MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES

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1 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES By JOHN M. SQUIRE,* HIND A. AL KHAYAT,* CARLO KNUPP,* AND PRADEEP K. LUTHER { *Biological Structure and Function Section, Biomedical Sciences Division, Imperial College London, London SW7 2AZ, United Kindom; { Biophysics Group, Department of Optometry and Vision Sciences, Redwood Building, Cardiff University, Cardiff CF10 3NB, Wales I. Introduction II. Hierarchy A. Components and Organization of the Sarcomere B. Actin Filaments C. Vertebrate A Band Lattices D. The Sliding Filament Model and the Crossbridge Cycle III. Actin Filament Structure and the Z Band A. The Actin Monomer B. F Actin C. The Thin Filament and Troponin D. Filament Organization in the Contractile Units of Different Muscle Types E. The Z Band F. Filament Organization in the Vertebrate I Band IV. Myosin Filament Structure and the M Band A. The X Ray Diffraction Approach to Myosin Filament Structure B. Myosin Head Organization in Relaxed Vertebrate Myosin Filaments C. Further A Band Analysis: C Protein, Titin, and the Vertebrate M Band D. Invertebrate Myosin Filaments E. Crossbridge Arrangements on Different Myosin Filaments: Variations on a Theme F. Conclusion: Implications about the Crossbridge Mechanism References I. Introduction The pioneering days of molecular biology in the 1950s and 1960s saw the determination of a variety of unknown molecular structures using an array of then novel techniques. The double helix of DNA was solved using high angle X ray fiber diffraction data (Watson and Crick, 1953; Wilkins et al., 1953). The technique of protein crystallography came of age when structures of key globular proteins were determined for the first time (Kendrew, 1963; Muirhead and Perutz, 1963). At the same time, electron microscopy and low angle X ray fiber diffraction helped to define the ADVANCES IN 17 Copyright 2005, Elsevier Inc. PROTEIN CHEMISTRY, Vol. 71 All rights reserved. DOI: /S (04) /05 $35.00

2 18 SQUIRE ET AL. Fig. 1. (A) Schematic illustration of the hierarchy of muscle. Skeletal muscle is composed of fibers about 20 to 100 mm in diameter and very long. Fibers in the light microscope appear cross striated and the muscles they come from are known as striated

3 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 19 structure of the muscle sarcomere in terms of separate actin and myosin filaments that slide past each other when a muscle shortens and that interact through cross connections (Huxley, 1957, 1969). Since then the muscle story has progressed in leaps and bounds so that in some ways it is now one of the best understood tissues. But this progress has been a story of using all of the techniques mentioned above in a correlated way; each method has illuminated the application of the other techniques and without any single one of them, our knowledge of muscle would be much the poorer. This article summarizes current knowledge about the major muscle components, the actin and myosin filaments. We also briefly discuss a third set of filaments, the titin filaments, which have remarkable properties and play a central role in integrating sarcomere structure (see Granzier and Labeit, 2005) We further describe how these filaments are organized in the muscle repeating unit, the sarcomere, through the cross linking M band and Z band structures. Finally, we discuss the filament arrangements in invertebrate muscles. This articles serves as an introduction to the detailed articles on titin (Granzier and Labeit, 2005), on muscle regulation (Brown and Cohen, 2005), and two articles on the contractile mechanism (Geeves and Holmes, 2005; Squire and Knupp, 2005). II. Hierarchy Anyone eating a steak or a slice carved from roast beef knows that meat is fibrous in texture. These fibers, 20 to 100 mm in diameter and very long, are the multinucleate muscle cells of which skeletal muscles are composed (Fig. 1A, B). Such fibers in the light microscope appear crossstriated and the muscles from which they are derived are known as striated muscles. The term striated also covers the muscles in animal hearts (Fig. 1C), but here the cells (the myocytes) are much shorter, they contain a single nucleus, and they are linked end to end by special structures known as muscles. Striations are from repeating units, the sarcomeres, with A band and I band regions. Each sarcomere extends between successive Z bands and is about 2.2 to 2.3 mm long in a resting muscle (Bloom and Fawcett, 1975). (B) Groups of muscle fibers (F), showing they are multinucleated (N ), and composed of the myofibrils (MF). Myofibrils may be about 2 to 5 mm in diameter. (C) Representation of the muscle cell arrangement in animal hearts, showing similar striations to those seen in skeletal muscles, but here the cells (the myocytes) are much shorter, they contain a single nucleus (N ), and they are linked end to end by special structures known as intercalated disks (D), which provide mechanical and electrical continuity between cells. (D) A typical smooth muscle that can be found surrounding the blood vessels and various hollow organs apart from the heart. These visceral muscles do not have cross striations. M, mitochondria, Z, Z band, N, Nucleus.

4 20 SQUIRE ET AL. intercalated disks, which provide mechanical and electrical continuity between cells. Other muscles in animals surround the blood vessels and various hollow organs apart from the heart. In vertebrates these visceral muscles are smooth muscles; they do not have cross striations (Fig. 1D). A closer look at striated muscle fibers shows that they themselves are assemblies of fine, hairlike structures known as myofibrils (Fig. 1A, B). Myofibrils may be about 2 to 5 mm in diameter, with cell organelles such as mitochondria and membranous systems called T tubules and the sarcoplasmic reticulum (SR) sandwiched between them (Fig. 2B). Vertebrate skeletal and cardiac muscles have a striated appearance because the myofibrils themselves are cross striated; they have a repeating unit along them known as the muscle sarcomere (Fig. 1A). In vertebrate muscles this repeat is approximately 2.2 to 2.3 mm long in a resting muscle but varies in length as the muscle stretches or shortens. The sarcomere, taken to extend between successive Z bands (Z discs) along the myofibril (Fig. 1A), is really the business part of the muscle where force is generated. Large muscles are assemblies of millions of sarcomeres all working together. Understanding the basic contractile mechanism in muscle therefore requires an understand how the sarcomere itself works. Sarcomere structure and function are the main topics of this article and those by Granzier and Labeit, Brown and Cohen, Geeves and Holmes, and Squire and Knupp in this volume. Before going into such detail, a description of some of the properties of muscle as a whole is warranted. Muscle innervation mechanisms and the kinds of contractile response that are produced are the topics of the rest of this section. Muscle contraction in the so called voluntary muscles, of which most skeletal muscles are examples, is initiated when a nerve action potential arrives at the nerve muscle (neuromuscular) junction (Fig. 2A). This in turn causes a depolarization of the muscle outer membrane, the sarcolemma, which causes the release of calcium ions into the interior of the muscle. However, muscle fibers are relatively large in diameter and it would take far too long for calcium to diffuse to the muscle interior to activate the centrally located myofibrils. For this reason, there are invaginations of the sarcolemma into the interior of the fiber. These invaginations are the T tubules (Fig. 2B). Thus, when the sarcolemma is depolarized, the depolarization is also propagated down the T tubules, which in turn interact with the terminal cisternae of the SR (Fig. 2B) to trigger the release of calcium ions locally into the adjacent myofibrils. The induced calcium release is mediated through the ryanodine receptor in the SR and subsequent sequestration of calcium when the muscle relaxes is accomplished by calcium pumps (Franzini Armstrong, 1999).

5 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 21 Fig. 2. (A) A set of fibers in a motor unit, activated by a single nerve axon also known as a motor nerve or motor neuron. (B) A group of muscle myofibrils showing the T tubular network, which is continuous with the fiber membrane (sarcolemma). When the sarcolemma is depolarized, this depolarization is also propagated down the T tubules, which in turn interact with the terminal cisternae (tc) of the sarcoplasmic reticulum to trigger the release of calcium ions locally into the adjacent myofibrils (Peachey, 1965). (C) Normalized tension records (twitch responses, see D) showing different contractile characteristics seen in different muscles and different fibers in the same muscle. (D) The tension response (pulse A) if a muscle is stimulated electrically by a very short pulse. In this twitch response the tension rises rapidly to a low value and then decays rapidly back to zero. If a second pulse arrives (pulse B) before the first twitch has finished, then there is a build up of tension so that the peak level in the second pulse is higher than the first. With repetitive pulses, the tension achieved gradually increases to a plateau level (D), which may be the maximum tension that the muscle can produce. This kind of maximal, sustained contraction is known as a tetanus and such a fused plateau response is known as a tetanic contraction. If the frequency is not quite high enough (C), the top tension level may still be bumpy and is known as an unfused tetanus. Only if the pulse train has sufficiently high frequency will successive peaks fuse to give a steady tension plateau (D).

6 22 SQUIRE ET AL. A nerve axon activating a particular muscle fiber is known as a motor nerve or motor neuron (Fig. 2A). This axon can in fact be just one branch of a large nerve that also interacts with many other fibers in the muscle. The set of fibers activated by a single nerve is known as a motor unit. Fibers in a motor unit need not be adjacent to each other, but may be distributed through a muscle. If a muscle is stimulated electrically by a very short pulse, then the response is known as a twitch (Fig. 2D). The tension rises rapidly to a certain value and then decays rapidly back to zero. However, if a second pulse arrives before the first twitch has finished, then there is a buildup of tension so that the peak level in the second pulse is higher than the first (wave B in Fig. 2D). With repetitive pulses the tension achieved can gradually increase to a plateau level, which may be the maximum tension that the muscle can produce. However, the top tension level may still be bumpy (trace C in Fig. 2D). Only if the pulse train has sufficiently high frequency will successive peaks fuse to give a steady tension plateau (wave D). This kind of maximal, sustained contraction is known as a tetanus. The bumpy version at a lower frequency (C) is an unfused tetanus. A fused plateau response is known as a tetanic contraction. Note that in muscle jargon a contraction can occur without the muscle changing length. This is then an isometric contraction. Muscles can also be studied while shortening under constant load an isotonic contraction. In everyday use the load and length of our muscles vary continuously, so the isometric and isotonic contractions often used by muscle researchers are useful steady state simplifications of what normally would be a constantly varying process. Finally, note in this quick overview of muscle properties that different muscles and different fibers in the same muscle may have different contractile characteristics (Fig. 2C). There are so called fiber types that can confer on individual muscles the particular responses that are needed for their differing functions (McComas, 1996). Some muscles are needed for posture, so they need to use energy slowly, they fatigue slowly, and they change length rather little. Other muscles are used for rapid spurts of activity and it may not matter that they tire quickly. The energy supply comes from the muscle mitochondria or from metabolism of glycogen granules. The speed of a muscle also depends on the particular isoforms of the main muscle proteins that they express. Different fibers may contain different isoforms of the same proteins (e.g., myosin or titin), which confer different contractile properties to their sarcomeres. In general, postural muscles are slower in their contractile response (e.g., with slow myosins), they have more mitochondria (they have a greater supply of the muscle oxygen carrier myoglobin which gives them a reddish colour), and they fatigue relatively slowly. Muscles required for rapid action have faster

7 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 23 isoforms of myosin and other sarcomeric proteins, fewer mitochondria, more glycogen, and they fatigue relatively rapidly. They are much less red in colour than slow fibers. Between these two extremes are wide gradations of fiber properties, and many muscles also contain a mixture of fiber types so that their responses are fine tuned to their functional role. A. Components and Organization of the Sarcomere 1. Introduction The muscle sarcomere contains the principal contractile proteins myosin and actin (Fig. 3A to C), which on their own can produce force and movement, together with a number of cytoskeletal and regulatory proteins. The latter include titin, C protein (MyBP C), tropomyosin, troponin, a actinin, myomesin, M protein, and so on. Some of these help to organize the myosin and actin filaments in the sarcomere, some to define the filament lengths and structure, some to regulate activity, and some to modulate the actin myosin interaction when the muscle is active. 2. Myosin Filaments Myosin filaments are composed of myosin molecules (Fig. 3B, C). It is now known that there are many varieties of myosin (currently about 17 types; c lmb.cam.a c.uk/m yosin/t rees/tree s.html), which are used in a diverse panoply of cellular roles (see Squire and Parry, 2005; and Geeves and Holmes, 2005). Muscle myosin is myosin type II. Myosin II has a long two chain, parallel, coiled coil a helical rod about 1500 Å long and about 20 Å in diameter with a globular region or head on the N terminal end of each chain (Fig. 3C). One chain of the rod part of myosin, together with the rest of the same chain that forms the bulk of one of the heads, is known as the heavy chain (Fig. 4A). Two light chains, the so called essential light chain (ELC) and the regulatory light chain (RLC), are associated with each myosin head. The globular domain is an ATPase; it catalyses the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate (P i ). This region of myosin is also the part that binds to actin and this binding enhances the ATPase activity of the head; the ATPase of myosin is said to be activated by actin. Studies of the myosin head, the globular part of the heavy chain together with the ELC and RLC, were dramatically transformed when the isolated myosin head from chicken skeletal muscle myosin was crystallized and its structure solved using protein crystallography by Rayment et al. (1993b; Fig. 4A). This showed that the head consists of a globular

8 24 SQUIRE ET AL. Fig. 3. (A) Actin filament composed of actin molecules (A), two tropomyosin stands (TM ), and troponin molecule complexes (TN ). (B) Myosin filament composed of myosin molecules shown in (C) with the rod of the myosin molecules forming the backbone of the filament and the myosin heads are arranged on the surface of the filament backbone. (D) The bipolar packing of the myosin molecules showing the antiparallel arrangement giving rise to a heads free bare zone region at the centre of the filament. This is also illustrated in (E). (F) Sarcomere structure extending between two successive Z bands (M, myosin, A, actin). (G J) Cross sectional views through different parts of the sarcomere, showing (G) the square lattice of actin filaments in the I band; (H) the hexagonal lattice between overlapping arrays of actin and myosin filaments in the A band; (I, J) the hexagonal lattice of myosin filaments in the M band (I) and barezone ( J ) regions, with the extra M protein density linking the myosin filaments at the M region in the center of the sarcomere (I).

