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Muscle assembly: a titanic achievement?
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Muscle assembly: a titanic achievement? Carol C Gregorio*, Henk Granzier†, Hiroyuki Sorimachi‡ and Siegfried Labeit§ The formation of perfectly aligned myofibrils in striated muscle represents a dramatic example of supramolecular assembly in eukaryotic cells. Recently, considerable progress has been made in deciphering the roles that titin, the third most abundant protein in muscle, has in this process. An increasing number of sarcomeric proteins (ligands) are being identified that bind to specific titin domains. Titin may serve as a molecular blueprint for sarcomere assembly and turnover by specifying the precise position of its ligands within each halfsarcomere in addition to functioning as a molecular spring that maintains the structural integrity of the contracting myofibrils. Addresses *Departments of Cell Biology and Anatomy, The University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724-5044, USA; e-mail: [email protected] †Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA, 991646520, USA; e-mail: [email protected] ‡Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; e-mail: [email protected] §European Molecular Biology Laboratory, Meyerhofstrasse 1, PO Box 102209, 69012 Heidelberg, Germany; e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:18–25 http://biomednet.com/elecref/0955067401100018 © Elsevier Science Ltd ISSN 0955-0674 Abbreviations FNIII fibronectin type III Ig-domain immunoglobulin-like domain KSP Lys–Ser–Pro PEVK Pro, Glu, Val, Lys SH3 Src-homology-3-like domain
mechanisms may have developed, since the thick filaments in their obliquely striated muscles are much longer than those in vertebrates, and no molecule of titin-like size has yet been identified. Several previous reviews have focused on the general properties, isoforms and order of assembly of the known sarcomeric components [2–4]. Here we emphasize the more recent developments in our knowledge of the possible roles that titin has in myofibril assembly. We first discuss the molecular structure of the titin filament and its detailed layout within the sarcomere. Next, we review progress in identifying specific protein ligands of titin. Finally, we speculate on possible mechanisms involved in orchestrating myofibril assembly and maintenance.
Molecular structure of the titin filament A decade ago, there was much resistance to the idea that a eukaryotic cell could translate megadalton-sized ‘titanic’ polypeptides; however, it is now firmly established that this is possible as a full-length titin cDNA was isolated that encodes a polypeptide of 27,000 residues [5]. A single gene locus for titin was located on the long arm of chromosome 2 in humans (e.g. [6•]). This gene is transcribed to produce a full-length mRNA of up to 100 kilobases, which encodes a titin polypeptide of up to 3.0 MDa.
The sarcomere is the contractile unit of striated muscle. Its formation is a unique example in biology where thousands of molecules assemble into a supramolecular structure with almost crystalline order. As a result of this precise assembly, many different classes of proteins function together to efficiently convert the molecular-level movements produced by actin and myosin into macroscopic movements of muscle.
The titin protein is extensively modular in structure (see Figure 2). It is composed of 132 copies of 100-residue repeats belonging to the fibronectin type III (FNIII) superfamily, and up to 166 copies of 90–95 residue repeats from the immunoglobulin (Ig) superfamily. Together, the Ig and FNIII repeats account for 90% of titin’s mass. Analysis of the three-dimensional structure of selected Ig and FNIII repeats from the I-band, A-band and M-line region (see Figure 1) has revealed that all of the repeats form stable globular domains composed of seven antiparallel β-sheets ([7•,8••], and references therein). The titin filament can therefore be thought of as a microscopic necklace made up of about 300 pearls (the Ig and FNIII domains), which extends across the entire half-sarcomere in both skeletal and cardiac muscle.
