The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling
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- Luther, P.K. J Muscle Res Cell Motil (2009) 30: 171. doi:10.1007/s10974-009-9189-6
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The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30–50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.
KeywordsZ-lineZ-bandMuscle proteinsActinAlpha-actininElectron microscopy
Two giant polymer proteins, titin and nebulin/nebulette, like actin, also overlap within and form important parts of the Z-disc (Clark et al. 2002). Titin spans half sarcomeres between the M-band and Z-disc and forms the template for the sarcomere. It has important elastic regions (PEVK regions) in the I-band (Granzier and Labeit 2004; Tskhovrebova and Trinick 2003). Nebulin runs along the thin filament and forms the template for thin filament assembly (McElhinny et al. 2003). CapZ caps the barbed ends of the actin filaments and interacts strongly with α-actinin and nebulin (Papa et al. 1999; Pappas et al. 2008). The atomic structures of a few Z-disc components, including actin, α-actinin (from homologous domains), CapZ (Yamashita et al. 2003) and the titin-telethonin complex (Zou et al. 2006) have been solved recently and there is the exciting possibility of fitting them into high resolution 3D electron tomograms of the Z-disc in the near future. The filaments and proteins mentioned above will be discussed in detail in this review.
Passive transmission of tension through the Z-disc structural assembly is an important role of the Z-disc. However, the Z-disc has additional important roles as it houses or anchors an amazing number of additional proteins. These proteins have various roles, including involvement with stretch sensing and signalling (Epstein and Davis 2003; Pyle and Solaro 2004). Like all cells, muscle fibres and cardiomyocytes respond to stretch. For this response, stretch sensors are required which stimulate signalling proteins that eventually communicate with the nucleus. The identity and mechanism of the stretch sensors is not known, but they are believed to be located in the Z-disc and M-band (Knoll et al. 2002). Mutations in many of the Z-band proteins lead to disease. There have been excellent reviews recently on Z-disc proteins and their role in signalling and disease (Faulkner et al. 2001; Frank et al. 2006; Sheikh et al. 2007), so this topic will be discussed only briefly.
Reviews on the structure of the Z-disc include the comprehensive review written nearly 15 years ago by Vigoreaux (1994) and parts of sarcomere reviews since then (Clark et al. 2002; Craig and Padron 2004; Squire et al. 2005). There have been significant and exciting advances in our knowledge of the structure and function of the Z-disc in recent years and these form the main part of the current review.
Z-disc lead players
The structure and symmetry of the actin filament and of α-actinin are important factors for building a tetragonal lattice Z-disc in vertebrate striated muscles; interdigitating actin filaments and α-actinin form different number of layers of crosslinks between the antiparallel actin filaments in different Z-disc types. In insect flight muscle the rather different Z-disc is based on a hexagonal lattice (Deatherage et al. 1989) but it will not be discussed further here.
Structure of actin filaments
Structure of α-actinin
The mode of α-actinin binding on actin was investigated by McGough et al. (1994) using cryo-electron microscopy. They showed that α-actinin binds over two neighbouring actin monomers along the long helix, namely with residues 348–355 in the first actin monomer and with residues 87–96 in the second. In their study of α-actinin binding to actin on lipid layers, Hampton et al. (2007) observed that the binding can occur at a range of angles, with a preference for 60 and 120°. The variation in binding angles may be due to varying torques, and the authors suggest that the variation in angle of attachment could be a tension sensing mechanism.
In addition to its structural role, it is now known that α-actinin has a major role in the docking of signalling and other proteins at the Z-disc (see reviews by Otey and Carpen 2004; Sjoblom et al. 2008).
Absence of tropomyosin and troponin from the Z-line
Structure of the Z-disc
Longitudinal section views
The Z-disc has characteristic appearances in longitudinal and transverse sections. The image in the electron microscope is a projected view of the structure through the depth of the sample. With different components at different depths in the section what is seen in the projected image can be quite blurred. In a typical section of ~100 nm thickness there are about 3–5 layers of unit cells of the Z-disc lattice (lattice size ~25 nm) within the depth of the section. Only if a section is oriented so that the appropriate lattice lines are aligned along the direction of view is the characteristic zigzag view of the Z-disc obtained (right side Z-disc in Fig. 1b and Fig. 7a). In most orientations, the appearance of the Z-disc resembles a fuzzy dense band (left side Z-band in Fig. 1b).