9 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 25 region, all part of the myosin heavy chain, containing the ATP binding site and the actin binding face, from which extends the next part of the heavy chain in the form of a very long a helical region, linking to the junction between the head and the myosin rod. Wrapped around this long a helix are the ELC and RLC, which serve both to stiffen the otherwise unsupported main chain a helix and in some muscles to regulate contractile activity. Because of its ATPase and actin binding properties, the globular part of the myosin head is sometimes called the catalytic domain, sometimes the motor domain. The thinner light chain binding region that joins the motor domain to the myosin rod is sometimes referred to as the neck region, or, for reasons that will become apparent later, the lever arm. In striated muscles, myosin molecules aggregate to form bipolar myosin filaments (see Fig. 3D). In the middle of each myosin filament the myosin rods pack in an anti parallel fashion, whereas outside this central region the rods pack parallel to each other. This gives the bipolar myosin filaments a head free region in the middle (the bare zone) on each side of which the heads are arrayed with opposite polarity on the filament surfaces in the bridge regions (see Fig. 3D, E). In different kinds of striated muscles, especially between vertebrate muscles and the striated muscles found in invertebrates, the size and symmetry of the bipolar myosin filaments can vary. However, in all vertebrate striated muscles so far studied, the myosin filaments all appear to be very similar; they are about 1.6 mm long, the bare zone length is just slightly longer (1600 Å) than the length of the myosin rod, and the myosin filament backbone is 140 Å in diameter with the heads projecting out from this. It was shown in the early studies of H. E. Huxley by electron microscopy and low angle X ray diffraction that there is a periodicity along the vertebrate myosin filaments that seemed to be associated with a helical array of myosin projections, now known to be the myosin heads (Huxley, 1963; Huxley and Brown, 1967). The repeat of the helical array was found to be 429 Å, with a sub periodicity of 143 Å. This was deduced from the spacings associated with layer lines in the X ray diffraction pattern from frog muscle (for details of muscle diffraction, see Squire and Knupp, 2005). The layer lines (ML1, ML2, ML3, etc.) appear as a set of parallel lines of intensity running at right angles to the muscle long axis in recorded diffraction patterns (Fig. 4B). The myosin heads were thought to give rise to layer lines with spacings that were orders of a repeat of 429 Å, with the third order (ML3) at 143 Å having particularly strong intensity on the central vertical axis of the diffraction pattern known as the meridian. This meridional reflection on the third layer line has been labeled the M3 reflection and is the subject of further discussion in Geeves and Holmes (2005) and Squire and Knupp (2005).

10 26 SQUIRE ET AL. Fig. 4. (A) Structure of the myosin head, S1, determined by X ray crystallography for chicken skeletal muscle without nucleotide bound. It consists of a heavy chain forming a globular part where the actin binding site is located, and also a long a helical chain

11 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 27 Hux ley and Brow n (19 67) o riginally sugg ested that th e surface array of heads wa s desc ribed by two coaxia l heli ces, each of pi tch Å, with a subunit axial translatio n along the heli x of 143 Å and six head pairs in one pitch. Thinking of th e heli x as a spira l stairc ase, th e subu nit axial translation is the verti cal rise on each step and the pitch is the height up the staircases where one is immed iately over the low est step. For further definitions of these terms and an introducti on to heli cal diffracti on th eory, see the documen tation for the HELIX or M uslabel pro grams at 3. ac.uk ( Knup p & Squir e, 2004; Squ ire & Knup p, 2004 ). For more detai ls of helical diffracti on th eory, see Squ ire (20 00) or Chandr asekaran and Stubbs (2001). Aft er this enormou s st ep forward in myosin filam ent analysi s, it was soon realized by Squ ire (1971, 1972), by compariso n of myosin head arrays from differ ent m uscle ty pes, that this postulate d symmet ry was likely to be wrong. In due course the evide nce pointed to a three start (or three stranded) heli x of head pairs ( Squir e, 197 2; see Fig. 3B, E ), each helix having a pitch of Å, the same subu nit axial tran slation of 143 Å as Huxley and Brow n had suggested, but wit h nine head pairs in one pitch. Because th e number of strands in the heli x and the num ber of head pairs in one heli x repeat are both divisib le by 3, the axial rep eat of the new structure is still 429 Å as required by the X ray diffr action data. So, vertebrate muscle myosi n filam ents are three stra nded and electro n micrographs show that they have threefold rotation al symmet ry in any cro sssection ( Luther et al., 1981 ). The term crown is used to desc ribe the ring of myosin head s within each 143 Å subu nit axial trans lation. We show later that myosi n filam ents in inverteb rate muscles have differ ent rotati onal symmetri es from th reefold and in so me cases differ ent axial rep eats, although all show the common crown subunit axial translation of 143 to 145 Å, which appears to be a signature of molecular packing between myosin II molecules. In the myosin containing part of the sarcomere, the A band, the myosin filaments are cross linked at the M band by various additional proteins ( Fig. 3F, I)). Details of th ese are given later in Sec tion II. C. Along the myosin filaments there is also part of the titin molecule (Fig. 5B, D). Titin is the largest known protein with a molecular weight of 3 MDa. It is anchored with its C terminus at the M band and its N terminus at the Z band. Titin shown in red surrounded by two light chains, the essential light chain (ELC) and regulatory light chain (RLC). (B) X ray diffraction pattern recorded at Spring-8 by Dr. J. J. Harford from relaxed fish muscle showing the myosin layer lines (ML) at orders of 429 Å repeat. The third order myosin layer line has a reflection on the meridian referred to as M3 at a spacing of 143 Å. Layer lines are sampled by the hexagonal lattice arrangement of actin and myosin filaments giving rise to (vertical) row lines (arrows).

12 28 SQUIRE ET AL. Fig. 5. (A) Electron micrograph picture showing a whole sarcomere from fish muscle in relaxing conditions (Z to Z distance about 2.3 mm). (B) Schematic diagram showing the sarcomere with titin molecules (green and blue) with the N terminus of each titin molecule located at the Z band and the C terminus at the M band. (C) Arrangement of the C protein within the A band showing seven stripes on each

13 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 29 stretches across half a sarcomere or 1.1 to 1.2 mm at the muscle rest length. In the A band it is closely associated with the myosin filaments. It then extends from the A band, through the I band part of the sarcomere, to the Z band (Fig. 5D). In the I band region titin displays remarkable elastic properties; it confers on the sarcomere much needed structural integrity and helps to maintain the central location of the A band in the sarcomere. Particular regions of the I band titin of interest are the PEVK region and the N2 region (Fig. 5D; see Granzier and Labeit, 2005). At the ends of the myosin filaments, at the A band I band junction, titin forms the so called end filaments (Fig. 5D). Also in the A band, in the middle of each of the bridge regions, the myosin filaments carry the extra protein originally known as C protein (now often called myosin binding protein C: MyBP C; Offer et al., 1973), which occurs in two sets of 7 to 9 stripes in the C zones in each half of the A band (Fig. 5C; Bennett et al., 1986; Sjostrom and Squire, 1977). The structure, interactions, and role of C protein are discussed in Section IV.C. B. Actin Filaments Actin filaments were first visualized by Hanson and Lowy (1963) in one of the pioneering applications of the negative staining technique for electron microscopy. The filaments appeared as two strings of beads that gradually twisted around each other, the spacing between crossovers being 370 Å (Fig. 3A). Each bead was in fact one actin monomer of molecular weight 42 kda. Since then actin filament structure has been studied in some detail as described later. At an early stage it was shown that the actin filaments in striated muscles carry the additional proteins tropomyosin and troponin, which are involved in regulating sarcomere activity (also shown in Fig. 3A). The tropomyosin molecule is rather like a short version of the rod part of the myosin molecule; it is a two chain, parallel, coiled coil a helical rod 400 Å long. It was also found that there is one tropomyosin molecule to one troponin complex and seven actin monomers (Ebashi et al., 1969). Since the axial repeat of the actin monomers along the filament was known from X ray diffraction to be 55 Å, it was suggested that the tropomyosin rods might lie along each of the twisting strings of actin beads. One troponin complex would then be associated with each tropomyosin molecule, giving an axial repeat of 7 55 Å or 385 Å half of the myosin filament. (D) Magnified version of the area between the Z band and I band showing the PEVK and N2 domains of titin and the end filament at the tip of the myosin filament.

14 30 SQUIRE ET AL. for the whole tropomyosin/troponin assembly. Further details of this structure are given in Section III and in Brown and Cohen (2005). C. Vertebrate A Band Lattices In a cross section through the A bands of vertebrate striated muscles the myosin filaments lie on a hexagonal lattice, and the actin filaments are at the so called trigonal points, midway between three mutually adjacent myosin filaments (see Fig. 3H J). However, an early suggestion by Huxley and Brown (1967) was that there is actually a more complicated arrangement than this, a so called superlattice. This means that adjacent myosin filaments do not all have the same orientations. Since the symmetry that they perceived for the myosin filaments was two stranded, the superlattice they suggested was not quite right. A careful study of muscle cross sections in electron micrographs of frog sartorius muscle by Luther and Squire (1980) showed that in fact the threefold symmetric myosin filaments occur in one of two possible orientations 60 apart (Fig. 6B), forming a statistical superlattice. It was found to be impossible to generate the superlattice in a regular way, but, by applying two packing rules, Luther and Squire found that a statistical superlattice of the required size was automatically produced, even though it contained a large amount of disorder. About the same time it was also found that some vertebrate striated muscles, particularly those of bony fish, do not have a superlattice at all; Fig. 6. (A) The simple lattice myosin filament arrangement that occurs in all the teleost (bony fish) muscles so far studied, in some muscles of cartilaginous fish and also in some primitive fish such as sturgeons and bowfin. (B) The superlattice arrangement present in the muscles of all the higher vertebrates, namely mammals (including humans), amphibians, birds, reptiles, and some muscles of cartilaginous fish.

