Titin-like Protein Research Articles

Titin(S): Towards an Atomic Understanding of Mechanosensory Events in the Elastic Scaffolds of the Muscle Sarcomere

The giant intra-sarcomeric filaments of the titin-like family are key orchestrators of stretch-sensing pathways that regulate muscle responses to mechanical load. Despite acute variations in the length and domain organization of these filamentous proteins across the animal biodiversity, they all comprise numerous Ig/FnIII domains linked in series and one or two kinase domains invariably located near their C-terminus. Combining the 3D-structural elucidation of multi-domain components at atomic level, in silico simulations, molecular engineering and in vivo transgenic muscle technologies, we are revealing the molecular events taking place during mechanosensing in titin-like proteins.Our findings show that the sensory role of titin is enabled by a subtle inter-domain order in the chain, imposed by short linkers that sterically govern domain packing and dynamics. This modular design is sensitive to mechanical deformations but affords a “chain memory” mechanism for molecular recovery. The local disruption of such domain arrangements by genetic mutation leads to human myopathy. Our data show that also the kinase domains of titin-like proteins undergo elastic deformations in their regulatory, flanking segments during muscle activity in vivo. Contrary to expectations, these kinases are catalytically-dispensable for muscle function and development. Instead, they act as regulated scaffolds for the recruitment of turnover/signaling proteins onto kinase-based signalosomes. This talk will provide a molecular perspective on the stretch-induced mechanics and signaling of titin-like proteins.Zacharchenkov T, et al. (2015). Biochem Soc Trans. In press.Bogomolovas J, et al. (2014). Open Biology. 4(5):140041.Von Castelmur E, et al. (2012). P.N.A.S. 109(34):13608-13.

Elastic proteins in the flight muscle of Manduca sexta

The flight muscles (DLM1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ∼500 and ∼700kDa, but also to bands at ∼1.6 and ∼2.1MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and sallimus isoforms were expressed to higher levels in ventrally located DLM1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle.

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.

Spontaneous Dimerization of Titin Protein Z1Z2 Domains Induces Strong Nanomechanical Anchoring

Muscle elasticity strongly relies on the mechanical anchoring of the giant protein titin to both the sarcomere M-band and the Z-disk. Such strong attachment ensures the reversible dynamics of the stretching-relaxing cycles determining the muscle passive elasticity. Similarly, the design of biomaterials with enhanced elastic function requires experimental strategies able to secure the constituent molecules to avoid mechanical failure. Here we show that an engineered titin-mimicking protein is able to spontaneously dimerize in solution. Our observations reveal that the titin Z1Z2 domains are key to induce dimerization over a long-range distance in proteins that would otherwise remain in their monomeric form. Using single molecule force spectroscopy, we measure the threshold force that triggers the noncovalent transition from protein dimer to monomer, occurring at ∼700 piconewtons. Such extremely high mechanical stability is likely to be a natural protective mechanism that guarantees muscle integrity. We propose a simple molecular model to understand the force-induced dimer-to-monomer transition based on the geometric distribution of forces occurring within a dimeric protein under mechanical tension.

A new property of twitchin to restrict the “rolling” of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin–myosin interaction by “freezing” tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.

Single-Molecule Force Spectroscopy Reveals a Stepwise Unfolding of Caenorhabditis elegans Giant Protein Kinase Domains

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30–150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

Molecular basis of the catch state in molluscan smooth muscles: a catchy challenge

The catch state (or 'catch') of molluscan smooth muscles is a passive holding state that occurs after cessation of stimulation. During catch, force and, in particular, resistance to stretch are maintained for long time periods with low (or no) energy consumption at basal intracellular free [Ca2+]. The catch state is initiated by Ca2+-stimulated dephosphorylation of the titin-like protein twitchin and is inhibited by cAMP-dependent phosphorylation of twitchin. In addition, catch is pH sensitive, but the reason for this is unknown. According to a traditional model, catch is due to slower cross-bridge cycles where myosin heads remain longer attached to the actin filaments after force generation, possibly caused by a hindered release of ADP from the myosin heads. However, this model was disproved by recent findings which showed that (i) inhibitors of myosin function, such as vanadate, do not affect catch force; (ii) factors which terminate the catch state do not accelerate myosin head detachment kinetics and (iii) a catch-like high resistance to stretch is still inducible when force development is prevented. Thus, catch probably involves passive linkage structures interconnecting the myofilaments (catch linkages). For example twitchin could (i) tie myosin heads to the thin filaments, (ii) mechanically lock them in a stretch resistant state or (iii) interconnect thick and thin filaments directly. However, it is questionable if these mechanisms are sufficient since twitchin seems to be about 15-times less abundant than myosin. Therefore, in addition, interconnections between thick filaments could exist, which could involve e.g. paramyosin or twitchin. Catch could even involve changes in the compliance of thick filaments. The function of myorod, found specifically in catch muscles in equal abundance with myosin, is not known. The suggestion is made here that catch linkages are present already during active contraction either as ratchet-like elements resisting stretch and not opposing shortening or in some kind of 'standby' mode ready to transform suddenly into the working mode by stretches or after Ca2+ removal following cessation of stimulation.

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber

We previously discovered a large titin-like protein-c-titin-in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures.