Early models of the Z-disc
Variation of Z-disc structure with fibre-type
Vertebrate muscle Z-bands have precise widths that depend on fibre type (slow and fast) and muscle type (cardiac and skeletal). Fast muscles typically have narrow Z-bands and slow muscles have wide Z-bands. This is certainly an adaptation to fine tune the muscle to its function. Fast muscles have higher contraction velocities and produce high force. A detailed analysis of electron micrographs of the different types of Z-band was shown by Rowe (1973). The typical image of the Z-band in longitudinal sections is depicted as zigzag links between the opposing ends of actin filaments from adjacent sarcomeres, with a relative half unit cell shift between the two opposing arrays (Fig. 1). Rowe (1973) showed that the fast muscles had narrow Z-discs with a double chevron appearance and that slow muscles had wide Z-discs with 3 or 4 chevrons.
3D structure of the Z-disc
Depicting the structure of the Z-disc on paper is difficult because the main components do not lie in a single plane. For example, in the [1, 0] lattice view actin filaments on either side of the Z-disc are arranged in different planes. Hence the schematic longitudinal view of a zigzag link between actin filaments from adjoining sarcomeres is a composite (projection) as the layers of actin filaments from adjacent sarcomeres and the Z-links occur in different planes. The Z-links are composed mainly of α-actinin, but other proteins may contribute like nebulin, titin and myotilin. The nomenclature for describing the different projections is shown in Fig. 6. The main lattice views are referred to as the [1, 0] and [0, 1] views or projections.
3D structure of the 2-layer Z-disc: the building block of vertebrate Z-discs
Franzini-Armstrong (1973) first reported a very narrow Z-band in fish white muscle which is a fast muscle. She noted that in certain orientations in longitudinal sections, the actin filaments ends of two adjacent sarcomeres were connected by a single zigzag link (Fig. 1b). Luther (1991) undertook a detailed 3D study of this muscle and showed that the zigzag appearance was one of the two main lattice views and that rotating the Z-band by 90° along the myofibril axis gave a different view that resembled overlapping spikes (Fig. 7a). We note that the image is symmetrical about the centre of the Z-band (strictly there is glide plane symmetry relating the upper and lower halves. The actin filaments from the adjacent sarcomeres were found to be cross-linked with two pairs of orthogonal links separated along the actin filament by ~10 nm (Luther 1991). This is probably 19 nm in light of new data described below.
Luther also described “polar” links at the periphery of the Z-band between actin filaments of the same sarcomere. Such links have been reported in other studies (Schroeter et al. 1996; Trombitas et al. 1988; Tskhovrebova 1991). As described in the section on α-actinin, Taylor and colleagues have shown that α-actinin can crosslink polar as well as bipolar actin filaments (see Fig. 3).
3D structure of a 3-layer Z-disc
A 3-layer Z-disc was first observed in fish fin muscle (Luther 2000). The two orthogonal views are shown in Fig. 7b. The appearance comprises one zigzag layer and bulbous densities which occur on different sides in the orthogonal [1, 0] and [0, 1] views. Unlike the symmetrical appearance of the simple 2-layer Z-disc relating the upper and lower halves of the Z-disc (Fig. 7a), the 3-layer has quite different appearances. This difference occurs because of the underlying even and odd number of layers of Z-links. Hence, for an unknown Z-band, one can determine whether it comprises even or odd layers by looking for a symmetrical appearance relating the Z-disc parts in adjoining sarcomeres.
3D structure of a 4-layer Z-disc
A formal 3D reconstruction of a 4 layer Z-disc has not been done. Examination of frog sartorius (fast) muscle Z-disc in longitudinal sections (Luther et al. 2003) revealed Z-discs between two sarcomeres with regions of two different widths. The narrow one had the same pattern as the three layer Z-disc. The second one, wider by ~18 nm, had symmetrical appearance about the centre of the Z-disc and was therefore inferred to comprise four layers (Fig. 7c).
