Organization of the Actomyosin Apparatus

Interdigitation of thick, bipolar, myosin filaments and thin actin filaments in the sarcomeres of living muscle cells is so precise (Fig. 39-3) that it yields an X-ray diffraction pattern (Fig. 39-13) revealing the spacing of the filaments and the helical repeats of their subunits to a resolution of about 3 nm. Z disks at both ends of the sarcomere anchor the barbed ends of the actin filaments, so their pointed ends are near the center of the sarcomere. Myosin heads project from the surface of thick filaments, whereas their tails are anchored in the filament backbone. Thick and thin filaments overlap, with the myosin heads only a few nanometers away from adjacent actin filaments. The alignment and interdigitation of the filaments facilitate the sliding interactions required to produce contraction.

Figure 39-3 electron micrographs and drawings of sarcomeres. A, Longitudinal thin section showing the array of thin filaments anchored to Z disks and overlapping bipolar thick filaments cross-linked in the middle at the M line. B, Longitudinal freeze-fractured, etched, and shadowed sarcomere showing myosin cross-bridges attached to thin filaments near the bare zone in the center (right) of a sarcomere. C–D, Cross-sections of insect flight muscle and vertebrate skeletal muscle showing the double hexagonal arrays of thick and thin filaments. E, Drawings indicating the polarity of the thick and thin filaments.

(A and C, Courtesy of H. E. Huxley, Brandeis University, Waltham, Massachusetts. B and D, Courtesy of J. Heuser, Washington University, St. Louis, Missouri.)

Figure 39-13 cross-bridge dynamics revealed by x-ray diffraction patterns of whole muscle. A, Electron micrograph showing the orientation of the muscle in the X-ray beam. B–C, Fiber diffraction patterns from relaxed and contracting skeletal muscles with interpretive drawings of cross-bridges in each state. Reflections from myosin heads arranged on the thick filament helix are strong in relaxed muscle. Reflections from the actin helix are stronger than the thick filament helix in contraction. The myosin and actin reflections are each labeled in only one of four equivalent quadrants. During contraction, a few myosin heads attach transiently to actin, increasing the strength of the actin helix reflections, but most are disordered.

(Micrograph and X-ray patterns courtesy of H. E. Huxley, Brandeis University, Waltham, Massachusetts.)

An important, simplifying architectural feature is that sarcomeres are symmetrical about their middles (Fig. 39-3). Consequently, the polarity of myosin relative to the actin filaments is the same in both halves of the sarcomere, allowing the same force-generating mechanism to work at both ends of the bipolar myosin filaments. Sarcomeres are organized end to end into long, cylindrical assemblies called myofibrils (Fig. 39-2) that retain their contractility even after isolation from muscle.

Thin Filaments

Thin filaments consist of actin and the tightly bound regulatory proteins troponin and tropomyosin (Fig. 39-4). When the concentration of Ca²⁺ is low in cytoplasm, troponin and tropomyosin inhibit the actin-activated ATPase of myosin. Tropomyosin, a 40-nm long coiled-coil of two α-helical polypeptides (see Fig. 3-10), binds laterally to seven contiguous actin subunits as well as head to tail to neighboring tropomyosins, forming a continuous strand along the whole thin filament. Troponin (TN) consists of three different subunits called TNC, TNI, and TNT (Table 39-1). TNT anchors troponin to tropomyosin. Like calmodulin (see Fig. 3-12 and Chapter 26), TNC is a dumbbell-shaped protein with four EF-hand motifs to bind divalent cations. In resting muscle the C-terminal globular domain of TNC binds two Mg²⁺ ions and an N-terminal α-helix of TNI, while the two low-affinity sites in the N-terminal globular domain of TNC are empty. Ca²⁺ binding to the low-affinity sites during muscle activation exposes a new binding site for TNI. The resulting conformational change in TNI allows tropomyosin to expose myosin-binding sites on the actin filament.

