The cytoskeleton in skeletal, cardiac and smooth muscle cells

The muscle cell cytoskeleton consists of proteins or structures whose primary function is to link, anchor or tether structural components inside the cell. Two important attributes of the cytoskeleton are strength of the various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. In striated muscle cells, extramyofibrillar and intramyofibrillar domains of the cytoskeleton have been identified. Evidence of the extramyofibrillar cytoskeleton is seen at the cytoplasmic face of the sarcolemma in striated muscle where vinculin- and dystrophin-rich costameres adjacent to sarcomeric Z lines anchor intermediate filaments that span from peripheral myofibrils to the sarcolemma. Intermediate filaments also link Z lines of adjacent myofibrils and may, in some muscles, link successive Z lines within a myofibril at the surface of the myofibril. The intramyofibrillar cytoskeletal domain includes elastic titin filaments from adjacent sarcomeres that are anchored in the Z line and continue through the M line at the center of the sarcomere; inelastic nebulin filaments also anchored in the Z line and co-extensible with thin filaments; the Z line, which also anchors thin filaments from adjacent sarcomeres; and the M line, which forms bridges between the centers of adjacent thick filaments. In smooth muscle, the cytoskeleton includes adherens junctions at the cytoplasmic face of the sarcolemma, which anchor beta-actin filaments and intermediate filaments of the cytoskeleton, and dense bodies in the cytoplasm, which also anchor actin filaments and intermediate filaments and which may be the interface between cytoskeletal and contractile elements.     

Binding of paratropomyosin to beta-connectin from chicken skeletal muscle

We found that paratropomyosin bound to beta-connectin, in examining binding of paratropomyosin at the junction of A- and I-bands of sarcomeres. The turbidity of a mixture of beta-connectin and paratropomyosin was greater with more paratropomyosin added, but high concentrations of Ca2+ suppress this increase. These results suggest that paratropomyosin is released from connectin filaments at the A-I junction region by increased concentrations of calcium ions in postmortem skeletal muscles.     

Myosin heavy chain phosphorylation sites regulate myosin localization during cytokinesis in live cells

Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.     

Broad A bands of striated muscle in Leber's congenital amaurosis: a new congenital myopathy?

A 2-year-old boy with Leber's congenital amaurosis, hypotonia, depressed myotatic reflexes, and delayed motor development had numerous foci of broadened or smeared A bands, loss of distinct I bands, and near-normal Z lines in biopsied muscle. The thick filaments in these lesions appeared misaligned, suggesting an abnormality of the M line or of the structural protein connectin. This unique alteration represents the first described morphologic abnormality of muscle in a patient with Leber's congenital amaurosis.     

The chicken muscle thick filament: temperature and the relaxed cross-bridge arrangement

Although chicken myosin S1 has recently been crystallized and its structure analysed, the relaxed periodic arrangement of myosin heads on the chicken thick filament has not been determined. We report here that the cross-bridge array of chicken filaments is temperature sensitive, and the myosin heads become disordered at temperatures near 4 degrees C. At 25 degrees C, however, thick filaments from chicken pectoralis muscle can be isolated with a well ordered, near-helical, arrangement of cross-bridges as seen in negatively stained preparations. This periodicity is confirmed by optical diffraction and computed transforms of images of the filaments. These show a strong series of layer lines near the orders of a 43 nm near-helical periodicity as expected from X-ray diffraction. Both analysis of phases on the first layer line, and computer filtered images of the filaments, are consistent with a three-stranded arrangement of the myosin heads on the filament.     

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Increase in dendritic cell numbers, their function and the proportion uninfected during AZT therapy

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, beta-filagenin, and then identified a gene that encodes a novel protein of 201-amino acid residues from databases using these sequences. beta-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four beta-strands but no alpha-helices. Western blotting using an affinity-purified antibody showed that beta-filagenin was associated with the cores. beta-Filagenin was localized by immunofluorescence microscopy to the A bands of body-wall muscles, but not the pharynx. beta-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, beta-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that beta-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.     

Automation system of X-ray filter in digital subtraction angiography

The giant muscle protein titin/connectin plays a crucial role in myofibrillogenesis as a molecular ruler for sarcomeric protein sorting. We describe here that the N-terminal titin immunoglobulin domains Z1 and Z2 interact specifically with telethonin in yeast two-hybrid analysis and protein binding assays. Immunofluorescence with antibodies against the N-terminal region of titin and telethonin detects both proteins at the Z-disc of human myotubes. Longer titin fragments, comprising a serine-proline-rich phosphorylation site and the next domain, do not interact. The interaction of telethonin with titin is therefore conformation-dependent, reflecting a possible phosphorylation regulation during myofibrillogenesis.     

Elastic bending and active tilting of myosin heads during muscle contraction

Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.     

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.     