3D structure of a 6-layer Z-disc
Slow and cardiac muscles typically have wide Z-discs, ~100–140 nm wide (Fig. 7d). The striking difference between the orthogonal lattice views is that in one of them, e.g. the [1, 0] view, the centre has distinct bars, whereas the [0, 1] view has a fuzzy region. The 3D reconstruction of a wide Z-disc from bovine neck slow muscle (Luther et al. 2002) was found to comprise a 6-layer Z-disc. With 6 layers of α-actinin crosslinking the overlapping actin filaments from adjacent sarcomeres this must form a relatively rigid Z-disc that should be most resistant to distortion during muscular activity.
3D structure of the nemaline rod Z-band
The 3D structure of the nemaline rod Z-band was investigated by Morris et al. (1990) in a classic paper in Z-band research. Stereo views of the 3D reconstruction and a schematic view of the model are shown in Fig. 8. As the appearance of nemaline rods in transverse view is the small-square type, this reconstruction gives insight into the architecture of such Z-discs. Morris et al. found that the crosslink comprises three parts, a central part that runs axially and two perpendicular parts that link neighbouring antiparallel filaments (Fig. 8f). They proposed that the axial part is composed of the rod part of two α-actinin molecules which link across to four actin filaments. Recently myotilin has been identified as a component of nemaline rods (Schroder et al. 2003), so there may be more components to fit in the 3D architecture of the nemaline and other Z-bands.
Loss of fixed-width in the Z-disc
As mentioned before, vertebrate striated muscle Z-discs have precisely defined widths for particular muscle types. To date we have identified widths that correspond to 2, 3, 4 and 6 layers of Z-link/α-actinin. There may be other Z-discs of different number of layers yet to be found. Z-bands with patches of two different widths within a single sarcomere were reported by Luther et al. (2003) and they were identified as 3 and 4 layer Z-bands. The cause for the variation within a single sarcomere is not certain but it may be a snapshot of transition of a fibre from one type to another.
Loss of Z-band width definition occurs in a variety of circumstances. As discussed earlier, nemaline myopathy is characterised by large isolated nemaline rods, but it also has regions of striated fibres with irregular width Z-bands (Fig. 8). In mice muscles deficient of muscle LIM protein (MLP; description later), the Z-bands in cardiac muscle were frequently irregular and widened (Knoll et al. 2002). Electron microscopy of human cardiac tissue from myectomy operations of hypertrophic cardiomyopathy patients sometimes shows irregular Z-bands that are often spindle shaped (author’s unpublished observations). Since cardiac patients are frequently quite old, we may ask whether the Z-disc defects are due to cardiac pathology or due to old age. Examination of the hearts in very old mice (nearly 2 years) has shown scattered regions with irregular or spindle shaped Z-discs (author’s unpublished observations). Zolk et al. (2000) have reported reduced levels of MLP in failing hearts and this reduction of MLP may also be the cause of irregular Z-bands observed in very old mice.
Massive loss of Z-disc integrity occurs following muscle injury (Jones et al. 2004; Lieber and Friden 2002). It is characterised by Z-line “streaming” in which Z-disc structure is disrupted and Z-disc material appears ripped out across a large part of a sarcomere. This occurs particularly after eccentric exercise. Z-line streaming is also common in several muscle disorders (Goebel 2002).