Figure 39-4 thin filament structure. A, Three-dimensional reconstruction from electron micrographs of a thin filament from vertebrate skeletal muscle showing actin and the position of tropomyosin in relaxed muscle. B, Drawing of a model of a thin filament from active muscle. Each tropomyosin is associated with seven actin subunits. The structure and binding sites of troponin and tropomodulin have been inferred from biochemical experiments. C, Ribbon diagrams of the atomic structures of troponin C, free and bound to a troponin I peptide. Two divalent cation-binding EF-hands are found at each end, separated by a long α-helix. In cells, two high-affinity sites at the C-terminal end are permanently occupied with Mg²⁺. Two low-affinity sites at the other end are unoccupied in relaxed muscle but bind Ca²⁺ when muscle is activated.

(A, Courtesy of W. Lehman, Boston University, Massachusetts. C, PDB files: 1AX2 and 1TROP.)

A protein meshwork in the Z disk anchors the barbed end of each thin filament (Fig. 39-5). Some cross-links between actin filaments consist of α-actinin, a short rod with actin-binding sites on each end (see Fig. 33-16). At least a half dozen structural proteins stabilize the Z disk through interactions with α-actinin, actin, and titin in the Z disk.

Figure 39-5 z disk structure. A–B, Electron micrographs of thin sections perpendicular to and in the plane of the Z disk. C, Three-dimensional reconstruction, based on electron micrographs of the Z disk, showing the network of protein cross-links that anchor the barbed ends of the yellow actin filaments.

(Courtesy of J. Deatherage, National Institutes of Health, Bethesda, Maryland; modified from Cheng NQ, Deatherage JF: Three dimensional reconstruction of the Z disk of sectioned bee flight muscle. J Cell Biol 108:1761–1774, 1989, by copyright permission of The Rockefeller University Press.)

Proteins cap both ends of thin filaments. Cap-Z, the muscle isoform of capping protein (see Fig. 33-14), binds the barbed ends of thin filaments with high affinity, limiting actin subunit addition or loss. Tropomodulin associates with both tropomyosin and actin to cap and stabilize the pointed end of thin filaments (Fig. 39-4B).

Tropomyosin and a gigantic filamentous protein, nebulin, stabilize thin filaments laterally. Nebulin consists of about 185 imperfect repeats of 35 amino acids that interact with each actin subunit, tropomyosin, and troponin along the length of thin filaments. Interactions with tropomodulin and Z disk proteins anchor nebulin at the two ends of the thin filament. Acting as a ruler and a cap, nebulin and tropomodulin help to set the length of thin filaments.

Thick Filaments

The self-assembly of myosin II (see Fig. 5-7) establishes the bipolar architecture of striated muscle thick filaments (Fig. 39-6). Some features of thick filaments are invariant, such as a backbone consisting of myosin tails, a surface array of myosin heads, the 14.3-nm stagger between rows of heads, and a central bare zone formed by antiparallel packing of tails. Filaments may vary in length, diameter, and organization of the helical array of heads in various species. Invertebrate thick filaments have a core of paramyosin, a second coiled-coil protein, which is not found in vertebrates.

Figure 39-6 structure of bipolar thick filaments. A, Electron micrograph of a thick filament isolated directly from skeletal muscle and prepared by negative staining. A myosin molecule is shown at the same magnification at the lower left. The myosin tails form the backbone of the thick filament and allow the two myosin heads to swing out from the side (see the enlarged inset on the right). B, Reconstruction from electron micrographs of part of a rabbit skeletal muscle thick filament. The surface bumps are myosin heads. C, Cross section of vertebrate skeletal muscle showing the double hexagonal arrays of thick and thin filaments. D, Electron micrograph of a highly stretched sarcomere with the M line in the middle. E, Drawing of protein links between thick filaments in the M line.

