Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of alpha-actinin.

The sarcomeric Z-disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by alpha-actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of alpha-actinin into the Z-disk. Titin interacts via a single binding site with the two central spectrin-like repeats of the outermost pair of alpha-actinin molecules. In the central Z-disk, titin can interact with multiple alpha-actinin molecules via their C-terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and alpha-actinin in vitro, and are expected to constrain the path of titin in the Z-disk. In thick skeletal muscle Z-disks, titin filaments cross over the Z-disk centre by approximately 30 nm, suggesting that their alpha-actinin-binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z-disk, where overlapping titin filaments and their interactions with the alpha-actinin rod and C-terminal domain can account for the essential ultrastructural features.     

A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.     

The interaction of titin and alpha-actinin is controlled by a phospholipid-regulated intramolecular pseudoligand mechanism

The assembly of stable cytoskeletal structures from dynamically recycled molecules requires developmental and spatial regulation of protein interactions. In muscle, titin acts as a molecular ruler organizing the actin cytoskeleton via interactions with many sarcomeric proteins, including the crosslinking protein alpha-actinin. An interaction between the C-terminal domain of alpha-actinin and titin Z-repeat motifs targets alpha-actinin to the Z-disk. Here we investigate the cellular regulation of this interaction. alpha-actinin is a rod shaped head-to-tail homodimer. In contrast to C-terminal fragments, full-length alpha-actinin does not bind Z-repeats. We identify a 30-residue Z-repeat homologous sequence between the actin-binding and rod regions of alpha-actinin that binds the C-terminal domain with nanomolar affinity. Thus, Z-repeat binding is prevented by this 'pseudoligand' interaction between the subunits of the alpha-actinin dimer. This autoinhibition is relieved upon binding of the Z-disk lipid phosphatidylinositol-bisphosphate to the actin-binding domain. We suggest that this novel mechanism is relevant to control the site-specific interactions of alpha-actinin during sarcomere assembly and turnover. The intramolecular contacts defined here also constrain a structural model for intrasterical regulation of all alpha-actinin isoforms.     

The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9

Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 muM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.     

Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain

Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.     

Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161).

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0 X 10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.7 X 10(7) M-1. The corresponding constants for native troponin C are 5.9 X 10(7) M-1. and 2.9 X 10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.     

Smooth muscle alpha-actinin interaction with smitin

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. In striated muscle sarcomeres, interactions between the myosin-binding protein titin and alpha-actinin in the Z-line provide an important structural linkage. We previously discovered a titin-like protein, smitin, associated with the contractile apparatus of smooth muscle cells. Purified native smooth muscle alpha-actinin binds with nanomolar affinity to smitin in smitin-myosin coassemblies in vitro. Smooth muscle alpha-actinin also interacts with striated muscle titin. In contrast to striated muscle alpha-actinin interaction with titin and smitin, which is significantly enhanced by PIP2, smooth muscle alpha-actinin interacts with smitin and titin equally well in the presence and absence of PIP2. Using expressed regions of smooth muscle alpha-actinin, we have demonstrated smitin-binding sites in the smooth muscle alpha-actinin R2-R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF-hand structures. These smitin-binding sites are highly homologous to the titin-binding sites of striated muscle alpha-actinin. Our results suggest that direct interaction between alpha-actinin and titin or titin-like proteins is a common feature of actin-myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for alpha-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.     

Structure of the alpha-actinin-vinculin head domain complex determined by cryo-electron microscopy

The vinculin binding site on alpha-actinin was determined by cryo-electron microscopy of 2D arrays formed on phospholipid monolayers doped with a nickel chelating lipid. Chicken smooth muscle alpha-actinin was cocrystallized with the beta1-integrin cytoplasmic domain and a vinculin fragment containing residues 1-258 (vinculin(D1)). Vinculin(D1) was located at a single site on alpha-actinin with 60-70% occupancy. In these arrays, alpha-actinin lacks molecular 2-fold symmetry and the two ends of the molecule, which contain the calmodulin-like and actin binding domains, are held in distinctly different environments. The vinculin(D1) difference density has a shape very suggestive of the atomic structure. The atomic model of the complex juxtaposes the alpha-actinin binding site on vinculin(D1) with the N-terminal lobe of the calmodulin-like domain on alpha-actinin. The results show that the interaction between two species with weak affinity can be visualized in a membrane-like environment.     

The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding.

The three-dimensional structure of alpha-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. alpha-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpretation of the 15 A-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of alpha-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca(2+)-dependent actin binding of non-muscle isoforms.     

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha- actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha- actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.     

Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.     

Targeting of nebulin fragments to the cardiac sarcomere

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.     

Distribution of tryptophan groups in molecules of troponin T, troponin I-troponin T complex, and alpha-actinin

The method of fluorescence quenching was used to experimentally determine the distribution of tryptophan residues in molecules of troponin T, troponin T-troponin I complexes, and alpha-actinin. Iodide and cesium ions, and acrylamide were used as quenchers. It was shown that cesium ions decrease the fluorescence intensity of troponin T and its complex with troponin I by the mode of dynamic quenching. For alpha-actinin such a dynamic quencher is anionic iodide. By using the modified Stern-Volmer equation, the quenching was found to be about 90% of total fluorescence intensity for troponin T, approximately 70% for the troponin T-troponin I complexes, and 20% for alpha-actinin. The penetration of cesium ions to tryptophan 206 (tryptophan 204) in the troponin T-troponin I complex is hindered, probably due to the participation of this tryptophan in the formation of bonds between troponin subunits.     

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein-protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229-233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.     

Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils

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.     

Mutations of hydrophobic residues in the N-terminal domain of troponin C affect calcium binding and exchange with the troponin C-troponin I96-148 complex and muscle force production

Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C(F29W) with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin C(F29W) complexed with the regulatory fragment of troponin I (troponin I(96-148)) and (b) the calcium sensitivity of force production. Troponin I(96-148) was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C(F29W)-troponin I complexes. The calcium affinities of the troponin C(F29W)-troponin I(96-148) complexes varied approximately 243-fold, whereas the calcium association and dissociation rates varied approximately 38- and approximately 33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C(F29W)-troponin I(96-148) complexes than from that of the isolated troponin C(F29W) mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C(F29W) for troponin I(96-148). However, the Phe(26) to Gln and Ile(62) to Gln mutations led to >10-fold lower affinity of calcium-saturated troponin C(F29W) for troponin I(96-148), causing a drastic reduction in force recovery, even though these troponin C(F29W) mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.     

The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF- hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.