Important structural proteins in the Z-disc
Nebulin is an 800 kDa protein that runs along the length of the thin filaments (Labeit and Kolmerer 1995; Wang and Wright 1988). Although not fully understood, nebulin plays an important role in the assembly, structure and function of the Z-disc in skeletal muscle (McElhinny et al. 2003). In particular it may be that nebulin helps to define the thin filament length in many skeletal muscles (Littlefield and Fowler 2008; Pappas et al. 2008; Witt et al. 2006). In cardiac muscle there may be a miniscule amount of nebulin or possibly none (McElhinny et al. 2003) and this may be related to the variable lengths of thin filaments in cardiac muscle (Burgoyne et al. 2008). Nebulette is a 107 kd nebulin homologue present in the cardiac muscle Z-disc (Moncman and Wang 1995). During myofibrillogenesis nebulin appears in I-Z-I bodies, precursors of Z-discs, before thin filaments attain full length (McElhinny et al. 2003). Nebulin is composed of repeating 35-residue α-helical domains with the conserved motif SDxxYK (Labeit et al. 1991) labelled M1 to M185. Nebulin runs along the full length of the thin filaments with the C-terminus located in the Z-disc. A single nebulin module interacts with each actin monomer along the actin filament (Pfuhl et al. 1994). Along most of its length, the repeating modules are organised into super-repeats of seven domains and this is thought to reflect the binding of tropomyosin and troponin to the thin filament (Labeit and Kolmerer 1995). The super-repeat is absent from the 100 kDa region from module M162 to the C-terminus. Previously it was thought that nebulin terminated at the near edge of the Z-band and did not overlap the Z-disc width (Millevoi et al. 1998). However, a new study by Pappas et al. (2008) on chick skeletal myotubes showed that nebulin is required for the localisation of CapZ in the Z-disc; they found that domains M160 to M164 bind to CapZ and that the remainder of the C-terminus interacts with the Z-band. Therefore, they proposed a model in which a nebulin molecule binds along thin filaments up to the near Z-disc boundary, then moves to the end of a neighbouring antiparallel actin filament where it interacts with its CapZ terminus. It then continues along this filament to the distal edge of the Z-disc (Fig. 1a). As a consequence nebulin has the potential to contribute to the tension bearing role of the Z-disc.
The effect of nebulin depletion has been investigated by two groups (Bang et al. 2006; Witt et al. 2006). Nebulin-knockout mice were growth retarded at birth and 90% died within 2 weeks. The mice had normal levels of CapZ, but the myopalladin levels (another Z-disc component) were much reduced. Both groups found that the thin filaments were shorter by 15–20%. Witt et al. found no nebulin in cardiac muscle, contradicting suggestions that there may be a small amount present that regulates the thin filament length in some fashion. Both groups observed widened Z-bands and the nemaline rod phenotype. They suggested that impaired thin filament capping contributes to a severe nemaline myopathy pathology resulting from thin filaments growing beyond the normal Z-width.
Titin is a giant 3 MDa protein that spans half sarcomeres from the Z-disc to the M-band [see reviews (Granzier and Labeit 2004; Linke 2008)]. The C- and N-terminii span the full width of the M-band and Z-disc, hence consecutive titins form a continuous structure from one end of the sarcomere to the other. The N-terminus 90 kDa of titin is located at the Z-disc (Gregorio et al. 1998; Young et al. 1998). Of this, the N-terminal 30 kDa region comprising the Ig domains Z1 and Z2 is located at the distal end of the Z-disc and the 60 kDa region comprising the so-called Z-repeats spans the width of the Z-disc (Gregorio et al. 1998).
CapZ is a heterodimer that binds tightly and caps the barbed ends of actin filaments in the Z-disc. It also strongly binds a spectrin domain of α-actinin (Papa et al. 1999) and the C-terminus (domain M162) of nebulin (Pappas et al. 2008). The crystal structure of CapZ (Yamashita et al. 2003) showed the arrangement of the α and β subunits. The C-terminal domains (~30 residues) of both subunits have α-helical regions and Yamashita et al. (2003) propose that these regions act like tentacles for binding actin. To determine the 3D localisation of CapZ on actin filaments, Narita et al. (2006) undertook cryo-electron microscopy of CapZ-bound actin filaments. Their 3D images at 2 nm resolution show distinct features of CapZ. From the known atomic structures of CapZ and the actin filament, they modelled the binding on their cryo-em maps (Fig. 10d).
Z-band proteins involved in signalling and disease
For just over a decade, the Z-band has been a treasure trove for the biochemist/geneticist, with a multitude of new proteins being discovered. These have been exciting discoveries as mutations in the genes for these proteins lead to muscle diseases and cardiomyopathies. Some of the proteins have domains that are well known in biology, like the PDZ and LIM domains, so their importance was immediately realised. Excellent reviews on these proteins and their role in signalling and disease have been written (Clark et al. 2002; Faulkner et al. 2001; Frank et al. 2006; Pyle and Solaro 2004; Sheikh et al. 2007) and only a brief summary is included here.