(A, Courtesy of John Trinick, University of Bristol, England. Reference: Knight P, Trinick J: Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle. J Mol Biol 177:461–482, 1984. B, Courtesy of M. Stewart, MRC Laboratory of Molecular Biology, Cambridge, England. C, Courtesy of J. Heuser, Washington University, St. Louis, Missouri. D, Courtesy of H. E. Huxley, Brandeis University, Waltham, Massachusetts.)

Several accessory proteins stabilize thick filaments (Table 39-1). Thick filaments in most striated muscles are girdled at intervals by semicircular bands of a protein that is, coincidentally, called C-protein. C-protein consists of fibronectin III and immunoglobulin domains. The “M line” in the center of the sarcomere is a three-dimensional array of protein cross-links that maintains the precise registration of thick filaments. At least three structural proteins and the enzyme MM-creatine phosphokinase (which transfers phosphate from creatine-phosphate to ADP) are located in the M line.

Titin Filaments

A third array of protein filaments lies parallel to the thin and thick filaments, connecting the Z disk to the thick filaments and the M line (Fig. 39-7). Hard to preserve for electron microscopy, these diaphanous filaments were neglected for years. Each filament is a single polypeptide named titin (after mythological giants), so named because of its remarkable size: more than 30,000 amino acids folded into a linear array of 300 immunoglobulin and fibronectin II domains measuring more than 1.2 μm long. Titin is thought to be the largest protein encoded by the human genome.

Figure 39-7 titin filaments. Upper panel, Electron micrographs of single, isolated titin molecules prepared by heavy metal shadowing. Titin molecules are long enough to extend from the Z disk to the M line. Middle panel, Drawing of a sarcomere, to the same scale as the electron micrograph, with the thick filaments removed from the bottom half to illustrate how titin molecules anchor thick filaments to the Z disk and extend to the M line. Lower panel, Drawing illustrating a model for the elasticity of titin. Modest stretches within the physiological range reversibly extend the chain of Ig domains in the I-band and the PEVK domain. Extreme extension can unfold immunoglobulin domains.

(Modified from Reif M, Gautel M, Oesterhelt F, et al: Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1090–1092, 1997. Reference: Leake MC, Wilson D, Gautel M, Simmons RM: The elasticity of single titin molecules using a two-bead optical tweezers assay. Biophys J 87:1112–1135, 2004.)

Titin molecules are elastic, and this accounts for the passive resistance to stretching of relaxed muscle. Their connections to the Z disk and thick filaments provide elastic continuity from one sarcomere to the next and keep the thick filaments centered in the sarcomere during contraction. If titin molecules are broken experimentally, thick filaments slide out of register toward one Z disk during contraction. Two features provide the elasticity during short (∼0.3 μm per titin), physiological stretches: The irregular chain of immunoglobulin domains in the I band straightens out, and a segment of the polypeptide rich in proline, glutamic acid, valine, and lysine (the PEVK domain) stretches. Stretching decreases entropy and provides the energy for elastic recoil. (See Fig. 29-12 for another example of an entropic spring in biology.) Extreme stretching unfolds Ig domains one by one.

Intermediate Filaments

Desmin intermediate filaments (see Chapter 35) help to align the sarcomeres laterally (Fig. 39-8) by linking each Z disk to its neighbors and to specialized attachment sites on the plasma membrane. Myofibrils near the cell surface are attached to the plasma membrane at specializations called costameres. In addition to desmin, costameres contain several cytoskeletal proteins (vinculin, talin, spectrin, and ankyrin) found in focal contacts and adherens junctions of nonmuscle cells (see Figs. 30-11 and 31-7). Desmin mutations in humans cause disorganization of myofibrils, resulting in generalized muscle failure.

Figure 39-8 desmin intermediate filaments in skeletal muscle. Desmin filaments connect Z disks laterally to each other and to the plasma membrane at specializations called costameres.

(Redrawn from Lazarides E: Intermediate filaments as mechanical integrators of cellular space. Nature 283:249–256, 1980.)

