The Initial Steps of Myofibril Assembly: Integrins Pave The

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The starting point for assembly opinion Electron microscopy observations indicate that Z-disks begin as small, membrane- The initial steps of myofibril associated aggregates called Z‑bodies, which mature into Z-disks19. Correlating immuno- assembly: integrins pave the way fluorescent and electron microscopy images reveal that Z-bodies are the sites of α-actinin and titin localization (two of the earliest John C. Sparrow and Frieder Schöck Z-disk markers), which further demonstrates 20 Abstract | Myofibril assembly results in a regular array of identical sarcomeres in that Z-bodies are precursors of Z-disks . The first myofibrils are always observed striated muscle. sarcomere structure is conserved across the animal kingdom, which close to the membrane19,21,22, which indicates implies that the mechanisms of myofibril assembly are also likely to be conserved. that sarcomere assembly begins at the cell Recent advances from model genetic systems and insights from stress fibre cell periphery. Immunofluorescent staining biology have shed light on the mechanisms that set sarcomere spacing and the further located and defined these myofibril 14 initial assembly of sarcomere arrays. We propose a model of integrin-dependent precursors, which are called premyofibrils . Premyofibrils use all of the sarcomeric com- cell–matrix adhesion as the starting point for myofibrillogenesis. ponents except for non-muscle myosin II, which is incorporated first but then replaced Muscle development is a multistep process skele tal muscle thin filaments additionally with muscle myosin II, as they mature that starts with the specification of certain contain nebulin, a long protein that regulates into myofibrils. It was recently shown in cells as muscle precursors, or myoblasts the length of thin filaments1–5. In the middle cardiomyocyte tissue culture and in intact (see Glossary). This is followed by either of the sarcomere, M-line proteins crosslink hearts that myofibril assembly initiates from myoblast fusion, which generates syncitial, and anchor the thick filaments to each other. a premyofibril stage14,23. multi nucleated myotubes, or by direct The thick filaments occupy the sarcomeric Premyofibrils resemble actin stress fibres differ entiation into cardiomyocytes. Muscle A‑band, whereas the region in which thin fila- of non-muscle cells because they exhibit the cells then attach to the extracellular matrix ments do not overlap with thick filaments is same irregular α-actinin and non-muscle (ECM) and to other muscle cells before known as the I‑band. Actomyosin interactions myosin II periodicity. In addition, premyo - assembling myo fibrils. Muscle cells typically gen er ate contractile force and sarcomeres fibril periodicity is considerably shorter contain dozens of myofibrils, each consisting shorten owing to the sliding of the two fila- than in mature sarcomeres, α-actinin and of many sarcomeres, the smallest functional ment systems. Molecules of the giant protein non-muscle myosin II spots are identical in contractile unit of muscle. These are highly titin span the half-sarcomere with their size, and titin and I-bands are absent. All organized macromolecular complexes that amino termini at the Z-disk and their car- of these features are also observed in actin consist of myosin II-containing thick fila- boxyl termini at the M-line6,7. Each titin exits stress fibres24–27. The precise role of transient ments and actin-containing thin filaments. the Z-disk close to a thin filament, crosses the non-muscle myosin II incorporation is still The initial step of myofibril assembly is the I-band and then binds along a thick filament unclear. Loss of non-muscle myosin IIB (also formation of a regular array of sarcomeres. as far as the M-line. The I-band region of titin known as MYH10) in transgenic mutant These sarco meres later grow in width and in forms an elastic element, which connects the mice causes defects in heart sarcomeres. some cases in length, and eventually align Z-disk to the thick filaments1 (BOX 1). However, initial sarcomere assembly still and attach to each other and the sarcolemma. Myofibril termini attach to the skeleton occurs. It is interesting to note that one-half An important question in muscle different- at myotendinous junctions or are connected of non-muscle myosin IIB-knockout iation is how the striated muscle cell pro- end-to-end at intercalated discs in cardiac mice showed upregulation of non-muscle duces these myofibrils with such regular muscle. Heterodimeric integrins, which myosin IIA (also known as MYH9) in the arrays of sarcomeres. connect thin filaments to ECM ligands, are heart, which might partly compensate for neighbouring sarcomeres share a Z‑disk, the