A molecular map of titin/connectin elasticity reveals two different mechanisms acting in series

In the I-band of skeletal muscle sarcomeres, the elastic region of titin consists of immunoglobulin (Ig) domains, and non-modular regions rich in proline, hydrophobic, and charged residues (PEVK). Using immunoelectron microscopy with sequence-assigned monoclonal antibodies, we demonstrate that extension of the Ig regions in M. psoas occurs largely at sarcomere lengths between 2 and 2.8 micron, decreasing in slope towards higher lengths. The Ig domains do not unfold. Above 2.6 micron, length changes are increasingly due to the PEVK-rich regions. We therefore propose that rubber-like properties of the PEVK-rich regions are mainly contributing to skeletal titin elasticity.     

Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.     

Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1alpha and monocyte chemotactic protein-1

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.     

Clinical manifestations and predictors of survival in older women infected with HIV

The structure and function of the giant elastic protein connectin/titin are described on the basis of recent investigations. The 3000 kDa protein links the Z line to the myosin filament in striated muscle sarcomeres. The NH2-terminal region of connectin filament is involved in the Z line binding, and the COOH-terminal region is bound onto the myosin filament with an overlap between the counter-connectin filaments at the M line. The PEVK region in the I band is shown to be mainly responsible for passive tension generation. The longitudinal continuity of myosin-, actin-free sarcomeres is explained by the linkage of freed connectin filaments extending from both sides of the Z lines in a sarcomere. The role of connectin in myofibrillar differentiation and the biodiversity of connectin-related proteins in the animal kingdom are briefly reviewed.     

Human autoantibodies reveal titin as a chromosomal protein

Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH2 termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.     

Actin-titin interaction in cardiac myofibrils: probing a physiological role

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Expression of sarcomeric proteins and assembly of myofibrils in the putative myofibroblast cell line BHK-21/C13

The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins.     

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.     

Marsupial light chains: complexity and conservation of lambda in the opossum Monodelphis domestica

The Ig lambda chains in the South American opossum, Monodelphis domestica, were analyzed at the expressed cDNA and genomic organization level, the first described for a nonplacental mammal. The V lambda segment repertoire in the opossum was found to be comprised of at least three diverse V lambda families. Each of these families appears to be related to distinct V lambda families present in placental mammals, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Based on framework and constant region sequences from full-length cDNAs and intron sequences from genomic clones, it appears that there are multiple functional J lambda-C lambda pairs in the opossum locus. The opossum J lambda-C lambda sequences are phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a large set of functional, expressed V lambda-J lambda recombinations is consistent with an unbiased, highly diverse lambda light chain repertoire in the adult opossum. Overall, the complexity of the Ig lambda locus appears to be greater than that found in the Ig heavy chain locus in the opossum, and light chains are therefore likely to contribute significantly to Ig diversity in this species.     

The evolution of titin and related giant muscle proteins

Titin and twitchin are giant proteins expressed in muscle. They are mainly composed of domains belonging to the fibronectin class III and immunoglobulin c2 families, repeated many times. In addition, both proteins have a protein kinase domain near the C-terminus. This paper explores the evolution of these and related muscle proteins in an attempt to determine the order of events that gave rise to the different repeat patterns and the order of appearance of the proteins. Despite their great similarity at the level of sequence organization, titin and twitchin diverged from each other at least as early as the divergence between vertebrates and nematodes. Most of the repeating units in titin and twitchin were estimated to derive from three original domains. Chicken smooth-muscle myosin light-chain kinase (smMLCK) also has a kinase domain, several immunoglobulin domains, and a fibronectin domain. From a comparison of the kinase domains, titin is predicted to have appeared first during the evolution of the family, followed by twitchin and with the vertebrate MLCKs last to appear. The so-called C-protein from chicken is also a member of this family but has no kinase domain. Its origin remains unclear but it most probably pre-dates the titin/twitchin duplication.     