15 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 31 in these muscles all the myosin filaments have exactly the same rotations, forming what is known as the simple lattice (Fig. 6A). Because this simple lattice is so regular, it makes structural studies of bony fish muscle relatively advantageous; it is much easier to analyze structural data in a rigorous way (e.g., Harford and Squire, 1986; Hudson et al., 1997; Luther and Crowther, 1984). Luther et al. (1996) conducted a systematic study of the occurrence of the simple lattice and superlattice across the vertebrate kingdom. Superlattices are present in the muscles of all the higher vertebrates, namely, in mammals (including humans), in amphibians, in birds, in reptiles, and in some muscles of cartilaginous fish. Simple lattices occur in all the teleost (bony fish) muscles so far studied, in some muscles of cartilaginous fish, and also in some primitive fish such as sturgeons and bowfin. D. The Sliding Filament Model and the Crossbridge Cycle At the time of writing it is exactly 50 years since the original major breakthrough in understanding sarcomere function, namely, the postulation of the sliding filament model whereby during muscle shortening myosin and actin filaments slide past each other without much change in length (Huxley and Hanson, 1954; Huxley and Niedergerke, 1954). Previously it had been assumed that actomyosin was some form of continuous filamentous structure extending throughout the sarcomere; a structure that would stretch or shorten in a similar way to the stretching of hair (keratin) under steam and the transition within the keratin chains from a short a helical structure to a longer b sheet structure. The idea of sliding filaments was revolutionary. The sliding filament model moved the central dilemma in muscle from how does actomyosin shorten? to what makes the actin and myosin filaments slide past each other? At an early stage it was realized that the myosin projections must be heavily involved in this process. Several studies then illuminated what must be happening. Reedy et al. (1965) showed that insect flight muscle (from Lethocerus) fixed for electron microscopy in two different states showed crossbridges between the myosin and actin filaments in the overlap part of the A band, but the angle of the crossbridges was different in the two states. Muscles fixed in the relaxed state showed crossbridges more or less at 90 to the myosin and actin filament long axes, whereas muscles fixed in the rigor state induced by the removal of ATP showed crossbridges at an angle closer to 45. It was known that in the absence of ATP the myosin heads became firmly attached to actin. Only on the addition of ATP to such a system would the heads come off actin. ATP

16 32 SQUIRE ET AL. Fig. 7. (A) The crossbridge cycle showing the way the myosin head attaches and detaches from actin filaments. In the rigor state (top right) heads are rigidly attached to actin in a specific conformation at a 45 angle forming the AM rigor complex. When ATP is added, the myosin head is released from actin (1) and hydrolysis of ATP into its products, ADP and P i, occurs, with both products still attached to the head (2). The hydrolysis of ATP is assumed to be accompanied by a conformational change of the heads from 45 to 90. It is at the M.ADP.P i state that can rebind to actin (step 3) with the heads still at a 90 angle and forming AM.ADP.P i. The transition from AM.ADP.P i to AM.ADP to AM, possibly with some isomerization steps within each state, is associated with force production and movement. The swinging of the elongated attached head

17 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 33 loss gives rise to the rigid state that occurs on death when the ATP supply stops, the state known as rigor mortis. The nucleotide free state of myosin heads on actin in the crossbridge cycle is therefore known as the rigor state. This and other evidence was put into a biochemical version of the crossbridge cycle by Lymn and Taylor (1971; shown in Fig. 7A), with a structural interpretation summarized by Huxley (1969) involving the 90 to 45 tilt of the actin attached myosin heads as seen by Reedy et al. (1965). Here the elongated myosin head (M; at that stage the detailed head shape in Fig. 4A was not known) is shown bound to actin (A) in the rigor, nucleotide free, state (AM). Subsequent binding of ATP brings the attached head rapidly off actin (the M.ATP state). The ATP on the detached head then hydrolyses to ADP and P i (M.ADP.P i ), but the products ADP and P i remain on the myosin. If the muscle is not switched off, a myosin head as M.ADP.P i can reattach to actin to give the AM.ADP.P i state. In Fig. 7A the head is shown attaching to actin at a 90 angle. It is the transition from AM.ADP.P i to AM. ADP to AM, possibly with some isomerization steps within each state as well, that is associated with force production and movement. Clearly a swinging of the elongated attached head from 90 to 45 causes relative sliding of the myosin and actin filaments. An early test of the sliding filament model was the very careful measurement by Gordon et al. (1966) of the active tension produced by the muscle at different sarcomere lengths (Fig. 7B D). If the myosin heads or crossbridges act as independent force generators, then, as the sarcomere length from 90 to 45 will cause relative sliding of the myosin and actin filaments. (C) The active tension produced by the muscle at different sarcomere lengths (B, C, D from Gordon et al., 1966) is shown in (B, D). If the myosin heads or crossbridges act as independent force generators, then, as the sarcomere length (S) is increased and the overlap of the actin and myosin filaments reduces, the tension produced by the muscle should gradually reduce in proportion to the overlap. A linear reduction in tension was observed as the sarcomere length changed from about 2.2 mm to about 3.6 mm labeled as (1). Since the actin filaments are about 1 mm long and separated by an estimated Z band thickness of 0.05 mm, and since the myosin filament length is about 1.6 mm, it would be expected that there would be zero overlap and hence zero tension when S 3.65 mm(¼ 1.6 mm þ 1.0 mm þ 1.0 mm þ 0.05 mm). As the sarcomere length is reduced the overlap will gradually increase until the two bridge regions of the myosin filaments are fully overlapped by actin. This will occur at a sarcomere length of about 2.25 mm (2 1.0 mm for the actin filaments plus 0.05 mm for the Z band, plus the size of the bare zone of be about 0.2 mm, labeled as (2). Reduction of S below this value would not increase the overlap any further so there will be an active tension plateau as observed between 2 and 3. After this there are complications to the simple analysis; first the actin filaments meet the M band, then there is overlap of anti parallel actin filaments, then the actin filaments start overlapping myosin bridge regions with the wrong polarity in the other half of the A band, and finally the myosin filaments bump up against the Z bands, so the observed tension gradually reduces below S ¼ 2.0 mm.

18 34 SQUIRE ET AL. (S) is increased and the overlap of the actin and myosin filaments reduces, the tension produced by the muscle should gradually reduce in proportion to the overlap. The tension in a muscle that is not stimulated, known as the resting tension, also changes with sarcomere length, so in the analysis by Gordon et al. the resting tension was subtracted from the total tension for a given sarcomere length to give the active tension. Their result is shown in Fig. 7C. They observed a beautifully linear reduction in tension as the sarcomere length changed from 2.2 mm to 3.6 mm. Since the actin filaments are 1 mm long and separated by an estimated Z band thickness of 0.05 mm, and since the myosin filament length is 1.6 mm, it would be expected that there would be zero overlap and hence zero tension when S 3.65 mm(¼ 1.6 mm þ 1.0 mm þ 1.0 mm þ 0.05 mm). As the sarcomere length is reduced, the overlap gradually increases until the two bridge regions of the myosin filaments are fully overlapped by actin. This occurs at a sarcomere length of 2.25 mm(2 1.0 mm for the actin filaments plus 0.05 mm for the Z band, plus the size of the bare zone taken by Gordon et al. to be 0.2 mm). Reduction of S below this value would not increase the actin overlap with myosin heads any further, so there will be an active tension plateau as observed (Fig. 7C). After this point, there are complications to the simple analysis. First, the actin filaments meet the M band; then there is overlap of anti parallel actin filaments; the actin filaments then start overlapping myosin bridge regions with the wrong polarity in the other half of the A band; and finally the myosin filaments bump up against the Z bands, so the observed tension gradually reduces below S ¼ 2.0 mm. (Note that these exact sarcomere length positions depend on the precise actin filament length, which can vary slightly between different vertebrate muscle types, and on the thickness of the Z bands and bare zones, which are discussed in later sections. The A band length always appears to be the same in vertebrate striated muscles.) In a later study, Huxley and Simmons (1971a,b) used tension records from single muscle fibers undergoing very rapid step changes in length to elucidate details of the crossbridge mechanism. The crossbridge properties that they observed also scaled exactly with sarcomere length. There could be little doubt that the myosin heads were acting as independent force generators. III. Actin Filament Structure and the Z Band A. The Actin Monomer As in the case of the myosin head, knowledge of actin filament structure, or thin filament structure as it is termed when tropomyosin and troponin are present, also progressed rapidly when the structure of the globular actin (G actin) monomer was determined by protein crystallography in

19 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 35 the work of Kabsch et al. (1990), who studied co crystals of actin bound to DNase 1. Since 1990 a number of other G actin crystal structures have been published (Chik et al., 1996; Graceffa and Dominguez, 2003; Otterbein et al., 2001), but the structures are essentially the same. The actin monomer (Fig. 8) is a four domain structure with two large domains known as 1 and 3 and two smaller domains 2 and 4. The N and Fig. 8. Illustration of the actin monomer structure solved by X ray crystallography (Kabsch et al., 1990) showing the four structural subdomains of the actin monomer, labeled, in the front (A) and back (B) view with N, C termini labeled 1 4. Also labeled is the loop linking residues 262 and 309 between subdomains 3 and 4.

20 36 SQUIRE ET AL. C termini of the protein chain are in subdomain 1. The chain then goes up into subdomain 2, back down to subdomain 1, across to subdomain 3, up to subdomain 4, back down through subdomain 3, and across to subdomain 1 again. In some ways the two halves of the monomer, subdomains 1 and 2 in one half and subdomains 3 and 4 in the other half, are rather similar in structure. The largest subdomains (1 and 3) contain a central five stranded b sheet structure with short a helices on each face. The smaller subdomains (2 and 4) are primarily b sheet with a small amount of a helix in subdomain 2 (the smallest subdomain) and slightly more a helix in 4. Centrally placed between the two halves of the monomer there is an ATP binding site. Actin is itself an ATPase, and the state of hydrolysis of ATP is an important factor in determining actin filament growth in cell motility and other general cell functions (see review in Sheterline et al., 1998). B. F Actin G actin monomers in muscle aggregate to form filamentous actin (or F actin) filaments as shown in Fig. 9B. This shows in molecular detail the twisting strands of globular beads seen originally by Hanson and Lowy (1963; cf. Fig. 3A). Holmes et al. (1990) modeled the G actin structure into filaments with the correct helical symmetry for F actin and tested such models against high angle fiber diffraction data from oriented gels of F actin to 8 Å resolution. The actin monomer is probably oriented with subdomains 3 and 4 on the inside of the filament where they would interact with subdomains 3 and 4 from other monomers. Subdomains 1 and 2 were placed toward the outside of the filament. It was later shown that the primary binding site for the myosin head is, in fact, on subdomain 1, so the exterior location of this subdomain makes good sense. This model for F actin, which must still be considered as a model since it has not been demonstrated at high resolution, has been tested in several studies (e.g., Al Khayat et al., 1995; Lorenz et al., 1993; Oda et al., 1998), and it is generally agreed that the Holmes et al. model is more or less correct. Subsequent studies have refined the structure slightly, but its essential features remain (Holmes et al., 2003). The structure of F actin can be described in terms of a helix of actin monomers with approximately 13 monomers in 6 left handed turns of the genetic helix (a 13/6 helix). The helix pitch is 59.6 Å, and the subunit axial translation is 27.5 Å. The fact that the pitch is only slightly different from twice the subunit axial translation gives rise to the very slow twist of the filament as observed by Hanson and Lowy (1963). The twist of this so called long period helix is right handed. The crossover repeat for a 13/6 actin helix is Å ¼ Å (the pitch of each long period

21 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 37 Fig. 9. (A) Actin monomer with each of its four structural subdomains shown in different colors: subdomain 1(red); subdomain 2 (green); subdomain 3 (blue); and subdomain 4 (yellow). (B) The helical arrangement of the actin monomers along the actin filament, F actin, according to Holmes et al. (1990). (C) Tropomyosin molecules consist of a two chain a helical coiled coil. (D) Crystal structure of Tn C (Herzberg and James, 1985, 1988). (E) Schematic of the whole troponin complex with a globular region composed of Tn C and Tn I and a rod region of Tn T. (Diagram modified from Squire and Morris, 1998.) strand is twice this value). Note that actin filaments can have different twists from this depending on their source and on which other proteins are bound to them. In insect flight muscle the actin filament symmetry is that of a 28/13 helix. The subunit axial translation is still 27.5 Å, but the crossover repeat is now 385 Å and the long period pitch is Å. Actin filaments with various non muscle actin binding proteins on them can have crossover repeats as little as 320 Å (e.g., McGough et al., 1997). Note also that even in a particular actin filament type the crossover position may be rather variable. Actin filaments have been observed to have what is

22 38 SQUIRE ET AL. known as random variable twist, meaning that they are azimuthally rather flexible and that the quoted crossover spacing is an average value (Egelman et al., 1982). One aspect of the Holmes et al. structure, which was not in the G actin structure but was modeled by hand since it seemed to make sense, is the loop between residues F262 and I309 situated at the interface between subdomains 3 and 4 (see Fig. 8). The F actin structure as originally modelled seemed to have rather few links across from one strand to the other, which would stabilize the structure. Holmes et al. suggested that this loop might remodel itself and insert between subdomain 4 of the monomer one below along the genetic helix (i.e., in the other long period strand) and subdomain 3 of the monomer one above in the genetic helix. These proposed cross linking loops can be seen across the gap between the two long period strands in the F actin model of Fig. 9B. C. The Thin Filament and Troponin The components of the full thin filament, including the regulatory proteins tropomyosin and troponin, are shown in Fig. 9. Tropomyosin (Fig. 9C) consists of two chains of molecular weight 32.8 kda (Sodek et al., 1972; Stone and Smillie, 1978) folded into a parallel coiled coil a helical structure. There is a pseudo repeat in the sequence along the tropomyosin chains, yielding reasonably equivalent sequences where it interacts with the seven actin monomers in the 385 Å repeat (Parry and Squire, 1973; Stewart and McLachlan, 1975). The sequence is such that there are 14 bands of acidic residues that occur in two alternating types called a and b (McLachlan and Stewart, 1976). Since the twisting tropomyosin coiledcoil is labeling seven actin monomers that themselves lie on helical tracks, it turns out that to an outside observer the tropomyosin strands only complete six half turns in the 385 Å repeat. This gives the tropomyosin coiled coil a pitch of 385/3 ¼ 128 Å, which is similar to those of other fibrous proteins such as paramyosin, a keratin, and honeybee silk (see discussion in Squire, 1981; Geeves and Holmes, 2005). The 385 Å thin filament repeat also contains a single copy of the troponin complex along each strand (Fig. 9E). This comprises troponin I (TnI) of molecular weight 23 kda, which on its own can inhibit contraction, troponin C (TnC) of molecular weight 18 kda, which reversibly binds to Ca 2þ ions in the physiology range of concentrations and was the first EF hand calcium binding protein to be solved by protein crystallography (Herzberg and James, 1985, 1988; Fig. 9D), and troponin T (TnT) of molecular weight 30.5 kda, which binds to tropomyosin and to TnC and TnI. The whole troponin complex is known to be elongated, 200 Å long, with a