main structural and functional compo- the absence of non-muscle myosin IIB28. in which thin filaments are anchored and nents of myotendinous junctions. Peripheral crosslinked by dimeric actin-binding myofibrils are also laterally anchored to the Integrin adhesion sites initiate assembly. All α-actinin molecules1,2. Thin filaments ECM at the level of the Z-disk in both verte- immunofluorescent observations support consist of filamentous (F)-actin and the brates and invertebrates8–11 (FIG. 1). These the view that the accumulation of muscle tropomyosin–troponin complex. The heads adhesion sites are termed costameres, and, as myosin II in myofibrils is one of the final steps of myosin II proteins in thick filaments bind with myotendinous junctions, they consist of after premyofibril formation and titin recruit- to the F-actin of thin filaments to produce many components that are typically found in ment14,20,29. By contrast, integrins, α-actinin contraction, a process that is regulated by the integrin adhesion sites11–13. and the integrin adhesion site components tropomyosin–troponin complex. Thin fila- Here, we review the initial steps of myo- vinculin and talin are the first proteins that ments are polar owing to the inherent polarity fibril assembly before presenting an exten- can be observed in periodic patterning at the of the actin monomers in the F-actin core. sion of the premyofibril model for myofibril plasma membrane22,30. we propose that these The thin filament ends, which are known as assembly14,15. This model is supported by early integrin adhesion sites, which we term plus and minus ends because of the relative pioneering genetic studies in Drosophila protocostameres, serve as nucleation sites for rates of monomer addition during F-actin melanogaster and Caenorhabditis elegans and α-actinin accumulation, which then causes polymeriza tion, are capped by CAPZ and highlights an important role for integrins in the independent assembly of premyofibril- tropomodulin, respectively. Vertebrate myofibril assembly9,16–18. associated Z-bodies. Finally, the maturation

Box 1 | Sarcomere structure Assembly of thin filaments. The initial step in the formation of an actin filament is actin Actin Tropomyosin–troponin complex M-line nucleation, in which a few actin monomers Nebulin Z-disk combine to form a nucleus or ‘seed’ on a Tropomodulin Tropomyosin Thin filament template protein. It is on this seed that a new F-actin filament can then polymerize. ZASP The elongation of thin filaments has Titin been studied in cultured muscle cells and D. melano gaster flight muscle, and, in con- CAPZ Myosin Thick filament trast to actin filament elongation in vitro, occurs at the minus end through regulation α-Actinin by tropomodulin (REFS 36–39). However, little is known about the nucleation and I-band A-band I-band initial assembly of thin filaments. Recently, Sarcomere leiomodin, a protein that is similar in structure to tropomodulin, has been shown to Thin filaments of opposite polarity are crosslinked by -actinin and anchored on either side of the α Nature Reviews | Molecular Cell Biology Z-disk, which forms the boundary of the sarcomere (see the figure). Thin filaments consist of accelerate actin filament nucleation in vitro filamentous (F)-actin and the tropomyosin–troponin complex, which are required for the proper and is also crucial for myofibril assembly in 40 interaction of thin filaments with myosin. Thin filaments also contain nebulin, a vertebrate thin muscle cell culture . Leiomodin localizes to filament length regulator. Thin filaments are capped at the plus end by CAPZ and at the minus end by the minus end of thin filaments, close to the tropomodulin. The PDZ–LIM domain protein ZASP organizes the Z-disk. Thick filaments (bipolar M-line in mature myofibrils40. Small inter- myosin) are anchored at the M-line. The area that is spanned by thick filaments is known as the fering RnA knockdown of leiomodin A-band. The area on both sides of the Z-disk that is spanned by thin filaments that do not overlap with severely disrupts sarcomere assembly, but a thick filaments is known as the I-band. Titin molecules in vertebrates extend from the M-line to the periodic α-actinin arrangement along actin Z-disk and provide elastic support and anchoring of thick filaments in the middle of the sarcomere. filaments is preserved. Therefore, it is poss- Striated muscle cytoarchitecture and many of its components are highly conserved from jellyfish to ible that another actin nucleator is involved humans68. Mutations in many costamere and sarcomere components cause myopathies, in particular in this process. Formins, which are proces- actin, myosin II, titin, α-actinin, nebulin family members, PDZ–LIM domain family members, troponin, tropomyosin, integrins, desmin and focal adhesion kinase2,69,70. Figure