PERSPECTIVES 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 NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 10 | APRIL 2009 | 293 © 2009 Macmillan Publishers Limited. All rights reserved PERSPECTIVES 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 struc- ture 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.