The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1- line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1- line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M- protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.
Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.
Myosin; Myomesin 3; M-line; Sarcomere
The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = −0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes’ cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-010-0131-2) contains supplementary material, which is available to authorized users.
Dilated cardiomyopathy; Heart failure; Sarcomere cytoskeleton; M-band; Myomesin
The expression of the myofibrillar M-band proteins myomesin and M- protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M- protein by 1.5 d. Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.
The muscle M-band protein myomesin comprises a 36 nm long filament made of repetitive immunoglobulin–helix modules that can stretch to 2.5-fold this length, demonstrating substantial molecular elasticity.
Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.
The contraction and relaxation cycles of active muscles generate substantial mechanical forces, both axially and radially, that place extraordinary stress on the molecular structures within the muscle fibers. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein that is thought to form bridges between the main contractile filaments of the muscle, providing the muscle structure with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, we determined the overall architecture of its complete repetitive domain array using a combination of four complementary structural biology methods. Our study reveals a long, dimeric tail-to-tail filament structure folded into an irregular superhelical coil arrangement of almost identical domain modules separated by short linkers. When we applied tension to these myomesin filaments, we found they could stretch to about 2.5 times their original length by unfolding these linkers, and then return to their original state when the tension was removed. Our findings explain how myomesin might adapt its overall length in response to the changing dimensions of the contracting and relaxing muscle, so acting as a highly elastic ribbon that maintains the overall structural organization of the muscle fibers. More generally, these findings demonstrate how repetitive domain modules, such as those in myomesin, can provide elasticity to highly organized biological structures.
The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH- terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.
The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2–Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9–Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.
To compare adult rat cardiomyocytes in primary culture for up to 28 days with those in primary culture for 10 days plus up to 18 days in a coculture (CC) system. The phenomenon of ‘second-floor’ cells in primary cultures and the behaviour of CC myocytes were studied over varying times with regard to protein content and attachment to underlying cells.
Qualitative confocal microscopy and quantitation using the Imaris program (Bitplane, Switzerland) for the measurement of the fluorescence intensity in the confocal microscope were used. The protein content of actin, myosin, desmin, tubulin, titin, myomesin, cadherin and connexin was determined. Cell death was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method for apoptosis and using propidium iodide applied to unfixed cultures to detect necrosis.
Compared with controls, second-floor cells contained only 14% of the actin and 4.9% of the tubulin at 10 days, whereas these proteins were well preserved in CC cells. All other proteins slowly declined in second-floor cells, whereas they were still present in normal amounts in CC cells. Cell death was evident in second-floor cells but absent in CC myocytes. Cellular attachment was still evident through original in vivo adherens junctions in second-floor cells but numerous newly developed cadherin- and connexin-containing junctions were visible in CC cells. It appears from the present study that second-floor cells are mummified dead cardiomyocytes, whereas CC myocytes survive and start to dedifferentiate.
The absence of actin and tubulin, together with nuclear changes, are indicators of loss of cell viability despite preservation of the cells’ rod shape and cross-striation, as observed in second-floor cells. In contrast, the establishment of a CC system of cardiomyocytes results in survival and organization of a three-dimensional cellular system, which may in the future be useful for tissue engineering attempts for replacement of lost tissue after myocardial infarction.
Cardiomyocytes; Cell death; Coculture; Protein content; Second-floor cells
A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A- bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross- bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15- nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.
The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.