is modified, with permission, sive nucleators that remain associated with from REF. 71  (2004) Birkhauser. the plus end, might be good candidates for the initial actin filament polymerization in muscle cells. of the Z-bodies forms the Z-disks. As differ- ZASP is required for Z-disk assembly and Intriguingly, in vitro, integrin adhesion entiation proceeds, many myofibrils are the maintenance of muscle attachments. sites from non-muscle cells have recently displaced to the interior, and the proto- ZASP has been placed genetically down- been shown to promote actin filament costameres mature into costameres in sub- stream of integrins, because it mislocalizes nucleation through the formin diaphanous41. sarcolemmal myofibrils and inter-Z-disk in the absence of integrins, and upstream These actin filaments are anchored with bridges between internal myofibrils. of α-actinin, because it is required for the their plus ends at the integrin adhesion site, Many lines of evidence point strongly to recruitment of α-actinin to the Z-disk32. which is the same orientation that is found at integrin adhesion sites as the starting point α-Actinin probably then recruits titin as muscle Z-disks. Furthermore, diaphanous- for myofibril assembly in vivo. In mice, it has separate titin- and ZASP-binding mediated polymerization of actin filaments D. melanogaster and C. elegans, genetic sites33. Z-disk-localized titin has also been from integrin adhesion sites occurs during studies have shown that integrins and their placed genetically downstream of ZASP32. actin stress fibre formation in tissue culture25. ECM ligands are required for sarcomere The genetic evidence from several model Stress fibres that have been polymerized from assembly and Z-disk formation9,16,18,31. More organisms argues strongly that myofibril different integrin adhesion sites can fuse importantly, integrins are found genetically assembly starts at the plasma membrane at laterally, probably through the alignment of upstream of all of the other components integrin adhesion sites. Indirect evidence antiparallel actin filament arrays and their that are required for myofibril assembly in from muscle cell differentiation in tissue connection by actin-crosslinking proteins. C. elegans and D. melanogaster. In C. elegans, culture supports this notion: inhibition of non-muscle myosin II incorporates both mutations in integrins and their ECM ligand integrins with function-blocking antibod- into newly polymerized stress fibres, perhaps perlecan have the most severe phenotypes. ies or the inability to form properly sized by re orientating actin polarity, and into Integrins are also required to recruit all of integrin adhesion sites inhibits myofibril laterally fused stress fibres25,42. Diaphanous the other proteins that are found at muscle assembly34,35. also increases non-muscle myosin II stability attachment sites and dense bodies (the and activity, thereby contributing to acto- C. elegans equivalent of Z-disks)17. Similarly, Assembly of filament systems myosin contraction of the actin cable at in D. melanogaster, integrins are the most In vitro, both myosin and actin can each the leading edge during dorsal closure in upstream component in muscle attachment polymerize into filamentous structures that D. melanogaster embryos43. The ability of sites and are required for Z-disk assembly16,18. resemble thick filaments and the F-actin core diaphanous to both polymerize actin fila- Another link between integrins and of thin filaments. In vivo, these pro cesses ments and, in an unknown manner, regulate Z-disk assembly was recently revealed by are regulated and involve many associated non-muscle myosin II activity might place it a study of the Zasp-mutant phenotype proteins. Here, we briefly discuss the initial in a central position in the initial assembly of in flies32. The PDZ–LIM domain protein assembly of thin and thick filaments. premyofibrils.

Assembly of thick filaments. non-muscle Costamere Connective tissue or other myofibres Sarcolemma myosin II can probably self-assemble into (cell membrane) Integrin short bipolar minifilaments that are found in the cortical actin cytoskeleton and stress Nucleus fibres, but recent work has shown that the Integrin assembly of muscle myosin II into thick filaments requires chaperones44. These chaperones are essential for thick filament assembly Actin T as mutations in zebrafish uncoordinated 45b endon or tendon Titin (unc45b) or heat-shock protein 90a (Hsp90a) Myosin completely lack thick filaments45,46. notably, this mechanism is just as important for main- Mitochondrion Inter-Z-disk bridge tenance and regeneration. Although these chaperones localize to the Z-disk in fully cell developed myofibres, they relocalize to the A-band during muscle damage. Here, reassembly of nascent myosin filaments occurs47.

