full length, native rabbit nebulin was investigated with an atomic force microscope by tethering, bracketing, and stretching full-length molecules via pairs of site-specific antibodies that were attached covalently, one to a protein resistant self-assembled monolayer of oligoethylene glycol and the other to the cantilever. Using this new nanomechanics platform that enables the identification of single molecule events via an unbiased analysis of detachment force and distance of all force curves, we showed that nebulin is elastic and extends to approximately 1 microm by external force up to an antibody detachment force of approximately 300-400 pN. Upon stretching, nebulin unravels and yields force spectra with craggy mountain range profiles with variable numbers and heights of force peaks. The peak spacings, analyzed by the model-independent, empirical Hilbert-Huang transform method, displayed underlying periodicities at approximately 15 and approximately 22 nm that may result from the unfolding of one or more nebulin modules between force peaks. Nebulin may act as an elastic strain gauge that interacts optimally with actin only under appropriate strain and stress. This stretch to match protein ruler may also exert a compressive force that stabilizes thin filaments against stress during contraction. We propose that the elasticity of nebulin is integral and essential in the muscle sarcomere.