In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

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

Heterogeneity of Z-band structure within a single muscle sarcomere: implications for sarcomere assembly

The vertebrate striated muscle Z-band connects actin filaments of opposite polarity from adjacent sarcomeres and allows tension to be transmitted along a myofibril during contraction. Z-bands in different muscles have a modular structure formed by layers of alpha-actinin molecules cross-linking actin filaments. Successive layers occur at 19 nm intervals and have 90 degrees rotations between them. 3D reconstruction from electron micrographs show a two-layer "simple" Z-band in fish body fast muscle, a three-layer Z-band in fish fin fast muscle, and a six-layer Z-band in mammalian slow muscle. Related to the number of these layers, longitudinal sections of the Z-band show a number of zigzag connections between the oppositely oriented actin filaments. The number of layers also determines the axial width of the Z-band, which is a useful indicator of fibre type; fast fibres have narrow (approximately 30-50 nm) Z-bands; slow and cardiac fibres have wide (approximately 100-140 nm) Z-bands. Here, we report the first observation of two different Z-band widths within a single sarcomere. By comparison with previous studies, the narrower Z-band comprises three layers. Since the increase in width of the wider Z-band is about 19 nm, we conclude that it comprises four layers. This finding is consistent with a Z-band assembly model involving molecular control mechanisms that can add additional layers of 19 nm periodicity. These multiple Z-band structures suggest that different isoforms of nebulin and titin with a variable number of Z-repeats could be present within a single sarcomere.     

Three-dimensional reconstruction of a simple Z-band in fish muscle

The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.     

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.     

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

The three-dimensional structure of the nemaline rod Z-band

In nemaline myopathy and some cardiac muscles, the Z-band becomes greatly enlarged and contains multiple layers of a zigzag structure similar to that seen in normal muscle. Because of the additional periodicity in the direction of the filament axis, these structures are particularly favorable for three-dimensional analysis since it becomes possible to average the data in all three dimensions and thus improve the reliability of the reconstruction. Individual views of the structure corresponding to tilted longitudinal and transverse sections were combined by matching the phases of common reflections. Examination of the tilted views strongly suggested that to the available resolution, the structure possesses fourfold screw symmetry along the actin filament axes. This symmetry could be used both in establishing the correct alignment for the combination of individual tilted views and to generate additional views not readily accessible in a single tilt series. The reconstruction shows actin filaments from one sarcomere surrounded by an array of four actin filaments with opposite polarity from the adjacent sacormere. The actin filaments show a right- handed twist and are connected by a structure that links adjacent filaments with the same polarity at the same axial level, then runs parallel to the filaments, and finally forms a link between two actin filaments whose polarity is opposite to that of the first pair. The connecting structure is probably composed of alpha-actinin which is located in Z-bands and cross-links actin filaments. The connecting structure may consist of two alpha-actinin molecules linking actin filaments of opposite polarity.     

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.     

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A- band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha- actinin and, if actin is added subsequently, the exogenous alpha- actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.     

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.     

Dynamics of Z-band based proteins in developing skeletal muscle cells

During myofibril formation, Z-bodies, small complexes of alpha-actinin and associated proteins, grow in size, fuse and align to produce Z-bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z-body and Z-band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green, or Cyan Fluorescent Protein linked to the Z-band proteins: actin, alpha-actinin, cypher, FATZ, myotilin, and telethonin. Each Z-band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z-bodies and Z-bands. Proteins that were present both early in development in Z-bodies and later in Z-bands had faster exchange rates in Z-bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z-band proteins that decrease their mobility and increase the stability of the Z-bands. A truncated construct of alpha-actinin, which localized in Z-bands in myotubes and exhibited a very low rate of exchange, led to disruption of myofibrils, suggesting the importance of dynamic, intact alpha-actinin molecules for the formation and maintenance of Z-bands. Our experiments reveal the Z-band to be a much more dynamic structure than its appearance in electron micrographs of cross-striated muscle cells might suggest.     