M-line The role of contractility Z-disk Sarcomere Tissue culture studies show that there is a Myotendinous junction requirement for contractility in myofibril Figure 1 | a schematic overview of striated muscle. A sagittal section through a skeletal myofibre assembly. Inhibition of contractility in skele- is shown, which depicts two myofibrils and their connections toNa eachtur eother Revie andws | theMol sarcolemma.ecular Cell Biolog Onlyy tal muscle by channel blockers, such as the three main structural components of sarcomeres — actin, myosin and titin — are shown. Myofibrils tetrodotoxin, nifedipine or verapamil, causes are aligned laterally at the level of the Z-disk. At least in vertebrates, myofibrils are connected to each myofibril disassembly48. Similarly, inhibi- other and to costameres by intermediate filament proteins (light blue) and other cytoskeletal proteins tion of cardiomyocyte contractility causes (yellow circles). At the myotendinous junction, transmembrane integrins connect myofibrils through the disassembly of costameres49. washing adaptor proteins (green ovals) to the tendon matrix (orange ovals). costameres connect Z-disks to the out these chemicals or stimulating contrac- surrounding connective tissue or other myofibres. in addition to integrins, dystroglycans (not shown) have an important role in transmembrane linkage at costameres. How M-lines are connected to each tility with electric pulses or isoproterenol other and the sarcolemma is unknown. cardiomyocytes have a similar basic structure, although instead re-establishes and accelerates myofibril and of a myotendinous junction they are connected end-to-end through cadherin-mediated adhesion at 30,48,50 costamere assembly or reassembly . the intercalated disc. Figure is modified, with permission, from REF. 1  (2002) Annual Reviews. It is particularly important to note that increases in contractility and myofibril assembly progress in parallel30. be anchored to protocostameres or terminal force is applied58. Growth and alignment of Z-bodies. Once a continuous array is protocostameres or Z-bodies may similarly Coordination of Z-disk assembly. Live established and muscle myosin II is be force-dependent processes, given that imaging of cardiomyocyte Z-disk assembly in corporated, Z-bodies will align in the they contain many of the same proteins as shows that α-actinin, which is initially evenly area of the future Z-disk. Sarcomere focal adhesions. distributed, aggregates into randomly dis- spacing then becomes increasingly regular tributed clusters. These clusters then become owing to the action of the contractile forces Muscle contractility and integrins. The regularly spaced by aligning in the area of the that the myofibrils experience, through the contractions that are required for sarco- future Z-disk and eventually coalesce later- signalling of elastic element proteins such mere formation are linked to integrin func- ally into Z-disks51,52. Many studies indicate as titin, along with thin and thick filament tion. The re-establishment of contractility that the transition from initial premyofibrils length-regulation mechanisms. in cardiomyocytes that have disassembled (which have poor regularity of α-actinin their myofibrils correlates with periodic staining) to myofibrils that have regular spac- Focal adhesion assembly. In tissue culture β integrin and vinculin accumulation49. ing of Z-bodies occurs almost simultaneously of non-muscle cells, force-dependent The first myofibrils that assemble de novo along the entire length or long sections of a maturation of integrin adhesion sites is well on electrical stimulation of contractility are muscle cell22,51,53. The overall process seems established. Small adhesion sites can mature found immediately below the sarcolemma30. to be similar in all of the systems that have into larger, more organized structures called Myofibrillogenesis can be accelerated by been studied so far: in C. elegans body wall focal adhesions, and the accumulation of plating on collagen and inhibited by plating muscles, in D. melanogaster flight muscles focal adhesion proteins correlates with local on RGD peptide, an integrin-binding site and in quail cardiomyocytes. traction forces54,55. Inhibition of actomyosin that is found in several extracellular The mechanism for the formation of contractility disassembles existing focal proteins. Collagen strengthens cell–matrix regular sarcomeres functions quickly and adhesions56,57. Most importantly, externally attachment, whereas the RGD peptide, over a long distance in a muscle cell. The applied force stimulates the growth of focal which binds to and blocks the ECM-binding contractile forces that are required for regu- adhesions, even at the level of individual domains of integrins, disrupts stable cell– lar sarcomere formation function across the focal adhesions58,59. Diaphanous has a cru- matrix attachment. This further indicates muscle cell and are probably responsible cial role in this process, because activated that integrin adhesion sites have a role in the for the formation of regular sarcomeres. diaphanous is necessary and sufficient for contractility-dependent assembly of myo- During this process, premyofibrils need to the growth of focal adhesions as external fibrils30. Finally, C2C12 skeletal myotubes

