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At least 145 records · Page 8Linked to original sources
PubMed1997

Fluorescence detection of calcium-binding proteins with quinoline Ca-indicator quin2.

We have established a fluorescence method to detect calcium-binding proteins by making use of the quinoline Ca indicator quin2. Authentic calcium-binding proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene difluoride membranes. Transfers were incubated with nonradioactive calcium ions, then with quin2 to detect the calcium-binding proteins as fluorescent bands by illumination with UV light. Calmodulin and parvalbumin of EF hand conformation calcium-binding type were clearly identified. Quin2 distinguished smooth muscle alpha-actinin from skeletal muscle alpha-actinin; the former was faintly fluorescent, having a low affinity for calcium ions. In whole myofibril preparations from skeletal muscles, troponin-C, connectin (titin), and nebulin were intensely fluorescent, being shown to have calcium-binding ability. The fluorescence method is an accurate, safe, and simple procedure to detect the binding of calcium ions to proteins following electrophoresis. The overlay technique described can be completed within 15 h and detects as little as 38 ng/well of troponin-C in the starting sample.

Actinin↗
PubMed1998

Hedgehog and wingless induce metameric pattern in the Drosophila visceral mesoderm.

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). We show here that the VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. As VM progenitors merge to form a continuous band running anterior to posterior along the embryo, expression of connectin (con) in 11 metameric patches within the VM reveals VM subdivisions analagous to ectodermal compartments. The VM subdivisions form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog (hh) and wingless (wg) and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Con in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers.

Animals↗
PubMed1996

Towards a molecular understanding of the elasticity of titin.

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.

Amino Acid Sequence↗
PubMed1999

Modularity and homology: modelling of the type II module family from titin.

We report the homology modelling of the structures of the 162 type II modules from the giant multi-domain protein titin (also known as connectin). The package MODELLER was used and implemented in an automated fashion using four experimentally determined structures as templates. Validation of the models was assessed in terms of divergence from the templates and consensus of the alignments. The homology within the whole family of type II modules as well as with the templates is relatively high (20-35% identity and ca 50% similarity). Comparison between the models of domains for which an NMR structure has been solved and the experimental solution gives an estimate of the quality of the modelling. Our results allow us to distinguish between a set of structurally relevant residues, which are conserved throughout the whole family and buried in the hydrophobic core, from the residues that are conserved and exposed. These latter residues are potentially functionally important. Comparison of exposed conserved patches for modules in different regions of the titin molecule suggests potential interaction surfaces. Our results may be tested directly for those modules whose binding partner is known.

Amino Acid Sequence↗
PubMed2002

The three-dimensional structure of a vertebrate wide (slow muscle) Z-band: lessons on Z-band assembly.

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.

Animals↗
PubMed1998

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.

Antibodies↗
PubMed2000

Titin-thin filament interaction and potential role in muscle function.

Titin (connectin) is a giant polypeptide that forms a single-molecule filamental structure extending from the M-line to the Z-line in the sarcomere of striated muscle. The primary structure of titin consists mainly of repeats of two types of approximately 100-amino acid modules (fibronectin type III and immunoglobulin-like motifs, respectively) and a Pro rich segment named the PEVK domain. The I-band region of titin shows an elasticity important to the passive properties of the myofibril. To investigate the biological function of titin, we cloned cDNA segments encoding single or linked structural modules of titin into expression vectors to produce non-fusion titin fragments in E. coli. High level expression of titin fragments was achieved and effective purification procedures were developed. We also developed specific monoclonal antibodies against the titin fragments and solid-phase protein-binding assays to investigate the interaction of the titin structural modules and other sarcomeric proteins. The results show that the immunoglobin-like module that are enriched in the I-band titin binds to F-actin. In contrast to the rigid association of A-band titin with the thick filament, the relatively weak titin-actin binding suggests that the I-band titin may reversibly interact with the thin filament during muscle contraction. This hypothesis is supported by an epitope similarity between the actin-binding site of caldesmon and the immunoglobulin-like module of titin, which suggests analogous functions of caldesmon and titin in organizing the contractile proteins. Together with data from mechanical studies demonstrating that the titin-actin interaction may contribute to the passive property of cardiac muscle in a Ca(2+)-dependent manner, we speculate that the Ca(2+)-mediated thin filament regulation may coordinate the function of titin during muscle contraction and relaxation.

Animals↗
PubMed2000

Mechanical manipulation of single titin molecules with laser tweezers.

Titin (also known as connectin) is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the vertebrate muscle sarcomere. The molecule is significant in maintaining sarcomeric structural integrity and generating passive muscle force via its elastic properties. Here we summarize our efforts to characterize titin's elastic properties by manipulating single molecules with force-measuring laser tweezers. The titin molecules can be described as an entropic spring in which domain unfolding occurs at high forces during stretch and refolding at low forces during release. Statistical analysis of a large number (> 500) of stretch-release experiments and comparison of experimental data with the predictions of the wormlike chain theory permit the estimation of unfolded titin's mean persistence length as 16.86 A (+/- 0.11 SD). The slow rates of unfolding and refolding compared with the rates of stretch and release, respectively, result in a state of non-equilibrium and the display of force hysteresis. Folding kinetics as the source of non-equilibrium is directly demonstrated here by the abolishment of force hysteresis in the presence of chemical denaturant. Experimental observations were well simulated by superimposing a simple domain folding kinetics model on the wormlike chain behavior of titin and considering the characteristics of the compliant laser trap. The original video presentation of this paper may be viewed on the web at http:¿www.pote.hu/mm/prezentacio/mkpres/++ +mkpres.htm.