23 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 39 globular region at one end that contains TnC, TnI, and part of TnT (known as T2), with the rest of TnT (T1) forming a long tail. The structure of the globular part of troponin has been the subject of many studies, including nuclear magnetic resonance (NMR; Lindhout and Sykes, 2003) and solution scattering (King et al., 2005; Stone et al., 1998) and has led in recent times to partial crystal structures (Takeda et al., 2003). The full troponin structure and the nature of its interactions with tropomyosin and actin are gradually emerging. Brown and Cohen (2005; this volume) present a discussion of the latest findings on troponin and tropomyosin structures. Note that it was shown by Huxley (1972), Haselgrove (1972), and Parry and Squire (1973) that the effect of Ca 2þ binding to troponin when calcium is released from the SR following arrival of a nerve stimulated action potential along the T tubules could be explained by a change in the position of tropomyosin on the long period actin strands, thus uncovering or at least modifying the binding site on subdomain 1 of actin where the myosin heads attach to go through their force generating cycle on actin. This model, known as the steric blocking model, with the implication that tropomyosin regulates by virtue of its changing position on actin, has gained general acceptance, although there are undoubtedly subtleties, foreseen in Parry and Squire (1973), such as the simultaneous propagation of information through the actin monomers, which have yet to be fully described. Vertebrate striated muscle sarcomeres contain two remarkable molecular rulers. One of these is titin, mentioned briefly earlier (see Fig. 5B and D), which runs from the Z band to the M band and provides the sarcomere with mechanical continuity. Titin is discussed further in Section III.F. The other is nebulin, which is an I band protein anchored in the Z band. Nebulin is largely a helical, has a repeating sequence along it with a 55 Å axial repeat (assuming it is 35 residues in an a helix), which may fit in with the axial repeat along the long period strands in actin filaments; it seems to run the whole length of skeletal muscle actin filaments, and it may define the thin filament length. The stoichiometry of nebulin to actin is not yet known, but there appears to be at least one and probably two chains to each actin filament (Zhang et al., 1998). Because of the two strands of the actin filament, the number of nebulins might also be expected to be two (or a multiple). However, as discussed later, neither the Z line nor the actin filament itself has exact twofold symmetry, so the number of nebulins need not necessarily be even. At the actin filament tip (the pointed end away from the Z band) there is a capping protein called tropomodulin (Krieger et al., 2002; Littlefield and Fowler, 1998) that also lacks twofold symmetry. Finally, there is at least one more molecular component of muscle thin filaments that should be mentioned. This is phalloidin, which does not

24 40 SQUIRE ET AL. bind to G actin, but binds to and stabilizes F actin, where it also inhibits release of P i from actin hydrolyzed ATP and it resists actin depolymerization (Dancker & Hess, 1990; Dancker et al., 1975; Estes et al., 1981). Phalloidin location on F actin was modeled by Lorenz et al. (1993) to lie at the contact region of three adjacent actin monomers between the two long period actin strands and thus to form a direct stabilizing link across the filament. Fiber diffraction studies of oriented F actin gels under various conditions and with different ligands have been performed by Oda et al. (19 98, 2005) and further work in press ( Oda et al., 2005 ). D. Filament Organization in the Contractile Units of Different Muscle Types The A band lattices in different kinds of striated muscles have distinct arrangements. As shown in Fig. 3 and reproduced in simpler form in Fig. 10A and B, vertebrate striated muscle A bands have actin filaments at the trigonal points of the hexagonal myosin filament array. As discussed prevously, this array also occurs in two types, the simple lattice and superlattice. The ratio of actin filaments to myosin filaments in each unit cell is 2:1. In both cases the center to center distance between adjacent myosin filaments is 470 Å, but this varies as a function of overlap, becoming smaller as the sarcomere lengthens, giving an almost constant volume to the sarcomere (April et al., 1971). Insect fibrillar flight muscles (IFM) have a highly developed stretchactivation mechanism and contract in an oscillatory fashion at a frequency determined by the resonance of the wing thorax assembly that is higher than the nervous stimulus (Pringle, 1957). For this reason they are sometimes referred to as asynchronous muscles. In IFM sarcomeres the filament arrangement is different from vertebrates. As shown later the myosin filaments have fourfold rotational symmetry, they are longer than vertebrate myosin filaments, about 2 mm, and although the myosin crown spacing (the subunit axial translation) is almost the same (145 Å), the filament repeat is 1160 Å rather than 429 Å (Al Khayat et al., 2003; Morris et al., 1990; Reedy, 1968). In the IFM A bands there is also a hexagonal array of myosin filaments, but the actin filaments are midway between two adjacent myosin filaments giving a thin to thick filament ratio of 3:1 (Fig. 10C). The centerto center myosin filament spacing is larger than in vertebrate muscles at 520 Å. Different kinds of insect asynchronous flight muscles have slightly different arrays of myosin filaments within the hexagonal lattice (e.g., between Drosophila [fly] and Lethocerus [water bug]), but the myosin filaments all seem to have fourfold rotational symmetry. In the water bug, the myosin filaments appear to have their crossbridge arrays in orientational

25 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 41 Fig. 10. (A F) A band filament lattices in different striated muscles showing the threefold myosin filaments in vertebrates, the fourfold myosin filaments in some invertebrates, and the sevenfold myosin filaments in scallop striated adductor muscle. The ratio of actin to myosin filaments within the different lattices is also shown. Two smooth muscle types are also illustrated; the face polar or side polar myosin filaments in vertebrate smooth muscle (G) and the large paramyosin containing filaments in molluskan smooth muscles (H).

26 42 SQUIRE ET AL. register at least locally (Freundlich and Squire, 1983; Schmitz et al., 1994), which, like bony fish muscle, gives the sarcomere a simple A band lattice with a high degree of three dimensional (3D) order. Note that in these insect muscles the Z band is also hexagonal, unlike vertebrate Z bands, which are tetragonal (see Fig. 3G). Like IFM, insect leg muscle myosin filaments also appear to have fourfold symmetry, but they often have many more actin filaments around them than given by the 3:1 ratio in the flight muscles. The filament ratio can be as high as 5:1 or 6:1 (Fig. 10D and E), and obviously the lattice spacing is larger than in IFM. This is also true of other arthropod leg and trunk muscles and some synchronous flight muscles. Many of these muscles also have myosin filaments with fourfold rotational symmetry, but the axial repeat of the crossbridge array, still with a 143 to 145 Å intercrown spacing, can vary. For example, four stranded myosin filaments occur in tarantula leg muscle (axial repeat 435 Å; Crowther et al., 1985; Padron et al., 1998) and in Limulus (horseshoe crab) telson muscle myosin filaments (axial repeat 435 Å; Stewart et al., 1981, 1985). The scallop striated adductor muscle has even larger myosin filaments with sevenfold rotational symmetry (axial repeat 1440 Å; Craig et al., 1991; Vibert, 1992; Fig. 10F). All of these myosin filaments in invertebrate muscles contain the protein paramyosin, which is rather like the rod part of myosin without the heads (Cohen et al., 1987). In fact, in some invertebrate muscles, particularly in mollusks, paramyosin is the major protein and forms a large central core on the surface of which is a layer of myosin molecules (Squire, 1971; Fig. 10H). In a given muscle the size of these paramyosin filaments can be variable, but they can reach more than 1000 Å in diameter. The actin filament arrays in this case are essentially rings around the thick filament circumference. The major factor here is that the interactin center tocenter spacing is often Å, presumably a spacing that more or less matches the lateral spacing of myosin crossbridges on the thick filament surface with which they interact (see Section II.D,E). The actin filaments are also placed Å from the thick filament surface, presumably to position them within easy reach of projecting myosin heads. A similar spacing occurs between actin filaments in vertebrate smooth muscles (Fig. 10G). E. The Z Band The vertebrate striated muscle Z band is a cross linking structure that links actin filaments of opposite polarity in successive sarcomeres along a myofibril. One of the curious things about it is that, unlike the A band,

27 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 43 which is base d on a hexagona l array of filamen ts, the Z band lattice is approxima tely square in cross section (see Fig. 3G ). This means that through the I band the ac tin filamen ts must gradual ly cha nge from a hexagonal arrangeme nt in th e A band to a sq uare arra ngement in the Z band. The st rands of titin must also change from m yosin linked and hexagonal ly arranged in the A band to anc horing in some syste matic way into th e tetragon al Z band. Befor e seeing how this mig ht work, th e main features and comp onents of the Z band itself ar e descri bed. The main cro ss linki ng pro tein between anti parallel acti n filamen ts is a acti nin. a Actinin is one of the spec trin fa milies of protei ns ( Brode rick and Winder, 2002 ). Each chain contai ns an actin bind ing gl obular end domain (two calponin hom ology doma ins) that conti nues into a rod shaped reg ion where th e chain is folded into a strin g of four 3 chain a heli cal coiled coi l domai ns, each one known as a spec trin rep eat ( Fig. 11A ). The end of the cha in then forms EF hands in a globular region which in some a acti nins is C a 2 þ binding. Two such chains interact in an anti pa rallel fa shion to give th e full a actinin molecule. In oth er words, in muscle, the a actinin molecule is an anti parallel dimer wit h acti n bind ing domains at each end. The structure of the a actinin rod has been defined by protein crystallography (Djinovic Carugo et al., 1999; Ylanne et al., 2001). The actin bind ing region in a actinin has also been determi ned (Fra nzot et al., 200 5), and is very similar to e quivalent domai ns in dystro phin and utrophin ( Keep et al., 1999 ). In the Z band, the a actinin molecules form cross links between anti parallel actin filaments. In muscle cross sections (Fig. 11B, C) the square Z band structure appears in two forms, the basketweave and the small square lattice. These appearances seem to depend on the shape of the cross links between adjacent anti parallel actin filaments (called up [U] and down [D] in Figs ). Some authors believe that the appearance depends on the physiological state of the muscle (e.g., Goldstein et al., 1988), although this is not certain. A possible mechanism relating the two may be that the small square lattice is simply a basketweave in which the a actinin cross links have become bent (Yamaguchi et al., 1985). Different muscles and fiber types have Z bands of different thicknesses (Fig. 12A H). This seems to depend on the exact number of levels of a actinin cross links that are present. In longitudinal section the Z band has a zigzag appearance, and different Z band types have different numbers of zigzags. However, detailed analysis of Z band structure (Fig. 12I) shows that the zigzags usually correspond to the overlapping of two levels of a actinin links (Luther et al., 2003). Other proteins in the vertebrate muscle Z band are parts of nebulin and titin. When the sequence of the Z band part of titin was analyzed, it was

28 44 SQUIRE ET AL. Fig. 11. The structure of a actinin and the two vertebrate Z band lattices. (A) The ubiquitous protein a actinin is an anti parallel homodimer. Each 100 KDa monomer comprises four central spectrin repeats (S1 to S4) an EF hand domain and two calponin homology domains (CH) at the N terminus. The EF hand domains bind calcium in non muscle cells. One a actinin molecule binds two actin filaments via the calponin homology domains. a Actinin binds titin via EF hand domains. (B, C) The Z band is the site where actin filaments from adjacent sarcomeres overlap in a tetragonal lattice and are crosslinked by a actinin molecules. The polarity and origin of the actin filaments is indicated by U (up) and D (down). The appearance of the Z band in cross section is typically basketweave like (B) or small square like (C). The appearance is reported to transform between the two appearances depending on the state of the muscle.