1 Integrin adhesion site protein, such as immunoglobulin, fibro- (protocostamere) nectin and kinase domains and the so-called Actin filaments 2 PEVK regions, are associated with different α-Actinin Minus end Plus end regions of the half-sarcomere — the Z-disk, the I-band and the A-band7. Titin isoforms vary in the number of repeated domains, and therefore protein length, in each of 3 these regions. This is thought to deter- mine I-band and A-band lengths. The titin n terminus is bound to α-actinin, actin and

Pr other proteins in the Z-disk. The extended Non-muscle myosin II emy 4 polypeptide chain exits the Z-disk close to a ofibril thin filament but then traverses the I-band to bind along the length of a thick filament (FIG. 1). As a link between the Z-disk and Titin Non-muscle myosin II 5 thick filaments, titin probably acts to organize the regular interdigitation of the thick and thin filaments in the myofibrillar lattice. Z-body This interaction is probably instrumental Muscle myosin II in the accumulation or assembly of thick 6

ation filaments, and its known association with

tur muscle myosin II isoforms might promote the replacement of non-muscle myosin II 7 in the early sarcomere. In addition, titin is well established as an elastic protein of the sarcomere, acting as an extensible ‘spring’ to absorb contractile forces and centre the ctility-dependent ma array of thick filaments in the sarcomere.

ontra Although mechanically its function is C passive, there is increasing evidence that Z-disk titin functions as a strain sensor that is 8 involved in signalling in the muscle cell. The C-terminal kinase domain of titin shows a strain-sensitive unfolding that is required for its ligand to gain access to the active site61. Other elastic domains of titin in the I-band have been proposed to function as a ‘tensiometer’62. Figure 2 | Model of myofibril assembly. integrin adhesion sites (protocostameres) that form at ran- dom positions along the muscle cell polymerize actin filamentsNa tur(stepse Re vie1,2).ws For | Mol simplicity,ecular Ce llonly Biolog twoy actin filaments per adhesion site are shown. Overlapping actin filaments of opposing polarity fuse Invertebrate titin. Titin mutations abolish 63–65 through α-actinin-mediated crosslinking (step 3). Laterally fused antiparallel actin filaments incor- sarcomere assembly in different species . porate non-muscle myosin ii, which displaces α-actinin (step 4). Note that several non-muscle Therefore, titin clearly has many important myosin ii bipolar filaments can be incorporated, depending on the number of actin filaments in the roles in sarcomerogenesis. Although titin or stress fibre. Protocostameres recruit titin and more α-actinin, thereby giving rise to electron-dense its homologues — Sallimus (also known as Z-bodies (step 5). Muscle myosin ii bipolar filaments are then incorporated, perhaps with the help of D-Titin) in D. melanogaster 63,66 and twitchin titin (step 6). correct spacing is mediated by both titin and further length maturation of thin fila- in C. elegans67 — are present in all striated ments. Z-bodies that are now connected to Z-bodies on the other side align longitudinally in the muscles, there are variations in function, future Z-disk plane through contractility-dependent maturation (step 7). Z-bodies then coalesce which suggests that the model presented laterally into Z-disks (step 8). Although not depicted, the distance between two protocostameres in steps 1–5 probably varies substantially. The final sarcomere length is mostly set from step 6 onwards, above does not apply to different species. although subtle modulations might occur, such as thin filament elongation. In D. melanogaster, although the sarcomeres of some muscles, including the flight muscles, are 50% longer than vertebrate sarcomeres, there is no gene that can encode develop myofibrils only when they adhere to Titin as a sarcomere template a protein of the length of vertebrate