Animals↗
PubMed1993

Partial titin cDNA sequence isolated from rabbit cardiac muscle RNA.

Two regions of the rabbit cardiac titin cDNA were amplified from rabbit cardiac muscle total RNA using primers based on rabbit skeletal muscle titin (connectin) cDNAs. These 1.7 kb and 1.5 kb RNA-PCR products were based on the 3' regions of the skeletal muscle titin clones CE12 and MS2, respectively. The cDNA sequence of the 1.7 kb product was extended an additional 1.5 kb by a novel 3' extension technique which used random primers in RNA-PCR. The cardiac titin cDNAs were 99% identical in nucleotide sequence to their skeletal muscle counterparts and predicted two types of 100-residue repeats. Southern blot analysis suggested that both cardiac and skeletal titin are encoded by the same gene. PCR amplification of human genomic DNA with titin specific primers indicated that there is strong sequence similarity between rabbit and human titin sequences. The successful amplification of a 907 basepair region from human genomic DNA suggested that titin contains large exons which span multiple motif borders. This may be particularly advantageous in the processing of such a large RNA transcript.

Animals↗
PubMed1996

Limits of titin extension in single cardiac myofibrils.

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from approximately 2-5 microns. Myofibril stretch up to sarcomere lengths of approximately 3 microns resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of approximately 2.7 microns. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond approximately 3 microns sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 microns. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

Animals↗
PubMed1996

Assembly of the cardiac I-band region of titin/connectin: expression of the cardiac-specific regions and their structural relation to the elastic segments.

The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.

Animals↗
PubMed1997

Expression of titin isoforms in red and white muscle fibres of carp (Cyprinus carpio L.) exposed to different sarcomere strains during swimming.

Titin (also known as connectin) is a striated-muscle-specific protein that spans the distance between the Z- and M-lines of the sarcomere. The elastic segment of the titin molecule in the I-band is thought to be responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscle types express isoforms of titin that differ in their molecular mass. To help to elucidate the relation between the occurrence of titin isoforms and the functional properties of different fibre types, we investigated the presence of different titin isoforms in red and white fibres of the axial muscles of carp. Gel electrophoresis of single fibres revealed that the molecular mass of titin was larger in red than in white fibres. Fibres from anterior and posterior axial muscles were also compared. For both white and red fibres the molecular mass of titin in posterior muscle fibres was larger than in anterior muscle fibres. Thus, the same fibre type can express different titin isoforms depending on its location along the body axis. The contribution of titin to passive tension and stiffness of red anterior and posterior fibres was also determined. Single fibres were skinned and the sarcomere length dependencies of passive tension and passive stiffness were determined. Measurements were made before and after extracting thin and thick filaments using relaxing solutions with 0.6 mol.l-1 KCl and 1 mol.l-1 KI. Tension and stiffness measured before extraction were assumed to result from both titin and intermediate filaments, and tension after extraction from only intermediate filaments. Compared to mammalian skeletal muscle, intermediate filaments developed high levels of tension and stiffness in both posterior and anterior fibres. The passive tension-sarcomere length curve of titin increased more steeply in red anterior fibres than in red posterior fibres and the curve reached a plateau at a shorter sarcomere length. Thus, the smaller titin isoform of anterior fibres results in more passive tension and stiffness for a given sarcomere strain. During continuous swimming, red fibres are exposed to larger changes in sarcomere strain than white fibres, and posterior fibres to larger changes in strain than anterior fibres. We propose that sarcomere strain is one of the functional parameters that modulates the expression of different titin isoforms in axial muscle fibres of carp.

Animals↗
PubMed1996

Calcium-dependent inhibition of in vitro thin-filament motility by native titin.

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 and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show -- using in vitro motility and binding assays -- that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin-thin filament interaction occurs in a calcium-dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.

Actin Cytoskeleton↗
PubMed1993

Triiodothyronine influences quail myoblast proliferation and differentiation.

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2,500 and 7,000 cells/cm2. Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and 3H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.

Animals↗
PubMed1992

Non-enzymatic weakening of myofibrillar structures during conditioning of meat: calcium ions at 0.1 mM and their effect on meat tenderization.

The tenderness of meat is set by the properties of connective tissue and myofibrils. Skeletal muscle connective tissues become firm with chronological aging concomitantly with the increase in intermolecular non-reducing cross-links of collagen, and this process toughens meat, however, connective tissues hardly change during conditioning of meat. Therefore, the tenderization of meat during post mortem aging, or to put it more precisely, during post rigor aging, stems for the most part from changes in myofibril structures. My research derives its origin on findings of two kinds of post mortem changes in myofibril structures; i) fragmentation of myofibrils; and ii) restoration of rigor-shortened sarcomeres. These results were published in 1967 [1], and were, thereafter proved by many workers to be closely related to meat tenderization. I report in this paper the essential molecular mechanisms of these phenomena, and of structural changes in connectin or titin filaments. All of them are non-enzymatically induced by 0.1 mM calcium ion, which is the ultimate concentration of sarcoplasmic calcium ion in post mortem muscles.

Calcium↗
PubMed2003

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding.

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

Adaptation, Physiological↗
PubMed1992

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.

Animals↗