29 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 45 found that there are repeating sequences of about 45 residues that have become known as Z repeats. These can occur in different numbers in titins from different muscles and fibers due to differential splicing (Gautel et al., 1996). There does seem to be a good correlation between the number of titin Z repeats, the thickness of the Z band, and the number of a actinin levels (Luther et al., 2002). It was shown by Atkinson et al. (2000) that the axial extent of a Z repeat is likely to be 120 to 150 Å. However, by careful measurement of Z band spacing in electron micrographs, Luther and Squire (2002) were able to show that the axial spacing between zigzag levels is 190 Å, which is too large to be spanned by a single Z repeat. It was concluded (Fig. 13) that there must be two Z repeats between zigzag levels, that the titin Z repeat region must span only half of the Z band, and that two anti parallel titins coming into the Z band from adjacent sarcomeres must be needed to define the whole Z band array. The nebulin strands that run along the actin filaments are anchored in the Z band and appear to start at the Z band edges (Millevoi et al., 1998). Other proteins in the Z band are telethonin (Mues et al., 1998) and PDZ LIM proteins, several of which bind to a actinin (Zhou et al., 1999). The presence of these proteins in the Z band suggests that they not only help to stabilize Z band structure, but that the Z band itself has more than just the passive role of transmitting tension from one sarcomere to the next along the myofibril. Also in the Z band is an actin filament capping protein, otherwise known as CapZ or b actinin in skeletal muscle (Papa et al., 1999) and as Cap32/34 in Dictyostelium (Haus et al., 1991). The crystal structure of CapZ has been determined (Yamashita et al., 2003). In a model of binding of CapZ to actin, a single molecule of CapZ binds to the barbed end of actin where it interacts with two or three actin monomers (Wear and Cooper, 2004). The most detailed Z band structure to date has come from the extended Z crystals found in muscles of patients with nemaline myopathy (Morris et al., 1990). This showed the unit cell of the structure to have the symmetry in which the actin filaments themselves have 4 3 screw symmetry (i.e., they are on a left handed 4/1 helix). In an earlier section the helical symmetry of typical actin filaments was described as a 13/6 helix of repeat Å. However, this is only one of a family of closely related symmetries. The actin filaments in insect flight muscle have 28/13 symmetry (28 actin subunits in 13 turns) and a repeat of 770 Å. The normal 13/6 helix could also be called a 26/12 helix (with a repeat of 715 Å), which shows the similarity between the two structures; one is a slightly unwound version of the other. One of the features of the 28/13 helix is that 28 is divisible by 4, which means that, starting from a given actin monomer, there must be three other monomers with exactly 90 azimuthal

30 46 SQUIRE ET AL. Fig. 12. Z band modular structures. The axial width of the Z band varies with fiber type: fast muscles typically have narrow Z bands of width 4070 nm, and slow and cardiac muscles have wide Z bands of width 100 nm. (A D) Electron micrographs of

31 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 47 separations around the actin filament from the first monomer and separated axially by 770 Å/4 ¼ Å. In fact, these four actin monomers on their own are related by 4 3 screw symmetry (they are on a left handed 4/1 helix). This, of course, is just what is needed to generate the Z crystal structure. The four actin subunits identified above will be the binding sites of four a actinin cross links pointing out at exactly 90 to the four neighboring anti parallel a-actin filaments. Since the actin molecules are themselves dimers, the Z crystal structure can also have twofold rotation axes perpendicular to the actin filament axis. Vertebrate muscle Z bands are clearly not extensive in the way that Z crystals are, but they do have interesting plane group symmetries. For a long time it was thought that the vertebrate Z band must have axial separations between the a actinin layers, which were directly related to the actin repeat measured from X ray diffraction patterns to be 360 Å. However, the careful measurements by Luther and Squire (2002) for comparison of the zigzag spacing with the size of the titin Z repeats showed that, as in the Z crystals, the axial separation of the layers of cross links in a variety of Z bands is 190 Å; much closer to one quarter of the 28/13 actin filament repeat than to one half of the 360 Å repeat found elsewhere in the vertebrate sarcomere. The implication is that the perfect fourfold helical symmetry intrinsic to a filament with 28/13 symmetry fits in much better with a actinin crosslinking in the Z band than does the 13/6 helix. It could even be that the a actinin and other Z band proteins pull the actin filament locally into the 28/13 geometry, whereas the rest of the actin filament, which does not make the same interactions, is closer to being that of a 13/6 helix. In summary, the actin and a actinin parts of the various vertebrate Z bands appear to be organized as subsets of the Z crystal structure. In each subset different numbers of layers from the Z crystal are present, the number presumably being determined by the titin Z repeats and possibly the Z band part of nebulin. Why the control of Z band thickness goes wrong when Z crystals are produced is not yet clear, but nemaline longitudinal sections of the Z band in (A) fish body white muscle (Luther, 1991); (B) fish fin muscle (Luther, 2000); (D) frog sartorius muscle (Luther et al., 2003); and (D) bovine neck (slow) muscle (Luther et al., 2002). The left and right panels show the primary orthogonal lattice views obtained by tilting the sections by 90 in the electron microscope. (E H) The corresponding schematic views of the electron micrographs in A D. Luther et al. (2003) proposed that these modular patterns are determined by the number of a actinin layers within the width of the Z band. (I) Stereo view of a model of a 6 a actinin layer Z band as found in slow muscle (Luther et al., 2003). Pairs of a actinin layers occur close together in longitudinal projection and give rise to three zigzag layers observed in electron micrographs, as shown by the model image superimposed on a corresponding electron micrograph (J).

32 48 SQUIRE ET AL. Fig. 13. The titin Z repeats and a Z band assembly model. The N termini of titin filaments from adjoining sarcomeres overlap in the Z band. This part of titin comprises a modular region of so called Z repeats, each about 45 residues long, the number of which is related to fiber type: 24 repeats occur in fast muscles, 57 occur in slow and cardiac muscles. This correlates with the Z band appearance since fast and slow fibers have narrow and wide Z bands, respectively, as shown in Fig. 12. The measured axial spacing between a actinin bridges is about 19.2 nm (Luther and Squire, 2002), a distance that is too long for a single Z repeat (A) to stretch to. Perhaps the bridge separation is related to two levels of Z repeats (C).

33 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 49 myopathy can be produced by mutations in actin, in troponin, in tropomyosin, and in nebulin (Wallgren Petterson and Laing, 2003). Since troponin is only found outside the body of the Z band, it could well be that it is the molecular organization at the Z band edges, where labeling of the actin filament by tropomyosin/troponin stops and nebulin insertion into the Z band edge occurs, that is important in controlling Z band thickening. F. Filament Organization in the Vertebrate I Band As mentioned earlier, vertebrate muscle sarcomeres have a strange geometric transition through the I band from the hexagonal A band lattice to the tetragonal Z band structure. It was Pringle (1968) who demonstrated that this kind of transition could be achieved by equal displacements of the actin filaments if the Z band lattice is not exactly square but has an included angle of 83. (Clearly an equivalent systematic change could occur if the Z band is in fact perfectly square, but the A band is not exactly hexagonal). The vertebrate A band has two actin filaments to one half myosin filament in a typical A band unit cell (see Fig. 10A and B). Since it has now been shown that there are probably six titin chains per half myosin filament (Liversage et al., 2001), the implication is that there are three titin strands per actin filament in the Z band. This number seems rather strange in view of the pseudo two strandedness of the actin filaments and the fact that in the Z band they seem to make a actinin crosslinks to four anti parallel actin filaments. However, the possible distribution of titin through the I band has been rationalized by Knupp et al. (2002) as illustrated in Fig. 14. Here the six titin strands from a single half myosin filament, which aggregate to form the end filaments at the A band edge, divide into two diametrically opposite pairs and two individual strands. The pairs pass to the Z band and interact with actin filaments of one polarity, whereas the single titin strands interact with actin of the opposite polarity in the Z band. This seems to be a plausible scheme and leads to an even distribution of titin interactions with every actin filament; all have two parallel titin strands and one anti parallel strand interacting with them in the Z band. It also would explain some of the lateral forces that are known to be present in the sarcomere. However, it is, of course, an oversimplification in that titin is known to make interactions with actin filaments in certain parts of the I band, possibly at the PEVK and N2 regions (Fig. 5D; and see Granzier and Labeit, 2005), and this will modify the simple scheme in Fig. 14.

34 50 SQUIRE ET AL. Fig. 14. Stereo pairs of the transverse structure (A) and the axial structure (B) of a 3D model relating successive half sarcomeres in vertebrate striated muscles. In both images, the wide blue and brown cylinders represent actin filaments, the gray cross links

35 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 51 IV. Myosin Filament Structure and the M Band A. The X Ray Diffraction Approach to Myosin Filament Structure As described in an earlier section, the myosin filaments in vertebrate striated muscles are bipolar assemblies of myosin molecules with threefold rotational symmetry. In the case of the filaments in bony fish muscle, they give the kind of X ray diffraction pattern shown in Fig. 4B. There is an axial repeat along the filaments of 429 Å and a subunit axial translation (the axial separation of myosin head crowns) of 143 Å. The shape of the myosin head was determined first by Rayment et al. (1993b; Fig 4A), but since then numerous other myosin head crystal structures have been determined (see Geeves and Holmes, 2005). A major feature of these is that the catalytic or motor domains are usually rather similar, and the neck regions or lever arms are also rather similar, but there may be a variety of angles between the two (Fig. 15). It is evident that there is a hinge within the myosin head, actually around residue 780 (Fig. 16A). It is this hinge that led to the idea of the neck region acting as a lever arm responding to ATP induced conformational changes in the catalytic domain (Rayment et al., 1993a). One of the tasks of structural biologists studying muscle contraction is to determine the organization and shapes of the myosin head in muscle under different physiological conditions. The technique of low angle X ray diffraction has unique advantages in this process, particularly since it can be applied to living muscle, which can be stimulated to produce active force or can be studied under a variety of different steady state conditions. The main problem with X ray fiber diffraction, as detailed in Squire and schematically represent a actinin bridges between anti parallel actin filaments, the narrow green and purple cylinders represent titin strands in one half sarcomere, and the narrow dark blue and red cylinders represent titin strands in the other half sarcomere. The narrow green and red cylinders represent single titin strands, whereas the narrow purple and dark blue cylinders represent paired titin strands. The myosin filaments are shown as brown cylinders. At the bottom of the structure (more obvious in B), there are seven myosin filaments, one central and six surrounding this, and at the top of the structure, for clarity, there is just a single myosin filament. Since the actin/aactinin Z band assembly appears to be a strong structure and is an early development in myogenesis, one can alternatively look on the 3D model as showing that there are balanced lateral forces keeping the myosin filament tips in the A band in the correct lateral positions relative to the actin filaments emanating from the Z band. At the same time the titin strands are held relatively clear of the actin filaments throughout most of the I band, thus allowing them to behave freely as parallel elastic elements. The opposite tilting of the titin and actin filament displacements in a given I band and the generally balanced 3D distribution of forces on the Z band actin filaments make this a very attractive model. (Modified from Knupp et al., 2002.)

36 52 SQUIRE ET AL. F ig. 15. Stereo views of the different myosin head, S1, structures showing their variable conformations in different crystal structures. (A) The heads with their motor domains superimposed and oriented as if interacting with a vertical actin filaments in the rigor conformation, Z band bottom and M band top. (B) The same structures in a view down the actin filament long axis, looking from the M band towards the Z band. Blue is the Dominguez et al. (1998) structure of S1 in chicken smooth muscle with ADP.AlF 4 bound, orange is the insect flight muscle S1 in the ADP.P i state (Al Khayat et al., 2003), yellow is scallop S1 crystal structure in the ADP.VO 4 state (Houdusse et al., 1999), and green is the chicken skeletal muscle with no nucleotide bound (Rayment et al., 1993a). Knup p (20 05 ; this volume), is one of interpr etation. Unl ike X ray crystal - log raphy, low angle fiber diffr action does not lead directly to image s of the di ffracting ob ject or to th e generat ion of elec tron den sity m aps. Setting up m odels with whatev er prior knowl edge is availa ble is requ ired, and then the unkn owns m ust be set up as pa rameters and searched for th e best values of th ese paramete rs to pro vide a mo del that satisfact orily accoun ts for the obs erved diffracti on patter ns.

37 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 53 Fig. 16. (A) Myosin head S1 showing the two domains referred to as the motor domain and the lever arm with a hinge at around residue 780 between the two domains that allows the relative movement between the two domains. (B) The arrangement of the myosin heads on the surface of the myosin filament backbone lying approximately on three coaxial helices of subunit translation 143 Å and repeat 429 Å. If the origins of each pair of myosin heads in each myosin molecule are labeled as black circles on a cylindrical piece of paper; when this is flattened out (C) a radial net is obtained (D) showing the three helices of myosin head origins in different colors, red, blue, and yellow.