Cultured cardiac myocytes were stained with antibodies to sarcomeric alpha-actinin, troponin-I, alpha-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, alpha-actinin is found as longitudinally aligned, irregular approximately 0.3-microns aggregates. Such aggregates are associated with alpha-actin, troponin- I, and titin. These I-Z-I-like complexes are also found as ectopic patches outside the domain of myofibrils in close apposition to the ventral surface of the cell. MHC is found outside of SMFs in the form of discrete fibrils. The temporal-spatial distribution and accumulation of the MHC-fibrils with respect to the I-Z-I-like complexes varies greatly along the length of the NSMFs. There are numerous instances of I-Z-I-like complexes without associated MHC-fibrils, and also cases of MHC-fibrils located many microns from I-Z-I-like complexes. The transition between the terminal approximately 1.7-microns sarcomere of any given SMF and its distal NSMF-tip is abrupt and is marked by a characteristic narrow alpha-actinin Z-band and vinculin positive adhesion plaque. A titin antibody T20, which localizes to an epitope at the Z-band in SMFs, precisely costains the 0.3-microns alpha-actinin aggregates in ectopic patches and NSMFs. Another titin antibody T1, which in SMFs localizes to an epitope at the A-I junction, typically does not stain ectopic patches and NSMFs. Where detectable, the T1- positive material is adjacent to rather than part of the 0.3-microns alpha-actinin aggregates. Myomesin and C-protein are found only in their characteristic sarcomeric locations (even in just perceptible SMFs). These A-band-associated proteins appear to be absent in ectopic patches and NSMFs.
Myofibrillogenesis in striated muscle cells requires a precise ordered pathway to assemble different proteins into a linear array of sarcomeres. The sarcomere relies on interdigitated thick and thin filaments to ensure muscle contraction, as well as properly folded and catalytically active myosin head. Achieving this organization requires a series of protein folding and assembly steps. The folding of the myosin head domain requires chaperone activity to attain its functional conformation. Folded or unfolded myosin can spontaneously assemble into short myosin filaments, but further assembly requires the short and incomplete myosin filaments to assemble into the developing thick filament. These longer filaments are then incorporated into the developing sarcomere of the muscle. Both myosin folding and assembly require factors to coordinate the formation of the thick filament in the sarcomere and these factors include chaperone molecules. Myosin folding and sarcomeric assembly requires association of classical chaperones as well as folding cofactors such as UNC-45. Recent research has suggested that UNC-45 is required beyond initial myosin head folding and may be directly or indirectly involved in different stages of myosin thick filament assembly, maintenance and degradation.
Myosin; chaperone; protein folding; UNC-45; motor domain; heat shock protein
Cardiac myosin-binding protein-C (MyBP-C), also known as C-protein, is one
of the major myosin-binding proteins localizing at A-bands. MyBP-C has three
isoforms encoded by three distinct genes: fast-skeletal, slow-skeletal, and
cardiac type. Herein, we are reporting a novel alternative spliced form of
cardiac MyBP-C, MyBP-C(+), which includes an extra 30 nucleotides, encoding 10
amino acids in the carboxyl-terminal connectin/titin binding region. This
alternative spliced form of MyBP-C(+) has a markedly decreased binding
affinity to myosin filaments and connectin/titin in vitro and does not
localize to A-bands in cardiac myocytes. When MyBP-C(+) was expressed in
chicken cardiac myocytes, sarcomere structure was markedly disorganized,
suggesting it has possible dominant negative effects on sarcomere
organization. Expression of MyBP-C(+) is hardly detected in ventricles through
cardiac development, but its expression gradually increases in atria and
becomes the dominant form after 6 mo of age. The present study demonstrates an
age-induced new isoform of cardiac MyBP-C harboring possible dominant negative
effects on sarcomere assembly.
We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail. Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background. The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes. We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA. We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.
In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact.
The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross- striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M- line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'- bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.