It has been speculated that, in vertebrates, the giant filamentous protein titin (also known as connectin), the only known structure that spans the entire half-sarcomere, may act as a molecular blueprint to direct sarcomere assembly (e.g.[1]; see Figure 1). In this role, titin would coordinate the precise assembly of the structural, regulatory and contractile proteins within the sarcomere, a prerequisite for efficient muscle contraction. In the invertebrate Caenorhabditis elegans, different assembly
The Ig and FNIII repeats are arranged in different patterns throughout the molecule (see Figure 1 and below for discussion). About 10% of titin’s mass is organized in nonrepetitive sequences which are situated between the Ig and FNIII repeats. A total of 17 such interdomain insertions are present, 16 of which have no significant homology to each other, or to other known protein sequences. One interdomain insertion that is located close to the carboxy-terminal end of titin is related in
Introduction
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Figure 1 I-band Z-disc Adjacent
A-band
Z-disc
Thick filament
M-line
Pi
Titin
Thin filament
sarcomere
NPi Pi
Pi
Titin kinase
Pi
-N
Pi
-C
Pi Pi
Pi
N
I-band
-N
Pi
T-cap
PEVK Pi Pi Pi
Pi Pi
Pi Pi Pi Pi
Phosphorylation motif
Nebulin Pi Pi Pi
C-protein
Pi
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-N C-
-C Pi
Pi Pi
Adjacent sarcomere
SH3 -C
Current Opinion in Cell Biology
Schema of a skeletal muscle sarcomere (a single contractile unit) with its A-bands that contain myosin thick filaments and I-bands that contain actin thin filaments. In the Z-lines, overlapping thin filaments from opposite sarcomeres are anchored, whereas thick filaments from opposite half-sarcometers are anchored in the M-lines. The layout of the four filament systems (thin, thick, titin and nebulin filaments) are shown. The amino-terminal regions of titin molecules from adjacent
sarcomeres fully overlap in Z-lines [10••]; the carboxy-terminal regions of titin molecules from opposite half-sarcomeres fully overlap in the Mlines [12]. In myofibrils composed of many sarcomeric units, the titin filaments (via their specific ligands) form a continuous filament system. The carboxy-terminal ends of nebulin molecules insert into the Z-disc [14••]; their amino-terminal ends are located at, or in close proximity to, the pointed ends of the thin filaments.
sequence to a serine/threonine kinase domain and is referred to as the ‘titin kinase domain’.
A-band titin
Layout of the titin filament in sarcomeres Initial immuno-electron microscopy studies, with a set of 10 nonrepetitive anti-titin monoclonal antibodies, revealed that a single molecule of titin extends from the Z-line to close to the M-line region [9]. More detailed information on the layout of the titin filament within the sarcomere has been obtained recently by immuno-electron microscopy studies using antibodies raised against specific bacteriallyexpressed domains of titin (see Figures 1 and 2). Z-disc titin
The labeling patterns obtained show that the amino-terminal 80 kDa of titin (or less, depending on the isoform) spans the entire Z-line region [10••]. Titin filaments from opposite sarcomeres therefore overlap within the Z-lines.
The carboxy-terminal 2 MDa of titin are located in the Aband; this region of titin is rendered inextensible by its tight association with the myosin thick filament. It contains two types of super-repeats, both consisting of FNIII and Ig repeats arranged into distinct patterns [5]. Within the central portion of the thick filament, seven FNIII and four Ig domains form a pattern, which is repeated 11 times and referred to as the 11-domain super-repeat (Figure 2). This super-repeat structure appears to correspond with the presence of the 11–43 nm crossbridge repeats of the thick filaments within the C-zone of the A-band; therefore, the 11 titin super-repeats are also referred to as ‘C-zone A-band titin’. More towards the amino-terminus within the A-band, five FNIII and two Ig repeats are arranged into a pattern that is repeated seven times. This part of the titin filament is referred to as ‘D-zone A-band titin’ (see Figure 2). M-line titin
I-band titin
Between 800 kDa and 1.5 MDa of titin are located within the I-band, depending on the particular isoform (see below). The central section of I-band titin — corresponding to its elastic region — is formed by tandemly arranged Ig repeats which are contained in two separate blocks, the proximal and the distal tandem Ig segments [11•]. Both of the tandem Ig groups are separated by up to 2200 residues that are unusually rich in P (proline), E (glutamate), V (valine), and K (lysine). This segment from the central I-band titin is referred to as the ‘PEVK domain’ [5].
The M-line region of titin corresponds to ~200 kDa of the final carboxy-terminal region of the protein. The kinase domain of titin is located at the periphery of the M-line region [12]. As in Z-lines, where titin filaments from opposite sarcomeres fully overlap [10••], titin filaments from opposite half-sarcomeres fully overlap within the M-line region [13••]. The overlapping ends are interconnected by their binding to other sarcomeric components (see below). Titin filaments with opposite polarity overlap in both the Z-lines and M-lines, forming a contiguous system within myofibrils (Figure 1).