38 54 SQUIRE ET AL. Fig. 17. (A) Principle of the searching routine showing various parameters describing the arrangement of the myosin heads on the myosin filament backbone and also changes in shape of the head by rotating the motor domain relative to the lever arm. (B) Illustration of the simulated annealing process showing the variation of R factor in a hypothetical parameter space. Points B and F are local minima; G and C are local maxima. The best parameter value is at point D, which is the so called global minimum,

39 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 55 A typical low angle diffraction pattern from relaxed bony fish muscle is shown in Fig. 4B. Much of the intensity that is seen comes from the organization of the myosin heads on the myosin filaments in the resting state (probably mainly M.ADP.P i ). We know that the myosin heads lie approximately on three co axial helices of subunit translation 143 Å and repeat 429 Å. This is most easily represented by the radial net shown in Fig. 16B D. The radial net in D is like an opened out surface view of the filament in B. Here the helical tracks become straight lines, and the black blobs represent the origins on the myosin filament surface of the pairs of myosin heads in each myosin molecule. From early studies it is known that the three crowns within the 429 Å repeat are not exactly the same and that there is a perturbation. Figure 17 illustrates the basis of the problem faced in modeling the observed X ray pattern as in Fig. 4B. We know the myosin head shape in that it appears to consist of two major domains that at low resolution appear to act as rigid bodies. We know the approximate arrangement of myosin head origins on the filament surface, taken initially to be perfectly helically organized, but we know that within a 429 Å repeat, successive crowns are not the same. It is therefore necessary to set up a model in which not only the relative orientations of adjacent heads can vary, but also their shape. The head organization must also be permitted to be different on the three crown levels within a 429 Å repeat. Figure 17A illustrates some of the parameters that are involved, such as the head tilt and the changes of shape produced by the catalytic domain slew, tilt, and rotation. In analysis of the bony fish muscle myosin filament structure, Hudson et al. (1997) set up a computer model of this structure with about 22 variable parameters; they stripped diffraction patterns such as that in Fig. 4B to give 56 independent X ray intensities and then used a simulated annealing search to define the best structure. For each set of parameters, they used the computer to generate model diffraction patterns to compare with the observed diffraction patterns. The term best above was defined as that giving the lowest R factor, where the R factor is an objective measure of agreement between the observed and calculated diffraction patterns; it is a kind of goodness of fit factor in which the smaller the value the better the fit. One possible way to conduct such a search is to systematically change every single parameter in appropriately small steps and to calculate the diffraction pattern in each case. However, with as many as 20 or so as opposed to local minima at B or F. The simulated annealing approach can be likened to heating up the system so that the parameter values can bounce around more freely in parameter space. It permits the search to jump out of local minima (B, F) and to find the global minimum (D).

40 56 SQUIRE ET AL. param eters and with many ste ps required for each pa rameter, th e comput ing time involved soon reaches many tens of year s. Another approa ch is nee ded. Prac tical appr oaches include refinemen t methods such as the Powe ll method and the downh ill simp lex method ( Brent, 197 3; Nel der and Mea d, 1965 ). Howe ver, th ese m ethods have a problem illustrate d in Fig. 17B. These refinemen t meth ods, which cha nge th e param eter values down loc al gradients in R fa ctor space, will take one from say poi nt G or poi nt A down to B at the bo ttom of the wel l that th ey are next to, but the refin ement then gets stuck. The best paramete r val ue is ac tually so mewhere else at point D, which is th e so calle d globa l minim um, as opp osed to local m inima at B or F. Another appr oach, known as simu lated annea ling ( Metr opolis et al., 1958 ), which can be likened to heati ng up the syste m so that the paramete r values can boun ce aroun d m ore freel y in paramete r spa ce, per mits the search to jump out of local minima and to find the gl obal mini mum. Sub sequent appl ication of one of th e other local refinem ent methods (Pow ell or simp lex above ) then ensu res that the poi nt D is reach ed at the bottom of the globa l well. B. Myosin Head Organization in Relaxed Vertebrate Myosin Filaments The results of modeling bony fish muscle myosin filaments using the simulated annealing approach (Hudson et al., 1997) are shown in Fig. 18. The top half of the observed X ray diffraction pattern, after data reduction and correc tion using CCP13 software ( 3.ac.uk ), is show n as Fig. 18A. The best model found after repeated simulated annealing searches (Fig. 18C) gave the computed diffraction pattern in Fig. 18B. In this structure the pairs of heads on one origin on the filament surface are relatively close to each other, but the three crowns are clearly different; the perturbation is evident. In fact, on two levels, the two heads in each pair lie one above the other, whereas on the third level the heads are side by side. On the face of it, this appears to be a very sensible structure, but there are two major problems in such analysis. The first is that vertebrate myosin filaments are known to carry extra proteins like C protein (MyBP C) and titin and the X ray model as described did not include these proteins. The second is that the diffraction pattern from the structure in Fig. 18C would be exactly the same if the structure was turned over top to bottom. In other words, there is no information in the X ray pattern about the polarity of the structure in Fig. 18C relative to the actin filaments with which the myosin interacts. Apart from testing the correctness of the X ray model, it is in these areas that electron microscopy comes into its own. Figure 19A shows a field of negatively stained isolated myosin filaments from bony fish muscle, together with actin filaments in the background.

41 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 57 Fig. 18. (A) Observed top half of the low angle X ray diffraction pattern from relaxed fish (see Fig. 4B) showing the three myosin layer lines (ML) at orders of 429 Å repeat and the third order meridional reflection, M3. (B) Calculated diffraction pattern from the best model shown in (C). The equator (Eq) and meridian (M) are labeled. (C) The model for the arrangement of myosin heads on the myosin filament that gave the best fit to the observed pattern shown in (A). (Based on Hudson et al., 1997.)

42 58 SQUIRE ET AL. Fig. 19. (A) Electron micrograph image of isolated myosin filaments and actin filaments from goldfish body muscle negatively stained and under relaxing conditions (scale bar ¼ 0.5 mm). (B) An isolated myosin filament from (A) showing two selected segments boxed in white and black rectangles and spaced at a multiple of the 429 Å repeat distance (arrows). The M band region is shown at the bottom. (C) A series of class averages from the particles selected and (D) reprojections of the final 3D reconstruction projected at the angles assigned to the corresponding classes shown in (C). (From Al Khayat et al., 2005a.)

43 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 59 One of the image processing techniques that has been applied to such myosin filaments is helical reconstruction (Eakins et al., 2002; Moore et al., 1970), where the computed Fourier transform (diffraction pattern) from a selected filament is processed to give a 3D reconstruction. As is clear from our need to use two eyes to get stereo vision, in order to generate a 3D reconstruction in electron microscopy it is necessary to obtain images of the same object viewed in different directions. Fortunately, with a helical structure (e.g., an actin filament or a perfectly helical myosin filament), a single filament image shows many subunits at different angles around the filament axis, so in this case a single image is sufficient to generate a 3D reconstruction. Such reconstructions of invertebrate myosin filaments are discussed later. The problem with vertebrate myosin filaments is simply that they are not exactly helical; there is the perturbation discussed above. For this reason, another approach is needed. In fact, the new technique of single particle analysis (e.g., van Heel et al., 2000) has been found to be very effective. Here the assumption is that there are many electron micrograph images available of exactly the same structure randomly oriented on the grid and therefore viewed in different directions. These images are then selected and a computer program is used to sort them into classes of views that look the same. The members of each class are then averaged. The problem that remains is to find the relative viewing directions of the different class averages. Computer programs such as IMAGIC (van Heel et al., 1996), SPIDER (Frank et al., 1996), or EMAN (Ludtke et al., 1999) make use of the common line projection theorem to estimate these angles. Once these have been determined, the particle structure can be put back together using a back projection procedure (Radermacher, 1992). This technique has been very successful for the analysis of globular particles, where remarkably high resolutions have been achieved: 10 Å in some cases and even higher with viruses that have a great deal of built in symmetry (van Heel et al., 2000). However, the method has a special problem when applied to filaments or other elongated particles (Paul et al., 2004). In these cases, the filaments normally lie down on the grid with their long axes parallel to the plane of the grid so the only rotations available are those around the filament long axis. Normally, tilts around more than a single axis are necessary for the computer algorithm that determines the particle viewing angles to work properly. However, with special precautions, evaluated by Paul et al. (2004) and Patwardhan et al. (2004), the approach can be applied successfully to elongated structures such as actin and myosin filaments. Figure 19B shows an isolated myosin filament from a field such as that in A and the rectangular boxes show particles that have been cut out from the filament images at 429 Å intervals to form a data set for single particle

44 60 SQUIRE ET AL. Fig. 20. (A) Cross sectional views of the three crowns of the X ray model shown in Fig. 18C. (B) Cross sectional views of the three crowns of the EM 3D map shown in E. (C) Cross sectional views of the densities between the crowns of the EM 3D map shown in E. (D) Stereo view of the X ray model of Fig. 18C reconstructed to 50 Å resolution. (E) Stereo view of the 3D reconstruction of the myosin filament structure obtained by single particle analysis using the classes shown in Fig. 19C (from Al Khayat et al., 2005a). The

45 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 61 analysis (Al Khayat, 2004, 2005a). Class averages from such particles are shown in C. These were then subjected to angle assignment and a 3D reconstruction generated as in Fig. 20E. This 3D density map can also be reprojected in the same directions as the original classes as a kind of test of how well the procedure is working. Figure 19D shows the reprojected classes for comparison with C; the correlation is good. Figure 20 also compares the results from this single particle analysis with the results from the X ray analysis of Hudson et al. (1997). Parts A and B show the projected densities in the three different crowns within a 429 Å repeat, A from the X ray model in Fig. 18B and C from the electron microscopic reconstruction in Fig. 20E. In both cases there is a striking feature. If the myosin filament was properly helical, there would be rotations of exactly 40 between the crowns and, apart from this rotation, the structures of each crown would be the same. However, in Fig. 20A and B it is clear that level 1 in both cases has a triangular profile pointing to the right. Level 2, on the other hand, points to the left and level 3 points to the right again. To a first approximation, levels 1 and 3 point in the same direction. Rather than having three equal angular steps of 40, there are two steps of 60 and one of 0. This is the azimuthal aspect of the crossbridge perturbation. The other feature, most obvious from the X ray structure (Fig. 20D) and the electron microscopic reconstruction in axial view (Fig. 20E), is that the crowns are not evenly spaced axially. This is the axial part of the head perturbation. The only other possible perturbation would be radial, but there seems to be very little in the way of a radial perturbation; all the crowns seem to have heads at about the same radius. Note finally that there is a difference between the X ray model, where only myosin is included, and the electron microscopic reconstruction, where everything is included. In part E the arrows highlight density features not seen in D that appear to lie between the levels of projecting heads. These positions are also shown in cross section in C. Presumably these features are associated with proteins such as titin and C protein that label the myosin filaments. The locations and properties of these proteins are briefly described in the next section. C. Further A Band Analysis: C Protein, Titin, and the Vertebrate M Band Titin is a very long string of immunoglobulin like (Ig) and fibronectinlike (Fn3) domains with some intervening unique sequences such as the three crown levels are labeled and extra density that could be attributed to non myosin density (e.g., titin or C protein) are shown by arrows. The M band is at the bottom in (D and E). In A to E, levels 1, 2, and 3 refer to the crown level.

