Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.

Making muscle elastic: the structural basis of myomesin stretching.

Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres--the structural units that produce contraction--and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.

Roles of titin in the structure and elasticity of the sarcomere.

The giant protein titin is thought to play major roles in the assembly and function of muscle sarcomeres. Structural details, such as widths of Z- and M-lines and periodicities in the thick filaments, correlate with the substructure in the respective regions of the titin molecule. Sarcomere rest length, its operating range of lengths, and passive elastic properties are also directly controlled by the properties of titin. Here we review some recent titin data and discuss its implications for sarcomere architecture and elasticity.

Shape and flexibility in the titin 11-domain super-repeat.

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of approximately 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 mum long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the 'large super-repeat'. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 mum long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerized when bound to the thick filament.

Giant proteins: sensing tension with titin kinase.

Recent studies at the single-molecule level show how signaling from the giant protein titin can be triggered by direct mechanical activation of its kinase domain.

Evidence for the oligomeric state of 'elastic' titin in muscle sarcomeres.

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an approximately 290 kDa ( approximately 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved 'distal' tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.

Persistence length of titin from rabbit skeletal muscles measured with scattering and microrheology techniques.

The persistence length of titin from rabbit skeletal muscles was measured using a combination of static and dynamic light scattering, and neutron small angle scattering. Values of persistence length in the range 9-16 nm were found for titin-II, which corresponds to mainly physiologically inelastic A-band part of the protein, and for a proteolytic fragment with 100-nm contour length from the physiologically elastic I-band part. The ratio of the hydrodynamic radius to the static radius of gyration indicates that the proteins obey Gaussian statistics typical of a flexible polymer in a -solvent. Furthermore, measurements of the flexibility as a function of temperature demonstrate that titin-II and the I-band titin fragment experience a similar denaturation process; unfolding begins at 318 K and proceeds in two stages: an initial gradual 50% change in persistence length is followed by a sharp unwinding transition at 338 K. Complementary microrheology (video particle tracking) measurements indicate that the viscoelasticity in dilute solution behaves according to the Flory/Fox model, providing a value of the radius of gyration for titin-II (63 +/- 1 nm) in agreement with static light scattering and small angle neutron scattering results.

Can the passive elasticity of muscle be explained directly from the mechanics of individual titin molecules?

Recent progress in understanding the role of titin/connectin in muscle elasticity has been heavily based on results from single molecule mechanical experiments. The shape of force-extension curves from such data is similar to curves from muscle fibres and it has been tempting to assume that muscle elasticity can be extrapolated directly from the single molecule data. In this paper we discuss some of the factors that act on titin in the sarcomere that are likely to preclude such a direct extrapolation.

Titin: properties and family relationships.

In striated muscles, the rapid production of macroscopic levels of force and displacement stems directly from highly ordered and hierarchical protein organization, with the sarcomere as the elemental contractile unit. There is now a wealth of evidence indicating that the giant elastic protein titin has important roles in controlling the structure and extensibility of vertebrate muscle sarcomeres.