For the newcomer it is a daunting prospect to understand all the new proteins. One could classify them by their binding partners; for example, several proteins bind α-actinin. However, the proteins have extensive interactions with each other and as Faulkner et al. (2001) state, they are not “islands unto themselves”. These interactions are illustrated schematically in the reviews above and in the review by Lange et al. (2006).
In a recent study, some of the Z-band proteins have been classified into three families, myotilin, FATZ and enigma (von Nandelstadh et al. 2009, and references therein). The myotilin family includes myotilin, palladin and myopalladin. These proteins have immunoglobulin domains and bind α-actinin, filamin and FATZ. The FATZ family includes FATZ (also called casarcin and myozenin) and their binding partners include myotilin, filamin, telethonin, α-actinin and ZASP. FATZ-1 and FATZ-3 occur in fast twitch muscles and FATZ-2 occurs in slow twitch and cardiac fibres. The enigma family of proteins is characterized by an NH2-terminal PDZ domain and nought to three LIM domains at the COOH terminal. PDZ domains are structural domains of 80–90 amino acids arranged in 5–6 β-strands and 2 α-helices and are involved in protein–protein interactions (Harris and Lim 2001). LIM domains are protein interaction domains of about 55 amino acids (Kadrmas and Beckerle 2004). They are composed of tandem zinc finger domains connected by a hydrophobic linker of two amino acids. Cypher/ZASP/Oracle is probably the most studied enigma member (Faulkner et al. 1999; Passier et al. 2000; Zhou et al. 2001). Cypher/ZASP may serve as a linker-strut by binding to α-actinin via its PDZ domain and may be involved in signalling as it binds protein kinase C via its LIM domains. Cypher-deficient mice die soon after birth and analysis of contracting and non-contracting (diaphragm) muscles suggests that cypher is essential for maintaining Z-band structure and muscle integrity (Zhou et al. 2001). Mutations in cypher lead to dilated cardiomyopathy and muscle myopathies now termed Zaspopathies.
Myofibrillar myopathy (MFM) is a muscle disorder especially relevant to the Z-disc. MFM is a morphologically distinct group of muscle pathologies of skeletal and cardiac muscle characterised by disintegration of the Z-disc and abnormal accumulation of proteins (Selcen and Engel 2004). It is caused by mutations in the genes for proteins involved in maintaining the structural integrity of the Z-disc. To date, these include desmin, αB-crystallin, myotilin and ZASP.
Muscle LIM protein (MLP) is a highly researched LIM only protein that has several binding partners including telethonin, α-actinin, Myo-D, N-RAP and β-spectrin (Gehmlich et al. 2008; Knoll et al. 2002). The MLP-telethonin-titin interaction was thought to be involved in mechanotransduction i.e. to be the elusive stretch sensor mechanism (Knoll et al. 2002). New research by Geier et al. (2008) using monoclonal antibodies against MLP suggest that MLP may not have specific a Z-band location as previously thought but has diffuse cytoplasmic location. Hence Gehmlich et al. (2008) suggest that MLP may be a downstream signal transducer in mechano-signalling cascades. The search for the stretch-sensor mechanism continues.
The image of the Z-disc as a sarcomere boundary marker passively involved in contraction has now moved more centre stage to that of a control watch tower. Teeming with proteins that can interact with each other, proteins with domains with established roles in biology for signalling and protein–protein interactions, the Z-disc is a fascinating assembly. There are important questions to address. What is the basis of the stretch sensing mechanism? What is the mechanism that goes awry in aging and disease that leads to loss of a fixed width in the Z-disc? Urgently needed now are high resolution tomographic studies that can identify the major members, actin and α-actinin, identify the recently solved structures of the titin Z1/Z2-telethonin complex and of CapZ and finally attempt to identify new members like myotilin and myopalladin.
Much of the work I have done on the sarcomere has been in collaboration with John Squire and I am greatly indebted to him. I have also collaborated with and had extensive discussions on the structure of the Z-disc with Ed Morris, John Barry, Tom Burgoyne, Roger Craig and Raul Padron. I would like to thank Cathy Timson for excellent technical help. I am grateful to John Squire, Roger Craig, Georgine Faulkner and Larissa Tskhovrebova for invaluable comments on the manuscript. I am grateful to the British Heart Foundation (Project PG/06/010) for their support of my research.
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