titin. substrates of tissue-like stiffness. Myotubes All vertebrate striated muscles contain the D. melanogaster titin homologues lack the sense an increase in substrate stiffness by giant protein titin. Proposals that titin could C-terminal kinase domains but they show increasing the strength of cell–matrix adhe- act as a one-dimensional template and ruler extensive sequence homology to the sions and therefore the size of integrin for the half-sarcomere are consistent with vertebrate n-terminal domains. adhesion sites34. This supports the notion observations that titin is present during the Immunogold studies66 show that these that properly sized basal integrin adhesion earliest stages of myofibril assembly20,22,60. D. melanogaster proteins extend from inside sites are starting points of myofibrillogenesis. Furthermore, specific domains of the the Z-disk, through the I-band, to the distal

glossary probably explains the simultaneous formation of evenly spaced Z-bodies along the A-band Myotendinous junction length of a muscle cell. Each sarcomere The area of the sarcomere that is spanned by thick An integrin adhesion site that connects myofibre termini to along an array will experience the same filaments. tendons or tendon cells. tension; local extension of elastic filament Cardiomyocyte Premyofibril proteins (for example, titin and Sallimus) A fully differentiated heart muscle cell with a single A myofibril precursor that partly resembles non‑muscle cell could signal to ensure the even length nucleus. stress fibres. Premyofibrils contain alternate sarcomeric or assembly of thick and thin filaments, α‑actinin and non‑muscle myosin II that originate at Costamere integrin‑mediated Z‑body precursors, called thereby ensuring an optimal spacing of An integrin adhesion site that connects the lateral protocostameres, in muscle cells. Z-bodies or Z-disks. Vertebrate titin is prob- sarcolemma to the surrounding connective tissue at the ably involved, along with other processes, in level of the Z‑disk. Protocostamere determining sarcomere length, as it reaches A small integrin adhesion site and Z‑body precursor, to from the Z-disk to the M-line. Eventually, Focal adhesion which the initial cortical premyofibrils are anchored. An integrin adhesion site on the basal side of tissue culture the arrays of Z-bodies will coalesce laterally cells to which stress fibres attach. Sarcolemma to form the Z-disks of myofibrils. The muscle cell membrane. Our model should provide a good start- I-band ing point for designing experiments that will The area on both sides of the Z‑disk that is spanned Sarcomere by thin filaments that do not overlap with thick The smallest contractile unit of muscle, which is bordered allow it to be further refined and altered. filaments. by Z‑disks. One prediction worth investigating is whether protocostameres precede Z-bodies Integrin adhesion site Stress fibre in all myofibrillar assembly systems. Any cell–matrix adhesion that is mediated by integrins, for A graded polarity actin filament bundle that has alternate example, a focal adhesion, a costamere or a myotendinous α‑actinin and non‑muscle myosin II, and is found in John C. Sparrow is at the Department of Biology junction. non‑muscle tissue culture cells. (Area 10), University of York, York, YO10 5YW, UK. Frieder Schöck is at the Department of Biology, McGill Intercalated disc Z-body University, 1205 Dr Penfield Avenue, Montreal, A cadherin adhesion site that connects myofibre termini An electron‑dense Z‑disk precursor that is assembled in Quebec H3A 1B1, Canada. end‑to‑end in cardiomyocytes. association with protocostameres. It contains Z‑disk proteins, including ZASP, sarcomeric α‑actinin and titin. e-mails: [email protected]; Myoblast [email protected] A muscle precursor cell with a single nucleus. Z-disk doi:10.1038/nrm2634 The boundary of the sarcomere, at which antiparallel thin Published online 4 February 2009 Myotube filaments are anchored to ‑actinin and many other α 1. Clark, K. A., McElhinny, A. S., Beckerle, M. C. & A multinucleated skeletal muscle cell. characteristic Z‑disk proteins. Gregorio, C. C. Striated muscle cytoarchitecture: an intricate web of form and function. Annu. Rev. Cell Dev. Biol. 18, 637–706 (2002). 2. Frank, D., Kuhn, C., Katus, H. A. & Frey, N. The regions of the thick filaments. This would is also found in stress fibres in non-muscle sarcomeric Z-disk: a nodal point in signalling and allow all titins to function as sarcomeric cells. Protocostameres will mature and disease. J. Mol. Med. 84, 446–468 (2006). 3. Bang, M. L. et al. 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