Studies on the interaction between titin and myosin.

This study examines the interaction of titin and myosin. In order to analyze the domains of myosin contributing to the binding for titin, we conducted a solid phase binding assay. Different portions of myosin (heavy chains, light chains and myosin fragments) were coated on the microtiter wells and reacted with biotinylated titin. Then the binding of biotinylated titin to these polypeptides was detected by using the avidinbiotin-peroxidase method. The results demonstrated that light meromyosin and subfragment 1 were the major domains of myosin interacting with titin. Titin fragments obtained by trypsin digestion were allowed to react with myosin in an affinity column, and the bound fragments were isolated by an acidic elution. Immunoblot analysis of myosin-bound titin fragments revealed that an A-band domain of titin was responsible for the binding of myosin. In addition, biotinylated titin labelled the outer A-bands and Z-bands in intact myofibrils, thus confirming the in situ binding of titin to myosin.     

The structural principles of multidomain organization of the giant polypeptide chain of the muscle titin protein: SAXS/WAXS studies during the stretching of oriented titin fibres

Elasticity of titin is a key parameter that determines the mechanical properties of muscle. These include reversibility, i.e., the muscle's capacity to change its length many-fold and return to its original state, and the transduction of passive tension generated by the stretched muscle. The morphology and elastic properties of oriented fibres of titin molecules were studied using SAXS and WAXS (small- and wide-angle X-ray scattering, respectively) and mechanical techniques. We succeeded in obtaining oriented filaments of purified titin suitable for diffraction measurements. Our X-ray data suggest a model of titin as a nanoscale, morphological, and aperiodical array of rigid Ig- and Fn3-type domains covalently connected by conformationally variable short loops. The line group symmetry of the model can be defined as SM with axial translation tau(infinity). Both tension transduction and high elasticity of titin can be explained in terms of crystalline polymer physics. Titin stretching experiments show that each individual titin macromolecule can adopt a novel two-phase state within the fibre. Conversion between high elasticity and strength can be explained as a phase transition under external tension. In the terms of the concept of orientational melting the origin of the functional heterogeneity along the titin strand becomes interpretable.     

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.     

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of alpha-actinin.

The sarcomeric Z-disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by alpha-actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of alpha-actinin into the Z-disk. Titin interacts via a single binding site with the two central spectrin-like repeats of the outermost pair of alpha-actinin molecules. In the central Z-disk, titin can interact with multiple alpha-actinin molecules via their C-terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and alpha-actinin in vitro, and are expected to constrain the path of titin in the Z-disk. In thick skeletal muscle Z-disks, titin filaments cross over the Z-disk centre by approximately 30 nm, suggesting that their alpha-actinin-binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z-disk, where overlapping titin filaments and their interactions with the alpha-actinin rod and C-terminal domain can account for the essential ultrastructural features.     

Titin mutations as the molecular basis for dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.     

On the titin isoforms

By our modified SDS gel electrophoresis and immunoblotting, the isoform composition of titin in skeletal and cardiac muscles of human and animals was studied to reveal new titin forms above 3700 kDa in size. The data obtained suggest that the new large-size titin species are the intact (original) isoforms of this protein, whereas the known N2A, N2B, and N2BA titin bands in electrophoregrams correspond to their fragments.     

Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres

The giant muscle protein titin is an essential structural component of the sarcomere. It forms a continuous periodic backbone along the myofiber that provides resistance to mechanical strain. Thus, the titin filament has been regarded as a blueprint for sarcomere assembly and a prerequisite for stability. Here, a novel titin-eGFP knockin mouse provided evidence that sarcomeric titin is more dynamic than previously suggested. To study the mobility of titin in embryonic and neonatal cardiomyocytes, we used fluorescence recovery after photobleaching and investigated the contribution of protein synthesis, contractility, and calcium load to titin motility. Overall, the kinetics of lateral and longitudinal movement of titin-eGFP were similar. Whereas protein synthesis and developmental stage did not alter titin dynamics, there was a strong, inhibitory effect of calcium on titin mobility. Our results suggest a model in which the largely unrestricted movement of titin within and between sarcomeres primarily depends on calcium, suggesting that fortification of the titin filament system is activity dependent.     

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 ∼ 290 kDa (∼ 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.     

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.     

Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts.

Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis.     

The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

Changes in the isoform composition of the cytoskeletal protein titn--adaptation process in hibernation

Changes in the isoform composition of the elastic protein titin from skeletal and cardiac muscles of hibernating ground squirrels were revealed for the first time. It was shown that, upon hibernation, the molecular mass of titin decreases and its functional properties change as compared with the active state of the animal. The physiological significance of the changes in titin isoform composition for the inhibition of muscle contractile activity upon hibernation is discussed in connection with similar changes during some cardiomyopathies.     

Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.     

The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.     

Titin isoform-dependent effect of calcium on passive myocardial tension

We studied the effects of Ca2+ on titin (connectin)-based passive tension in skinned myocardium expressing either predominantly N2B titin (rat right ventricle, RRV) or predominantly N2BA titin (bovine left atrium, BLA). Actomyosin-based tension was abolished to undetectably low levels by selectively removing the thin filaments with a Ca2+-insensitive gelsolin fragment (FX-45). Myocardium was stretched in the presence and absence of Ca2+, and passive tension was measured. Ca2+ significantly increased passive tension during and after stretch in the BLA. The increase was insensitive to the actomyosin inhibitor 2,3-butanedione 2-monoxime, supporting the conclusion that the effect is titin based. Passive tension did not respond to calcium in the RRV, indicating that passive tension developed by N2B titin is calcium insensitive. Western blot analysis and immunofluorescence studies indicated that N2BA titin expresses E-rich PEVK motifs, whereas they are absent from N2B titin, supporting earlier single molecule studies that reported that E-rich motifs are required for calcium sensitivity. We conclude that calcium affects passive myocardial tension in a titin isoform-dependent manner.     

Titin and the sarcomere symmetry paradox

Titin is thought to play a major role in myofibril assembly, elasticity and stability. A single molecule spans half the sarcomere and makes interactions with both a thick filament and the Z-line. In the unit cell structure of each half sarcomere there is one thick filament with 3-fold symmetry and two thin filaments with approximately 2-fold symmetry. The minimum number of titin molecules that could satisfy both these symmetries is 12. We determined the actual number of titin molecules in a unit cell from scanning transmission electron microscopy mass measurements of end-filaments. One of these emerges from each tip of the thick filament and is thought to be the in-register aggregate of the titin molecules associated with the filament. The mass per unit length of the end-filament (17.1 kDa/nm) is consistent with six titin molecules not 12. Thus the number of titin molecules present is insufficient to satisfy both symmetries. We suggest a novel solution to this paradox in which four of the six titin molecules interact with the two thin filaments in the unit cell, while the remaining two interact with the two thin filaments that enter the unit cell from the adjacent sarcomere. This arrangement would augment mechanical stability in the sarcomere.     

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin- dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.