The Ca2+/Mg2+ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca2+ was bound to the N-terminal regulatory Ca2+ -binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca2+. NEM-S1 activation was assayed at pCa 8.0-6.5 and in the presence of either 1mM or 30 μM free Mg2+. ATPase activity was maximal when sites III and IV were occupied by Mg2+ and it steadily declined as Ca2+ displaced Mg2+. The data suggest that in the absence of Ca2+ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg2+ rather than Ca2+. This finding could be relevant to the contraction-relaxation kinetics of cardiac muscle. As Ca2+ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca2+ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.
troponin; thin filament; myosin crossbridges; magnesium; NEM-S1
Myosin binding protein C (MyBP-C) is a thick filament protein involved in the regulation of muscle contraction. Mutations in the gene for MyBP-C are the second most frequent cause of hypertrophic cardiomyopathy. MyBP-C binds to myosin with two binding sites, one at its C-terminus and another at its N-terminus. The N-terminal binding site, consisting of immunoglobulin domains C1 and C2 connected by a flexible linker, interacts with the S2 segment of myosin in a phosphorylation-regulated manner. It is assumed that the function of MyBP-C is to act as a tether that fixes the S1 heads in a resting position and that phosphorylation releases the S1 heads into an active state. Here, we report the structure and binding properties of domain C1. Using a combination of site-directed mutagenesis and NMR interaction experiments, we identified the binding site of domain C1 in the immediate vicinity of the S1–S2 hinge, very close to the light chains. In addition, we identified a zinc binding site on domain C1 in close proximity to the S2 binding site. Its zinc binding affinity (Kd of approximately 10–20 μM) might not be sufficient for a physiological effect. However, the familial hypertrophic cardiomyopathy-related mutation of one of the zinc ligands, glutamine 210 to histidine, will significantly increase the binding affinity, suggesting that this mutation may affect S2 binding. The close proximity of the C1 binding site to the hinge, the light chains and the S1 heads also provides an explanation for recent observations that (a) shorter fragments of MyBP-C unable to act as a tether still have an effect on the actomyosin ATPase and (b) as to why the myosin head positions in phosphorylated wild-type mice and MyBP-C knockout mice are so different: Domain C1 bound to the S1–S2 hinge is able to manipulate S1 head positions, thus influencing force generation without tether. The potentially extensive extra interactions of C1 are expected to keep it in place, while phosphorylation dislodges the C1–C2 linker and domain C2. As a result, the myosin heads would always be attached to a tether that has phosphorylation-dependent length regulation.
MyBP-C, myosin binding protein C; FHC, familial hypertrophic cardiomyopathy; WTWT, wild type; IgI, immunoglobulin I; C1C2, C1–linker–C2; NOE, nuclear Overhauser enhancement; MD, molecular dynamics; domain C1; NMR spectroscopy; model building; mutagenesis; protein structure
The muscle M-band protein myomesin comprises a 36-nm long filament made of repetitive immunoglobulin–helix modules that can stretch to 2.5-fold this length, demonstrating substantial molecular elasticity.
Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres—the structural units that produce contraction—and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.
We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.
Sarcomeres, bundled into thick and thin filaments, are the units of contraction in the striated muscle. The thick filaments comprise several hundred hexameric myosin molecules, composed of 2 myosin heavy chain (MyHC) proteins, the molecular motor of contraction, and 2 regulatory and 2 essential light chains. The globular head of MyHC contains the binding domains for cardiac α-actin and adenosine triphosphate (ATP) and is attached to a hinge region, which when flexed, moves the globular head over the thin filaments. The thin filaments comprise the cardiac troponin C (cTnC), T (cTnT), and I (cTnI) complex, α-tropomyosin dimers, and cardiac α-actin, maintained in a tight 1:1:7 stoichiometry. Several additional sarcomeric proteins, such as myosin-binding protein C, titin, obscurin, and telethonin contribute to the stabilization and function of the sarcomeres.
Editorials; heart failure; myosin isoforms; troponins; genetics
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.
The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.
Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation.
Sarcomere; Mechanical strain sensor; Mechanobiology; Titin; Connectin; Twitchin; Myosin light-chain kinase; Autophagy; Obscurin; Myomesin; Nbr1; p62/SQSTM1; MURF; Telethonin/TCAP
Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation– dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH2-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.
myosin assembly; myosin binding; myosin filament; human p32; smooth muscle