The molecular elasticity of the insect flight muscle proteins projectin and kettin

Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force-extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F(u) approximately 50-150 pN) than Ig-domains (F(u) approximately 150-250 pN). Among Ig domains in Sls/kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s(-1) (25 degrees C)] and even under high forces (15-30 pN). Temperature affected the unfolding forces with a Q(10) of 1.3, whereas the refolding speed had a Q(10) of 2-3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism.

Smooth muscle α-actinin interaction with smitin

Actin–myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells also contain the actin filament-crosslinking protein α-actinin. In striated muscle sarcomeres, interactions between the myosin-binding protein titin and α-actinin in the Z-line provide an important structural linkage. We previously discovered a titin-like protein, smitin, associated with the contractile apparatus of smooth muscle cells. Purified native smooth muscle α-actinin binds with nanomolar affinity to smitin in smitin–myosin coassemblies in vitro. Smooth muscle α-actinin also interacts with striated muscle titin. In contrast to striated muscle α-actinin interaction with titin and smitin, which is significantly enhanced by PIP 2, smooth muscle α-actinin interacts with smitin and titin equally well in the presence and absence of PIP 2. Using expressed regions of smooth muscle α-actinin, we have demonstrated smitin-binding sites in the smooth muscle α-actinin R2–R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF-hand structures. These smitin-binding sites are highly homologous to the titin-binding sites of striated muscle α-actinin. Our results suggest that direct interaction between α-actinin and titin or titin-like proteins is a common feature of actin–myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for α-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Two-dimensional gel electrophoresis of Caenorhabditis elegans homogenates and identification of protein spots by microsequencing.

Employing isoelectric focusing on immobilized pH gradients followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have obtained a map of C. elegans proteins, from a mixed culture containing all developmental stages, presenting over 2000 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Edman microsequencing yielded successful results in 12 out of 24 analyzed spots. All but one of the N-terminal sequences retrieved C. elegans sequences in cosmid and/or expressed sequence tag clones. Structurally related protein sequences found in data banks included enzymes in energy metabolism (cytochrome oxydase, ATP synthase, enolase), a fatty acid-binding protein, a translationally controlled tumor protein, an unknown C. elegans protein, an acidic ribosomal protein, a titin-like protein, a G-protein beta chain, cyclophilin, and cathepsin D. Experimental determination of N-termini allowed us to define sites of signal cleavage providing further information on the physiological role of the newly found C. elegans proteins. This report demonstrates the possibility of two-dimensional gel electrophoresis and Edman microsequencing in the elucidation of C. elegans proteome.

Immunohistochemical study and western blotting analysis of titin-like proteins in the striated muscle of Drosophila melanogaster and in the striated and smooth muscle of the oligochaete Eisenia foetida.

The presence and distribution of titin-like proteins have been examined in transversely striated muscle of Drosophila melanogaster, in obliquely striated muscles (body wall and inner muscular layer of the pseudoheart) and smooth muscle (outer muscular layer of the pseudoheart) from the earthworm Eisenia foetida by means of Western blotting analysis, light microscopy immunohistochemistry, and electron microscopy immunogold labeling, using antibodies anti vertebrate (chicken) titin (3,000 kDa) and arthropod (D. melanogaster) mini-titin (twitchin or projectin) (700 kDa). To determine whether these antibodies immunoreact non-specifically against vertebrate titin, mouse skeletal muscle was also studied. As negative control, mouse smooth muscle was used. Immunoreaction to mini-titin was found in all the invertebrate muscles studied. For each of these muscles, Western blotting analysis of mini-titin showed a single band, at approximately 700 kDa. Electron microscopy immunolabeling to this protein was observed along the whole sarcomere length (A bands and I bands) in both transversely striated muscles of the insect and obliquely striated muscles of the earthworm, although the number of immunogold particles was more abundant in the insect muscles. Mini-titin immunolabeling was also observed in the smooth muscle cells that formed the outer layer of the earthworm pseudoheart although in lower amounts than in the obliquely striated muscle. The absence of true sarcomeres in the smooth muscle cells did not permit to determine the extension of mini-titin immunolabeling. No immunoreaction to this protein was found in the striated and smooth muscles of the mouse. Immunoreaction to titin was only observed in the mouse skeletal muscle, in which both A bands and I bands appeared immunolabeled. Present results show that mini-titin in the invertebrate muscles studied differs immunohistochemically from vertebrate titin and, in contrast with titin, mini-titin is also present in invertebrate smooth muscles.

The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments.

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.

Titin: major myofibrillar components of striated muscle.

Electrophoretic analyses of protein components of striated muscle myofibril purified from various vertebrate and invertebrate species revealed that proteins much larger than myosin heavy chain are present in significant amounts. To define possible roles of these heretofore unidentified proteins, we purified a combination of two uncommonly large proteins, designated as titin, from chicken breast myofibrils. Chemical and immunological studies indicated that titin is distinct from myosin, actin, and filamin. Specific titin anti body crossreacts with similar protein in both skeletal and cardiac myofibrils of many vertebrate and invertebrate species. Immunofluorescent staining of glycerinated chicken breast myofibrils indicated that titin is present in M lines, Z lines, the junctions of A and I bands, and perhaps throughout the entire A bands. Similar staining studies of myofibrils from other species suggest that titinlike proteins may be organized in all myofibrils according to a common architectural plan. We conclude that titin is a structurally conserved myofibrillar component of vertebrate and invertebrate striated muscles.