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.
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.
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
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.
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 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.
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.
Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30- h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament- associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.
We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform.
Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non-beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1-deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non-striated F-actin fibrils with α-actinin puncta dispersed irregularly along their lengths. αTM1’s binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin-containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1-deficient cardiomyocytes, α-actinin/F-actin adherens belts fail to assemble at apical cell-cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1-deficient cardiomyocytes exhibit failure of α-actinin/F-actin adherens belt assembly.
Absence of αTM1 resulting in unstable F-actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1-based pathway stabilizing F-actin at cell-cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis.
α-actinin; cell junction; cardiomyocyte; heart; myofibrillogenesis; myomesin; thin filament; titin; tropomodulin1
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 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.
Human myofibrillogenesis regulator 1, a novel 17-kDa protein, is closely involved in cardiac hypertrophy. We studied the molecular mechanism that links MR-1 to hypertrophic response. Hypertrophic hallmarks such as cell size and [3H]-leucine incorporation were significantly increased when MR-1 was transfected into cardiomyocytes for 48 h. However, sarcomere organization was promoted when MR-1 was transfected for 8 h. The finding that cardiac hypertrophy was induced long after increase of sarcomere organization indicates that the promoted sarcomere organization may be one of the crucial factors causing hypertrophy. Furthermore, when MR-1 was transfected into cardiomyocytes, the nuclear localization of myomesin-1 was shifted to the cytoplasm. Transfection with small ubiquitin-like modifier-1 (SUMO-1) mimicked the effect of MR-1 inducing translocation of myomesin-1. However, transfection with SUMO-1 in MR-1-silenced cardiomyocytes failed to induce translocation and sarcomere organization, even though SUMO-1 expression was at the same level. Overexpression of MR-1 may induce cardiomyocyte hypertrophy via myomesin-1-mediated sarcomere organization.
cardiomyocyte; hypertrophy; myofibrillogenesis regulator; sarcomere organization
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
Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.
Cardiac myocytes; Myofibrillogenesis; Myosin; Obscurin; Sarcomere; Sarcoplasmic reticulum; Z-disks
Monoclonal antibodies specific for the muscle protein titin have been used in conjunction with muscle-specific antibodies against myofibrillar myosin heavy chains (MHCs) and desmin to study myogenesis in cultured cells. Desmin synthesis is initiated in replicating presumptive myoblasts, whereas the synthesis of titin and MHC is initiated simultaneously in their progeny, the postmitotic, mononucleated myoblasts. Both titin and MHC are briefly localized to nonstriated and thereafter to definitively striated myofibrils. At no stage during myofibrillogenesis is either protein observed as part of a sequence of mini-sarcomeres. Titin antibodies bind to the A-I junction, MHC antibodies to the A bands in nascent, maturing, and mature myofibrils. In contrast, desmin remains distributed as longitudinal filaments until well after the definitive myofibrils have aligned laterally. This tight temporal and topographical linkage between titin and myosin is also observed in postmitotic, mononucleated myoblasts and multinucleated myotubes when myofibrillogenesis is perturbed with Colcemid or taxol. Colcemid induces elongating postmitotic mononucleated myoblasts and multinucleated myotubes to round up and form Colcemid myosacs. The myofibrils that emerge in these rounded cells are deployed in convoluted circles. The time required for their nonstriated myofibrils to transform into striated myofibrils is greatly protracted. Furthermore, as Colcemid induces immense desmin intermediate filament cables, the normal spatial relationships between emerging individual myofibrils is distorted. Despite these disturbances at all stages, the characteristic temporal and spatial relationship observed in normal myofibrils between titin and MHC is observed in myofibrils assembling in Colcemid-treated cells. Newly born postmitotic mononucleated myoblasts, or maturing myotubes, reared in taxol acquire a star-shaped configuration and are induced to assemble "pseudo- striated myofibrils." Pseudo-striated myofibrils consist of laterally aggregated 1.6-micron long, thick filaments that interdigitate, not with thin filaments, but with long microtubules. These atypical myofibrils lack Z bands. Despite the absence of thin filaments and Z bands, titin localizes with its characteristics sarcomeric periodicity in pseudo-striated myofibrils. We conclude that the initiation and subsequent regulation of titin and myosin synthesis, and their spatial deployment within developing sarcomeres are tightly coupled events. These findings are discussed in terms of a model that proposes interaction between two relatively autonomous "organizing centers" in the assembly of each sarcomere.
The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone 15N, 13C and 1H chemical shift and 13Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.
Muscle protein; Titin A-band; Large super-repeat unit; Fibronectin type III domain tandem
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
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.
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
Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.
Myosin-binding protein C (MyBP-C) is a ~130 kDa rod-shaped protein of the thick (myosin-containing) filaments of vertebrate striated muscle. It is composed of ten or eleven globular, 10-kDa domains from the immunoglobulin and fibronectin type III families, and an additional, MyBP-C-specific motif. The cardiac isoform, cMyBP-C, plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart disease. In addition to binding to myosin, MyBP-C can also bind to actin, via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and 3D reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25-180 mM KCl concentration. 3D helical reconstructions, carried out at the 180 mM KCl level to minimize non-specific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.
muscle structure; muscle regulation; cardiac contraction; cardiac regulation; cardiac muscle
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.
Despite early demonstrations of myosin binding protein C’s (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C’s ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C’s N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C’s C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for formation and/or maintenance of normal cMyBP-C sarcomeric distribution.
Myosin binding protein C; Contraction; Muscle; Sarcomere
Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.