The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere

The sarcomere of striated muscle is an efficient molecular machine, characterized by perfect structural organization of contractile filaments. This order is ensured by the sarcomere cytoskeleton, an important element of which is the M-band, believed to maintain the thick filament lattice. We review here recent progress in understanding the M-band function and its structural organization. We explain how the M-band might reduce the intrinsic instability of thick filaments and help titin to maintain order in the sarcomeres. The M-band molecular structure has been clarified recently by biochemical and biophysical approaches that focused on the properties of the prominent M-band component myomesin. These have shown that antiparallel myomesin dimers might link the thick filaments in the M-band, a role analogous to that of alpha-actinin in the Z-disc. Furthermore, similar to titin, myomesin is a molecular spring with complex visco-elastic properties that can be modified by alternative splicing. M-band protein composition correlates with the expression of titin isoforms and appears to be a reliable marker for biomechanical conditions in contracting muscle. We propose that the M-band is in fact a dynamic structure that monitors the stress appearing in the thick filament lattice during contraction and quickly reorganizes to meet new physiological requirements.     

Amorphin is phosphorylase; phosphorylase is an alpha-actinin-binding protein

In a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565-573] reported the isolation of a new, 85-kD Z-band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti-amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z-bands of skeletal muscles, we reacted biotinylated alpha-actinin with purified amorphin and with purified phosphorylase and found that alpha-actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha-actinin, and, with lower affinity, to F-actin. Negative staining of actin filaments demonstrated that alpha-actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha-actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z-bands accompanied by a diffuse pattern in the cytoplasm.     

Myofibrillogenesis in skeletal muscle cells in the presence of taxol

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

MODELS OF MUSCLE Z-BAND FINE STRUCTURE BASED ON A LOOPING FILAMENT CONFIGURATION

The fine architecture of skeletal muscle Z bands is considered in view of stereo electron microscopical evidence and current biochemical and immunological concepts, and a new Z-band model is proposed. This model is based on a looping, interlinking configuration, within the Z band, of strands which emanate from I-band (actin) filaments of adjacent sarcomeres. Two versions of the model seem presently feasible: one in which the Z-band lattice is composed of actin loops; and another in which the same pattern is derived from tropomyosin. Either version satisfies actual electron micrograph images as well as or better than prior Z-band models. Moreover, the principle of looping linkage in filament-to-filament attachment can be related to similar filament patterns seen in several adhesion sites where intracellular filaments insert on cell membranes.     

Terminal assembly of sarcomeric filaments by intermolecular beta-sheet formation

The contraction-relaxation cycle of muscle cells translates into large movements of several filament systems in sarcomeres, requiring special molecular mechanisms to maintain their structural integrity. Recent structural and functional data from three filaments harboring extensive arrays of immunoglobulin-like domains - titin, filamin and myomesin--have, for the first time, unraveled a common function of their terminal domains: assembly and anchoring of the respective filaments. In each case, the protein-protein interactions are mediated by antiparallel dimerization modules via intermolecular beta-sheets. These observations on terminal filament assembly indicate an attractive model for several other filament proteins that require structural characterization.     

Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments.     

PEVK domain of titin: an entropic spring with actin-binding properties

The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility. Since in situ, titin is in close proximity to the thin filaments, we also studied whether the PEVK domain of cardiac or skeletal titin may interact with actin filaments. Interaction was indeed found in the in vitro motility assay, in which recombinant PEVK titin constructs slowed down the sliding velocity of actin filaments over myosin. Skeletal PEVK titin affected the actin sliding to a lesser degree than cardiac PEVK titin. The cardiac PEVK effect was partially suppressed by physiological Ca(2+) concentrations, whereas the skeletal PEVK effect was independent of [Ca(2+)]. Cosedimentation assays confirmed the Ca(2+)-modulated actin-binding propensity of cardiac PEVK titin, but did not detect interaction between actin and skeletal PEVK titin. In myofibrils, the relatively weak actin-PEVK interaction gives rise to a viscous force component opposing filament sliding. Thus, the PEVK domain contributes not only to the extensibility of the sarcomere, but also affects contractile properties.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.