Localization of CapZ during myofibrillogenesis in cultured chicken muscle

Actin filaments undergo dramatic changes in their organization during myofibrillogenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to "cap" each actin filament of the sacromere. Double immunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, alpha-actinin, titin, and myosin during myofibrillogenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric alpha-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric alpha-actinin in young myotubes was not coincident; in older myotubes, both CapZ and alpha-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

The in vivo state of assembly of myosin in vertebrate smooth muscle is controversial. In vitro studies on purified smooth muscle myosin show that it is monomeric (10S) under relaxing conditions and filamentous under contraction conditions. Electron microscopic and antibody labelling studies of intact smooth muscles, on the other hand, suggest that myosin is filamentous in the relaxed as well as the contracting state and that 10S myosin occurs only in trace amounts. However, birefringence, conventional EM and X-ray diffraction evidence suggests that in certain smooth muscles in vivo (e.g. rat anococcygeus), while myosin filaments exist in the relaxed state, their number increases on contraction. Here, we have used low temperature electron microscopic techniques (rapid freezing followed by freeze-substitution), which preserve labile components in close to their in vivo state, to detect any change in filament number on contraction. The results from rat anococcygeus have been compared with those from guinea pig taenia coli, in which other techniques have revealed no change in filament number. In the anococcygeus, we find evidence for a 23% increase in filament density in transverse sections of contracting muscle compared with relaxed muscle. In the taenia coli we find no change. These results are in qualitative agreement with earlier findings. They provide evidence for polymerization of myosin in contracting rat anococcygeus, and suggest that this process is subtle and occurs only in some smooth muscles.     

A dysfunctional desmin mutation in a patient with severe generalized myopathy

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies.     

Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III

A monoclonal antibody, CML-1, raised against carrot (Daucus carota L.) nuclear-matrix proteins selectively labeled the nuclear periphery of carrot protoplasts when visualized by confocal and electron microscopy. To identify the constituent proteins of higher plant cells structurally homologous to the vertebrate nuclear lamina, we cloned overlapping cDNAs partially encoding a CML-1-recognized protein and determined the entire sequence including the open reading frame. When the deduced amino acid sequence was compared with other known protein sequences contained in major databases, no protein was found to show high sequence identity across the whole region of the protein, while the partial sequence showed strong similarities with myosin, tropomyosin, and some intermediate filament proteins. The protein, designated NMCP1, had an estimated molecular mass of 133.6 kDa and showed three characteristic domains. The central domain contains long alpha-helices exhibiting heptad repeats of apolar residues, demonstrating structural similarity to that of filament-forming proteins. The terminal domains are predominantly nonhelical and contain potential sequence motifs for nuclear localization signals. NMCP1 has many recognition motifs for different types of protein kinases, including cdc2 kinase and PKC. These results suggest that NMCP1 protein forms coiled-coil filaments and is a constituent of the peripheral architecture of the higher plant cell nucleus.     

Disproportionate loss of thin filaments in human soleus muscle after 17-day bed rest

Previously we reported that, after 17-day bed rest unloading of 8 humans, soleus slow fibers atrophied and exhibited increased velocity of shortening without fast myosin expression. The present ultrastructural study examined fibers from the same muscle biopsies to determine whether decreased myofilament packing density accounted for the observed speeding. Quantitation was by computer-assisted morphometry of electron micrographs. Filament densities were normalized for sarcomere length, because density depends directly on length. Thick filament density was unchanged by bed rest. Thin filaments/microm2 decreased 16-23%. Glycogen filled the I band sites vacated by filaments. The percentage decrease in thin filaments (Y) correlated significantly (P < 0.05) with the percentage increase in velocity (X), (Y = 0.1X + 20%, R2 = 0.62). An interpretation is that fewer filaments increases thick to thin filament spacing and causes earlier cross-bridge detachment and faster cycling. Increased velocity helps maintain power (force x velocity) as atrophy lowers force. Atrophic muscles may be prone to sarcomere reloading damage because force/microm2 was near normal, and force per thin filament increased an estimated 30%.     

Is nebulin truly a component of the thin filament?

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.     

Excitation-contraction coupling--cardiac muscle events in the myofilament

The interaction of myosin and actin is by intracellular Ca2+ concentration, which in turn is controlled by the sarcoplasmic reticulum. In muscle--including cardiac muscle--of vertebrates, and some invertebrates, the site of Ca2+ control is in the thin, actin-containing filaments. These filaments contain tropomyosin and troponin; the latter is a complex of three subunits. When Ca2+ combines with troponin C, the Ca-binding subunit, a shift occurs in the position of tropomyosin that makes it possible for the myosin heads to bind to actin. This process is inhibited by a conformational change in troponin C, resulting in the release of the troponin complex from one of the binding sites on the thin filament. This process exhibits cooperative aspects which have been analyzed in terms of the Ca-binding process and the effect of Ca2+ on actomyosin ATPase activity.     

Structural and functional responses of mammalian thick filaments to alterations in myosin regulatory light chains

The ordered array of myosin heads, characteristic of relaxed striated muscle thick filaments, is reversibly disordered by phosphorylating myosin regulatory light chains, decreasing temperature and/or ionic strength, increasing pH, and depleting nucleotide. In the case of light chain phosphorylation, disorder, most likely due to a change in charge affecting the light chain amino-terminus, reflects increased myosin head mobility, thus increased accessibility to actin, and results in increased calcium sensitivity of tension development. Thus, interactions between the unphosphorylated regulatory light chain and the filament backbone may help maintain the overall order of the relaxed filament. To define this relationship, we have examined the structural and functional effects of such manipulations as exchanging wild-type smooth and skeletal myosin light chains into permeabilized rabbit psoas fibers and removing regulatory light chains (without exchange) from such fibers. We have also compared the structural and functional parameters of biopsied fibers from patients with severe familial hypertrophic cardiomyopathy due to a single amino acid substitution in the regulatory light chains to those exhibited by fibers from normal relatives. Our results support a role for regulatory light chains in reversible ordering of myosin heads and suggest that economy of energy utilization may provide for evolutionary preservation of this function in vertebrate striated muscle.