Structural Proteins of the Plasma Membrane: Defects in Muscular Dystrophies

In addition to providing a permeability barrier, the plasma membrane of the muscle cell must maintain its integrity while being subjected to years of forceful contractions. Accordingly, the membrane is stabilized by a transmembrane complex of proteins, the dystroglycan-sarcoglycan complex, that links the internal membrane skeleton to the basal lamina outside (Fig. 39-9 and Table 39-2). Occasional breaches of the membrane are inevitable, so muscle cells also depend on a repair process that reseals holes. If membrane damage exceeds the repair capacity, muscle cells degenerate locally (segmental necrosis) or globally. Cell death beyond the ability of muscle stem cells to repair the tissue results in muscular dystrophy. The age of onset and clinical features of inherited muscular dystrophies depend on the molecular defect. Patients with severe defects develop progressive muscle weakness as children. Ultimately, failure of respiratory muscles is fatal.

Figure 39-9 dystrophin and associated proteins stabilize the plasma membrane of skeletal muscle. A–B, Fluorescent antibody staining of cross sections of human skeletal muscle showing the localization of dystrophin at the plasma membrane of a normal individual (B) and its absence in an individual with Duchenne’s muscular dystrophy (A). C, Model of the transmembrane complex of proteins that links dystrophin and actin filaments in cytoplasm to laminin in the basal lamina outside the cell.

(A–B, Courtesy of L. Kunkel, Harvard Medical School, Boston, Massachusetts. C, Based on a drawing by K. Amann and J. Ervasti, University of Wisconsin, Madison.)

Mutations in more than 40 human genes have been linked to muscular dystrophies (Table 39-2), so it is possible that the malfunction or lack of any molecule in the system that maintains the integrity of the plasma membrane will cause disease. Disease-causing mutations occur in genes for the proteins of the dystroglycan-sarcoglycan complex, extracellular matrix proteins, Golgi apparatus enzymes that process these structural proteins, and the membrane repair machinery. Some of these mutations also affect the nervous system. Mutations are usually autosomal-recessive and are not uncommon. About one in several thousand humans develops some form of muscular dystrophy, because they inherit mutations in both copies of one of the sensitive genes. The mechanism of disease in muscular dystrophies is similar to that in hereditary spherocytosis, in which deficiencies of the membrane skeleton make red blood cells susceptible to mechanical damage (see Fig. 6-10).

The proteins that stabilize muscle membranes escaped detection until the late 1980s, when x-linked mutations in the dystrophin gene were discovered to cause Duchenne’s muscular dystrophy, the most common human form of the disease. Dystrophin is an enormous member of the α-actinin superfamily of actin-binding proteins (see Fig. 33-16). The dystroglycan-sarcoglycan complex was found when it copurified with dystrophin after solubilizing the membrane with detergents. Loss of any protein of the dystrophin-dystroglycan-sarcoglycan complex typically leads to secondary loss of the other proteins from muscles.

Studies of mutations in muscular dystrophy patients led to the identification of most of the other genes in this system. a2 laminin is the extracellular ligand for dystroglycan in the basal lamina. Glycosylation of the transmembrane complex by Golgi apparatus glycosyltransferases is required for their mechanical functions. Proteins in cytoplasmic vesicles are used to repair damaged plasma membranes. The mechanical activity of muscle cells might make them more sensitive than other cells to deficiencies in proteins that support the nuclear envelope (lamin A/C and emerin).

Dystroglycans and a dystrophin homolog, utrophin, participate in clustering acetylcholine receptors at the neuromuscular junction, the chemical synapse between motor neurons and skeletal muscle (see Fig. 11-8). When, during development, a motor neuron arrives at the surface of its target muscle cell, the neuron secretes an adhesive protein called agrin, which is incorporated into the basal lamina, immediately adjacent to the nerve terminal. Dystroglycan binds agrin and positions associated acetylcholine receptors at the site where they receive acetylcholine secreted by the nerve in response to an action potential.