46 62 SQUIRE ET AL. Fig. 21. (A) The A band part of titin consists of a repeating pattern of 11 Ig and Fn3 domains grouped in 11 copies from C terminus to N terminus as (Fn Fn Fn Ig Fn Fn Fn Ig Fn Fn Ig) starting at the edge of the bare zone. It then continues as six copies of the repeat (Fn Fn Fn Ig Fn Fn Ig) plus a few extra domains out to the myosin filament tip. (B) C protein (MyBP C) in cardiac and skeletal muscle; common features are 10 domains, which are Fn3 like, and Ig like, numbered C1 to C10. The C terminal domains C8, C9, C10 are associated with myosin and titin binding. Between C1 and C2 is a sequence that binds to myosin S2. Cardiac C protein has an additional Ig domain (C0)

47 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 63 PEVK reg ion (see Fig. 5D ) in the I band part of titin and a titin kinase region close to the M band (s ee Granzier and Labeit, 2005 ). In the A band ( Fig. 21A ), titin occ urs in close associati on wit h the myosi n fila ments and through muc h of its A band part it has a rep eating pattern of 11 Ig and Fn3 domains grouped in 11 cop ies from C termi nus to N terminus as ( Fn Fn Fn Ig Fn Fn Fn Ig Fn Fn Ig ) startin g at the edg e of the bare zone. It then continues as six cop ies of the repeat (Fn Fn Fn Ig Fn Fn Ig ) to the m yosin filament tip. There are six titin molecul es per myosin fila ment in hal f of the sarcomere ( Liver sage et al., 2001 ). These six mo lecules converg e to form the so called end filamen ts at th e A band edge ( Tr inick, 1981 ), af ter wh ich the tit in stran ds cross th e I band th rough to the Z band, as desc ribed earlier (s ee Fig. 14 ). The I band part of titin is essent ially compos ed of Ig domains apart from the maj or insert ion rich in th e amin o acids P, E, V and K (prolin e, gl utamic acid, valine, and lysine). As descri bed in Granzier and Labeit (2005), th is part o f titin has remark able elastic pro pertie s. Becau se all the subd omains are of about the same siz e (40 Å ), the 11 subdomain repeat in the A band part of titin is 440 Å long. In fact, this is thought to coincide with the 429 Å repeat of the myosin head array, giving a titin subdomain repeat of 429/11 ¼ 39 Å. Such a repeat, the eleventh order of 429 Å, has in fact been observed in freeze fractures of muscle A bands (Cantino et al., 2002) and in single particle analysis of myosin filaments (A Khayat et al., 2005a), and the eleventh order meridional peak in muscle X ray diffraction patterns is also relatively strong (Oshima et al., 2003). Note, however, that some aggregates of parts of the myosin rod also give a strong eleventh order peak in their computed Fourier transforms (diffraction patterns) even in the absence of titin (Bennett, 1981). C protein (MyBP C) has a substructure that is in many ways similar to parts of titin. Some of its domains are Fn3 like and Ig like domains, but there are also unique sequences. The cardiac and skeletal forms of C protein are also different (Fig. 21B). The skeletal form has 10 domains in which the C terminal domains C8, C9, and C10 are associated with myosin and titin binding. C10 is the main myosin binding site, but C8 and C9 are needed to give C protein its proper location on the myosin filament. Between C1 and C2 is a sequence that can bind to myosin S2 at its N terminus. Phosphorylation sites are indicated by a rectangle in the S2 binding site and in C5. A pro Ala rich domain is present at the N terminus in both isoforms; it links C0 and C1 in the cardiac molecule. Also shown are the related structures of H- protein, myomesin, and M-protein. (C) Interactions can occur between domains 5 and 8 and between domains 7 and 10 of different C protein molecules (from Moolman Smook et al., 2002). These interactions may suggest that three C protein molecules form a collar around the myosin filament.

48 64 SQUIRE ET AL. (Kunst et al., 2000). At the N terminal end of skeletal C protein is a sequence rich in proline and alanine, and therefore known as the Pro Ala domain. Cardiac C protein has an additional Ig domain (C0) at its N terminus and has some sequence insertions both in the S2 binding region, which can also be phosphorylated, and in C5. It has been found that interactions can occur between domains 5 and 8 and between domains 7 and 10 (Fig. 21C) of different C protein molecules (Moolman Smook et al., 2002), and these interactions were used to suggest that three C protein molecules form a collar around the myosin filament. On the basis of some puzzling X ray diffraction and electron microscopy evidence, Squire et al. (2003) have now suggested that while the C terminal end of C protein binds to myosin, the N terminal ends may bind to actin filaments, at least in resting muscle. The existence of an actin interaction would explain the observation of a C protein peak at 442 Å rather than at the 429 Å repeat of the myosin filament (Haselgrove, 1975; Squire et al., 1982). Electron microscopy has also suggested the presence of two repeats in the C zones of vertebrate muscles (Sjostrom and Squire, 1977). Figure 22 shows how C protein molecules based on the myosin filament repeat, with three molecules at each 429 Å spaced level, would extend out to actin and bind actin binding sites which would give the actin end a rather different repeat from 429 Å. Since there is also good biochemical and physiological evidence for actin binding (Kulikovskaya et al., 2003; Moos et al., 1978) and now further X ray diffraction evidence (Squire et al., 2004), it does seem fairly certain that C protein can bind to actin as well as myosin. Note that this region of C protein also binds to myosin (Kunst et al., 2000). For reasons outlined in Squire and Knupp (2005), it also seems possible that the C terminal ends of C protein may run axially along the titin strands with which they interact, rather than forming a collar as suggested by Moolman Smook et al. (2002). By comparison of the sequence of the Pro Ala domain of C protein and the sequences of similar regions in one of the light chains of myosin, shown by Trayer and colleagues to bind to actin (Timson et al., 1998), it was suggested by Squire et al. (2003) that this region of C protein might be part of the actin binding site. This idea remains to be tested. In the central region of vertebrate myosin filaments is the bare zone (Fig. 3D). It is bare in the sense that there are no myosin heads there, but it is not actually bare. In muscle there is a strong cross linking structure, the M band, in the middle of this region and titin strands continue from the bridge region into the M band. Because it is not bare in the sarcomere, but contains the M band proteins and other structures, this region is sometimes called the M region (Sjostrom and Squire, 1977). In electron micrographs of muscle cross sections, the M band appears as dense myosin

49 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 65 Fig. 22. (A) Radial net of an array of six actin filaments (light blue) surrounding a myosin filament as seen from the axis of the myosin filament backbone looking radially outward. Open circles and lines represent the radial net of the MyBP C protein origin positions on the thick filament surfaces. This consists of seven levels of C protein molecules axially separated by 429 Å and with 3 C proteins 120 apart at each level. The orange circles show which actin monomers might be labeled by the N termini of the C protein molecules whose origins are represented by the open black circles. (B) A possible model for the C protein geometry within the sarcomeric A band of resting vertebrate striated muscle. A thick filament backbone (brown) is surrounded by six actin filaments (dark gray). C protein molecules with domains C7 to C10 running axially along the myosin filament backbone interact with actin filaments through the C protein N terminal Pro Ala rich domains (green ovals). A few myosin heads are drawn as transparent ghosts to indicate their proximity to the C protein array. (After Squire et al., 2003.) filament profiles arranged on a hexagonal lattice and cross linked to their six myosin filament neighbors by bridging structures (see Fig. 3I), which sometimes show an increased density halfway along them. In axial views, the M band can vary between muscles and fiber types, but there appears to

50 66 SQUIRE ET AL. be a symmetrical pattern of up to five lines of M bridges (Fig. 23A, B and D, and Figs. 24 and 25). These lines are separated axially by about 220 Å. Because of the presence of some weaker lines between them, these main M band lines were termed M6, M4, M1 (centrally located), M4 0, and M6 0 (Sjostrom and Squire, 1977). Of the weaker lines in different M bands, those that were particularly consistent and strong were the lines at M3, which were thought by Luther and Squire (1978) to possibly represent weaker bridging structures in the M band. The M region transverse lines observed by Sjostrom and Squire (1977) extended out to M9 and M9 0 at the edges of the M region, with the next observed densities thought to be the first level (P1) of myosin heads in the proximal zone of the bridge region (Fig. 23B). Different fiber and muscle types have M bands with different relative densities of the strong M band lines (Carlsson et al., 1990; Sjostrom and Squire, 1977). In particular, relatively fast fibers have rather weak lines at M6 and M6 0, giving a three line M band (Fig. 23D), whereas very slow fibers have relatively weak density at M1, giving a four line M band. Other muscles have line densities between these extremes, sometimes with all lines relatively strong, giving a five line M band. In all cases the M4, M4 0 lines are strong, suggesting that the structures giving rise to M4 are the primary elements defining M band strength. One intriguing aspect of the M band is that it presumably is the structure that defines the relative rotations of adjacent myosin filaments in the A band hexagonal lattice. In other words, it determines whether the A band has a superlattice or a simple lattice. In fact, the generation of the two lattice types can be rationalized in terms of the molecular interactions that might occur at the M4 level (Pask et al., 1994; based on Luther and Squire, 1980). At this position the myosin filament cross sectional profiles appear triangular (Fig. 23E and F). Since they effectively make six cross linking interactions with their six myosin filament neighbors, the binding sites for the cross links must either come from a triangle tip or a triangle side. If the tip interactions are called A or a and the side interactions are called B or b, then the lattice type is, in fact, defined by the interactions between these sites on adjacent filaments. If an A always interacts with a B (Fig. 23E), then all the triangles must have the same orientation and a simple lattice is automatically generated. On the other hand, if an a site prefers to bind to another a site, or a b to a b, then such interactions cannot be systematically generated across the myofibril, but optimization of the number of such interactions automatically generates the kind of statistical superlattice defined by Luther and Squire (1980; Fig. 6B). Figure 24A shows three cross sectional slices through the fish simple lattice M band reconstruction of Luther and Crowther (1984). Here it

51 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 67 Fig. 23. (A) 3D Reconstruction of fish muscle M band/bare zone obtained from single particle analysis (Al Khayat et al., unpublished data). (B) 1D Density profile of (A) showing the main stripes, M6 0,M4 0, M1, M4, and also M6, M8 (titin kinase?), M9, and P1 (the first myosin crown). (C) The anti parallel packing of myosin rods in the bare zone, together with the main M bridge levels. M1 and M6 are of variable density giving rise to different M band types (D). M band interactions at the M4 level can explain the generation of simple and superlattice A bands (E and F). (After Pask et al., 1994.)

52 68 SQUIRE ET AL. Fig. 24. M band structure from electron microscopy of both simple lattice and superlattice muscles. (A) 3D Reconstruction of fish muscle M band. Three distinct layers were observed in the reconstruction, at each of the M bridge levels: M4 0, M1, and M4 (M and B label the myosin filaments and M bridges, respectively.) The observed 32 point group symmetry has been imposed on the 3D map. (B) Part of the M band as modeled by Luther and Squire (1978). M1 and M4 bridges are seen connecting adjacent myosin filaments. Halfway along the M bridges and running parallel to the myosin filaments are the M filaments. M3 marks a further level of secondary Y shaped bridges. (C) A slice

53 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 69 can be seen that th e M1 pro file is rather sixfold, whereas the M4 and M4 0 profiles are rather tria ngular with the bridg e density on the poi nts of the triangle s rather strong er than on the triangle sides. The wh ole M band and bipolar myosin fila ment has th e symmet ry of the 32 point group ( Luther et a l., 1981 ). However, M4 and M4 0 do show a slight thickeni ng of the bridges halfway across, which has been interpr eted in terms of the pre sence of axially align ed densit y, the so called M filamen ts, running throu gh the M band hal fway be tween adjacen t myosin filamen ts. Fig. 24B summari zes the gene ral features of o ne half of the M band, exc luding M6 ( Luther and Squ ire, 1978 ). The myosin filamen ts, M filamen ts, M1 bridges, and M4 bridges form a 3D networ k, with densit ies at M3 poss ibly acti ng as secondar y links betwe en the M filam ents. The existence of axially aligned density betwe en the myosin filamen ts is support ed by a new rec onstru ction of the M band in frog muscle ( Fig. 24C ). An obliqu e view of a 3D rec onstruction of the supe rlattice M band in frog sar torius muscle is show n in Fig. 24D, where both lateral and axial den sities can be seen ( Luther et al., 2005 ). Apar t from the M band part of titin, the bridges in the M band appear to be comp osed of at least two pro teins, m yomesin and M pro tein. These two rath er simi lar proteins have a domain structu re show n in Fig. 21B, which is clearly assemb led from Ig and FN3 doma ins as in titin and C protein. Ther e are also unique sequenc es at th e N termin al ends. Antibody labeling studies have sugges ted the mo lecular ar rangement illustrated in Fig. 25F ( Ober mann et al., 1996, 1997). Here titin forms part of the axial ly aligne d density at the M fila ment positio n, and assoc iated wit h this is part of myom esin. The N termi nal part of m yomesin th en forms the M4 bridges. M pro tein, on th e other hand, appear s to be mainly confined to the M 1 positio n. Other pro teins in th e M band includ e the muscle form of creatine ki nase (MM CK; Horne mann et al., 2003 ). Since th e M region part of titin also contai ns a kinase doma in ( Ma yans et al., 1998 ) it is clear that, like th e Z band, the M band does not just have a stru ctural role but also has importan t metabol ic pro pertie s. Tha t the M band st ructure is clearly rel ated to functio n is demons trated by th e appear ance of the M band in the hear t muscles of di fferent animals with di fferent hear t rates ( Pas k et al., 1994 ). Figure 25A shows microg raphs and their dens ity pro files from a variety of cardiac muscles, goin g from through a 3D reconstruction of frog sartorius muscle. The M band featur es are noisier than in the fish case because of the statistical superlattice structure (see text). Myosin filaments (M) are observed with M bridges (B) and longitudinal running M filaments. (D) Stereo view of the 3D reconstruction of the frog muscle (superlattice) M band, showing M bridges and M filaments (Luther et al., 2005).