Early dating by ultrasound and perinatal outcome. A cohort study

The myofibrils of adult rat cardiac muscle cells in culture break down and later reorganize into mature myofibrils. The myofibrillar breakdown and reorganization processes have been investigated with electron microscopical and immunocytochemical studies. The immunocytochemical studies included antibodies to actin, myosin, titin, and alpha-actinin. In addition, rhodamine-labeled phalloidin has been used. These studies revealed that the myofibrils were disorganized into amorphous and/or other forms during breakdown process. Some of these myofibrils undergo degradation and finally extrusion through exocytosis. The reorganization of myofibrils takes place mainly with the participation of the existing myofibrillar proteins in myocytes. This remyofibrillogenesis showed the emergence of punctate alpha-actinin from the existing amorphous alpha-actinin along with the differentiation of titin periodicities, which remained attached to the alpha-actinin structures. The punctate alpha-actinin later differentiated into periodicities, forming Z-lines. The periodicities of actin were differentiated from the amorphous actin and associated with the Z-lines, giving rise to titin, alpha-actinin, and actin complexes. Later, myosin filaments became associated with these complexes, forming sarcomeres where other myofibrillar proteins participated in the formation of mature myofibrils. The temporal sequence of differentiation of periodicities of certain myofibrillar proteins varied among different myocytes and within a single myocyte. The dynamic role of adult cardiac myocytes in the reconstruction of myofibrils is a remarkable phenomenon, which stabilizes adult cardiac muscle cells in long-term culture.     

Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins

A size-selected genomic library from Elymus alaskanus was screened for the presence of (GA)n, (GT)n, (CAC)n, and (TCT)n microsatellite sequences. A total of 28 positive clones were found for the two dinucleotide repeats, whereas no positive clones were found for the trinucleotide repeats. Positive clones were sequenced to validate the presence of microsatellites and to generate polymerase chain reaction (PCR) primers, based on the sequences flanking the microsatellite. Primer pairs were designed and evaluated for 18 selected microsatellites. The resulting loci were analysed by PCR for their usefulness as molecular markers in an array of 18 accessions representing E. alaskanus and 10 other Elymus species. PCR amplification revealed alleles for the 18 loci, which varied in having 1-10 alleles in these accessions. In the 18 accessions tested, 7 of the 18 loci were polymorphic, with gene diversity values ranging from 0.54 to 0.80 among all species. Within E. alaskanus, gene diversity values ranged from 0.20 to 0.72, with a mean of 0.48. Polymorphism was also detected within accessions. No clear relationship between total repeat length and the degree of polymorphism was observed in this study. Primer pairs designed to amplify microsatellites in E. alaskanus can be used to generate polymorphism products in other species within the genus. Hence, microsatellites are useful markers for studying both inter- and intra-specific genetic variability within Elymus.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

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.     

Effect of postmortem storage on the Z-line region of titin in bovine muscle

Myofibrils were prepared from bovine muscles (cutaneous trunci, rectus abdominis, psoas major, and masseter) and compared between different aging periods at 4 degrees C (0, 1, 2, 4, 8, and 16 d). Myofibrils were stained with an antibody directed against a 56-kDa fragment (FE-RE) of titin located in the Z-line region. Unaged myofibrils from all four muscles showed a single stained band at the Z-line with similar intensities. Postmortem time did not significantly affect the total amount of fluorescence in the sarcomere, suggesting the titin FE-RE epitope was not degraded nor were titin fragments containing this epitope released during storage. However, the fluorescence patterns were altered. The relative fluorescence intensity at the Z-line decreased but that in the I-band increased gradually, showing the translocation of some titin FE-RE epitopes during the aging period. This suggested that a cleavage occurred in a region of titin very close to the Z-line during postmortem storage. Usually the position of maximum fluorescence remained at the Z-line, although about 1/3 of the myofibrils from rectus abdominis showed a two-band pattern around the Z-line after 16 d of aging. The titin changes observed may be related to the increased fragility of the myofibril and the improvement of meat tenderness during postmortem storage.     

IMGT, the International ImMunoGeneTics database

IMGT, the international ImMunoGeneTics database, is an integrated database specialising in Immunoglobulins (Ig), T cell Receptors (TcR) and Major Histocompatibility Complex (MHC) of all vertebrate species, created by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes three databases: LIGM-DB (for Ig and TcR), MHC/HLA-DB and PRIMER-DB (the last two in development). IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 23 000 Immunoglobulin and T cell Receptor sequences from 78 species. MHC/HLA-DB contains Class I and Class II Human Leucocyte Antigen alignment tables. An IMGT tool, DNAPLOT, developed for Ig, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including nucleotide and protein sequences, oligonucleotide primers, gene maps and other genetic data of Ig, TcR and MHC molecules, and to provide a graphical user friendly data access. IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is freely available at http://imgt.cnusc.fr:8104     

Complete unfolding of the titin molecule under external force

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch-release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.