54 70 SQUIRE ET AL. Fig. 25. Variety of cardiac M band structures and their molecular origin. (A E) Electron micrographs of M region segments from (A) cow, (B) guinea pig, (C) rabbit, (D) plaice, and (E) carp. The profile plots on the right show the main stripes, M1, M4, and M6. M1 is a strong stripe in A through C but missing from E through E the fish hearts. (F) Molecular arrangement of the major M band proteins, myomesin (left) and

55 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 71 one with a clearly prominent M1 line (A) to one with M1 almost absent (E). Presumably the M band in E has little M protein, A has much M protein, and presumably M4, which remains strong in all M bands, is the site of myomesin which is always present. MM CK probably also occurs in variable amounts on and around M1 to M4, but its location is relatively ill defined. D. Invertebrate Myosin Filaments Myosin filaments in muscles other than vertebrates appear to be relatively simple, in that they all appear to have their myosin heads arranged in regular helices, apart from at the edges of the bare zones where perturbations in the regular molecular packing are to be expected. Because of their helicity, analysis of electron micrographs of such filaments by helical reconstruction has been relatively successful. 3D reconstructions of the filaments in insect flight muscle (Morris et al., 1991), tarantula leg muscle (Crowther et al., 1985), limulus telson muscle (Stewart et al., 1981, 1985), and scallop striated muscles (Craig et al., 1991; Vibert, 1992) have been published. In the case of insect flight muscle, the myosin filament structure has also been determined from X ray diffraction modeling (Al Khayat et al., 2003). The muscle is so well ordered that, as in the case of fish muscle, the X ray diffraction patterns are beautifully sampled (Fig. 26A). The simulated annealing approach of Hudson et al. (1997) was adopted for this study, except that, since the structure was assumed to be perfectly helical, many fewer unknown parameters were needed than with vertebrate myosin filaments. Figure 26B and C compare the observed and calculated diffraction patterns for the insect case, and Fig. 26D shows the resulting model of the insect (Lethocerus) four stranded myosin filaments. In this case, the subunit axial translation (i.e., between crowns) is much like that in vertebrates at just below 145 Å. However, the repeat of the structure is after eight crowns at 1160 Å (see Fig. 27C), a number that fits in nicely with the Å crossover repeat of the 28/13 actin filaments in this muscle ( ¼ 1160) and helps to explain why this muscle is so well ordered in three dimensions. Unlike the vertebrate structure, here the heads are very closely confined axially to 145 Å spaced shelves of density. However, half of the heads are projecting from the filament surface and are apparently supported by the other heads, which are wrapped around the filament circumference. A circumferential head from one myosin M protein (right), elucidated using immunolabeling (Obermann et al., 1997). Fast muscles have both proteins, but slow muscles have only myomesin. This correlates with the observed M band stripes where M1 is missing in slow muscles.

56 72 SQUIRE ET AL. Fig. 26. (A) Observed X ray diffraction pattern recorded at the APS BioCAT Argonne Synchrotron Radiation Source for relaxed insect flight muscle (Lethocerus) showing a beautifully crystalline sampled pattern with the 145 Å meridional reflection labeled. (B) The bottom right quadrant of the observed pattern (OBS) shown in (A) with the myosin layer lines at orders of 1160 Å repeat. (C) The calculated diffraction pattern (CALC) for the best model that gave the best fit to the observed pattern (B). (D) 3D Model for the best X ray model for the myosin filament in relaxed insect flight muscle shown with its M band at the bottom and with the filament backbone shown simply as a cylinder (Al Khayat et al., 2003). (E and F) 3D Reconstruction of myosin filaments in relaxed scallop muscle obtained from single particle analysis (Al Khayat et al., 2005b) and viewed from different directions, M band bottom in E and F and viewed from the M band toward the Z band in G.

57 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 73 molecule supports a projecting head from a neighboring molecule, thus providing the potential for cooperative interactions, possibly involved in regulation (Al Khayat et al., 2003; Squire et al., 2005). Other four stranded myosin filaments occur in tarantula, in the leg muscles of other insects, and, for example, in the Limulus telson muscle (Fig. 27B). In these cases, the intercrown spacing is still 145 Å, but, as in vertebrate filaments, the axial repeats of the filaments are after three Fig. 27. Radial net of various crossbridge lattices from different species along with their corresponding computed Fourier transform. The myosin filament is threestranded in (A) vertebrate muscle, four stranded in invertebrates (B and C), and seven stranded in scallop muscle (D). This figure shows the similarity of the surface lattices of the myosin head origins on the myosin filaments in different muscles although the myosin heads have different slew, tilt, and rotations. Images were created using the program HELIX (Knupp and Squire, 2004).

58 74 SQUIRE ET AL. crowns at 435 Å. Reconstructions of these filaments show strong continuity of myosin head density along the long period strands of the helical surface net, once again showing interactions between the heads of different myosin molecules, but this time between crowns rather than within a crown as in insect flight muscle (e.g., Padron et al, 1998; Stewart et al, 1985). The myosin filaments in scallop striated adductor muscle are much larger in diameter than vertebrate myosin filaments and have seven co axial strands of myosin heads (Fig. 27D). As in the other cases of helical filaments, the scallop structure has also been determined by 3D helical reconstruction (Vibert, 1992). The reconstruction had a resolution of 70 Å, as for the other filaments reconstructed by this method. The single particle analysis technique described earlier for analysis of the nonhelical vertebrate myosin filaments can also be applied to purely helical structures and, even using the same micrographs, it has the potential to yield much higher resolution. This technique is being applied to electron micrographs of insect flight muscle thick filaments (Al Khayat et al., 2004) and also to scallop striated muscle filaments (Al Khayat et al., 2005b), in both cases achieving 50 Å resolution. The result in the latter case is shown in Fig. 26E G. Here it can be seen that, although the head density appears to be aligned along the long period helical tracks (part E), it does appear to be such that both myosin heads would be accommodated within the main density peaks (F and G). E. Crossbridge Arrangements on Different Myosin Filaments: Variations on a Theme One of the early suggestions about myosin filament structure made by Squire (1971, 1972) in his General Model of Myosin Filament Structure was that, since all muscle myosin filaments are made up from muscle myosin molecules that are rather similar in size, shape, and structure, perhaps myosin filaments, although different in different muscles, would have closely related packing arrangements. If this is considered in terms of the molecular packing of the myosin rods, then the implication is that, whatever the tilts, slews, and rotations of the heads, the underlying surface lattices of the myosin head origins on the different myosin filaments would be rather similar. As evidence has accumulated about different myosin filaments, this general theme appears to have been confirmed. The different filament lattices shown in Fig. 28 have different axial repeats, they have different numbers of strands of myosin heads, but if looked at locally, the relationships between adjacent heads are very similar. In each case, they are axially separated by Å axially and the lateral separation is in the

59 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 75 Fig. 28. Classification of crossbridge configurations in myosin filaments in different muscles. In each case, the axial separation is Å and the lateral separation is Å. There are three main classes: (A) Class I, where the interaction is between heads of the same molecule as in vertebrate striated muscles; (B) Class II, where interaction occurs between heads of adjacent myosin molecules in the same crown, as seen in insect (Lethocerus) flight muscles; and (C) Class III, where the interaction appears to be between heads in different crowns, as seen in tarantula and Limulus. region of 120 to 150 Å, a dimension that correlates well with the lateral separation of actin filaments in different muscles (see Fig. 10). The lateral separations would not be expected to be identical, because, if the flat layer of closely packed myosin rods, as envisaged by Squire (1973), was bent by varying amounts to produce filaments with different rotational symmetries, then the outer part of the layer, on which the heads are located, would open out by different amounts depending on the filament symmetry. Flat filaments in vertebrate smooth muscle (see Fig. 10G) or very large diameter filaments in molluskan smooth muscles (see Fig. 10H) would have relatively undistorted myosin layers and might have closer head separations than in other filaments where the layers are bent around to give small diameter filaments (see the filament backbone in Fig. 29A). It is in the muscles with flat or large myosin filaments that the actin filaments are relatively long and in excess over the myosin containing filaments and form pools of actin organized into actin lattices with an interactin filament spacing of 120 Å (Lowy et al., 1970; Small and Squire, 1972). In striated muscles the actin filament separations are much larger than this and the myosin heads radiate out toward them from tightly curved layers of rods. It is clear that

60 76 SQUIRE ET AL. F ig. 29. (A) View down the filament long axis of one full myosin 429 Å repeat of the X ray model of the fish muscle myosin filament shown in Fig. 18C along with the backbone mode l from Squire (1986) and Chew and Squire (1995). (B) View down the fiber axis of one full myosin 429 Å repeat in the fish muscle A band unit cell, in which two actin filaments have been included. The head perturbations create a local environment of actin binding sites (blue) on the myosin heads (yellow) around the actin filaments (green). m yosin fila ments as a family do inde ed have a gre at deal in common, as envi saged by Squ ire (19 71) and revie wed in Squire (1986). In the discussi ons abo ve, it wa s not ed that in some cases the intera ction betwe en head s in resti ng muscl e myosin filam ents is betwee n head s of the same myosin molec ule ( vertebra te), whereas in other cas es (insec t flight), it is betwe en adjacent m olecules in the same crown. In a third type

61 MOLECULAR ARCHITECTURE IN MUSCLE CONTRACTILE ASSEMBLIES 77 (tarantula and Limulus), the interaction appears to be between heads in different crowns. We have just seen that the underlying surface lattices of myosin head origins are rather similar (see Fig. 28). It is convenient in discussing the different head organizations on these relatively uniform underlying lattice geometries to call them Class I, Class II, and Class III, respectively, as in Fig. 28. The different classes may have a distinct role in defining the regulation or other functions of the heads in these muscles, and their classification should help to define and understand their different functional roles in different muscle types. Note that according to early published models (Vibert, 1992), scallop muscle myosin filaments would have been classified as Class III, but the reconstruction in Fig. 26E G may suggest that these are actually Class I filaments. F. Conclusion: Implications about the Crossbridge Mechanism Several of the articles that follow this one, particularly those by Geeves and Holmes (2005) and Squire and Knupp (2005), are concerned with the Fig. 30. Comparison of the X ray modeled myosin head arrays in relaxed fish muscle (A) and relaxed insect flight muscle (B), with the motor (catalytic) domain of outer myosin heads in each model circled to show the close similarity of their configurations in the two different species. The M band is at the bottom in both models.

62 78 SQUIRE ET AL. m yosin crossb ridge mechan ism in active m uscle. The present arti cle has set th e scen e in which this interesti ng m olecular mechan ism ta kes place. Many of the stud ies o f th e con tractile mecha nism have involved studies of vertebrate striated and insect flight m uscles, so this article summari zes what is known from these muscles. Figu re 29A show s that the filamen t backb one has the myosin rods in a tightly packed and physic ally plau sible arrangem ent that agrees with the high angle X ray diffr action patter ns from muscl e ( Squ ire, 1973, 1986 ) better than any oth er publis hed m odel ( C hew and Squ ire, 1995 ). On the surf ace of this rod array, th e myosin head arrangem ent is shown as determi ned by Hudson et al. (19 97) for the A band unit in resting fish muscle. The singl e partic le analysis work of Al Khayat et al. (20 04, 2005a) has confi rmed many of the features of this m odel (see Fig. 20 ). When the structure in Fig. 29A is put into th e fish muscle A band unit cel l ( Fig. 29B ; note that the analysi s of Hudson et al. [1997 ] defin ed the abso lute orien tation of the fila ment within the A band lattice ), it can be se en that the acti n bind ing sites on the myosi n head s are alread y cl ose to Fig. 31. Implications about the contractile mechanism in insect flight muscle. Blue is insect flight muscle S1 shape in pre powerstroke state (Al Khayat et al., 2003), and green is chicken skeletal muscle S1 in the rigor state with no nucleotide bound (Rayment et al., 1993a). The actin filament (right) is shown with the Z band at the bottom and M band at the top. A transition from the pre powerstroke/resting S1 shape to the rigor/end of post powerstroke shape would involve an axial swing of the lever arm by 100 Å, resulting in the sliding of the actin filaments past the myosin filaments and toward the M band.