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Figure 2
Half I-band Z-disc Tandem Ig region
PEVK-segment
NH 2 N2-A
P1
Half A-band A/I junction
P-zone
C-zone 11domain super-repeat
D-zone 7domain super-repeat
M-line P1
COOH Kinase domain Current Opinion in Cell Biology
Titin domains Ig-module
Titin ligands : :(by yeast two-hybrid) PEVK-segment
FN-module
Z-repeats
Unique sequence
Splice site
α-actinin
Calmodulin
p94
Titin-cap/ telethonin
Titin ligands: (by protein biochemical studies) C-protein P1
Myomesin Myosin
Phosphorylation site
Molecular structure of the titin filament, and location of differential splicing sites that result in different titin isoforms. Above the corresponding titin domains, blocks indicate ligands that have been
identified by yeast two-hybrid studies. Below, blocks indicate putative titin ligands identified by in vitro studies using expressed domains (see text).
Comparison of the titin and nebulin filament layouts: functional implications
Z-lines vary in width in different tissue types (for reviews see [17•,18]). Interestingly, both Z-disc titin and nebulin contain differentially expressed repeat families, referred to as Z-repeats. We speculate that together, the differential expression of nebulin’s carboxy-terminal and titin’s amino-terminal Z-repeats may account for the variability of vertebrate Z-lines. Regulating the amount of titin and actin filament overlap in the Z-line and the degree of Z-line insertion of nebulin and tropomyosin is likely to be physiologically important as the width of the Z-line is likely to be correlated to the mechanical properties of muscle; fast and slow-twitch fibers have narrow and wide Z-lines, respectively.
A fourth structural filament system of striated muscle is formed by nebulin, an 800 kDa thin-filament-binding protein specific to skeletal muscle. This protein is sugegsted to function as a molecular ruler to specify the length of thin filaments. The SH3-containing carboxy-terminal domain of a single molecule of nebulin inserts into the Zdisc, associate with the thin filament along its length. The amino-terminus whereas the central regions of nebulin locates in close proximity to the pointed (free) ends of the thin filaments (see Figure 1). In cardiac muscle, nebulette — a 109 kDa nebulin-related protein, sharing extensive structural homology with nebulin’s Z-disc (carboxy-terminal) region — appears to substitute for the Z-disc integrative function of nebulin (see below), but not to share a role in thin filament specification, as it is predicted to extend along only ~25% of the thin filament [14••,15]. In contrast to actin and titin, which span the entire Z-line, nebulin filaments appear to insert only within the periphery of the Z-line [14••]. Similarly, polymers of tropomyosin, which coil around and stabilize actin filaments, are predicted to be absent from the central Z-disc region of the thin filament, as α-actinin, a major structural component of the Z-line, inhibits tropomyosin binding to actin filaments [16]. It is possible that the amino-terminal region of titin may specify the principal amount of thin filament overlap, while the carboxy-terminal region of nebulin (or nebulette) may specify where tropomyosin binding on the actin filament terminates.
Interaction of titin with other sarcomeric components Here, we describe the proteins that interact with titin — starting from its amino-terminal Z-disc to its carboxy-terminal M-line end — and identify their potential binding sites in human titin sequences (see Figure 2). Interactions of titin’s Z-disc region
Using the yeast two-hybrid system to search for binding partners of the amino-terminal Z-line portion (~80 kDa) of titin, three distinct interactions have been identified. First, the extreme amino-terminal titin Ig repeats Z1 and Z2 (residues 1–200) bind to a novel Z-line protein [10••,19], which we suggest calling the ‘titin cap’ (T-cap) [10••]. Both the Z1 and Z2 repeats are required for binding. Further away from the amino terminus, the Z-disc titin residues 450–750 contain up
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Figure 3
Z-disc 50nm
50nm *
*
*
Zr1
Zr7
Z-repeats COOH
Zr7
*
*
Zr1
*
* Zr1
*
To M-line
* Zr7
To pointed end
NH2
Current Opinion in Cell Biology
Nebulin: SH3-domain
Titin: Ig-domains
Z1/Z2
Z-repeats *(differentially expressed)
Nebulin repeats *(differentially expressed)
Unique sequence
Ser-rich domain
Titin-cap protein
Actin (thin) filament decorated with tropomyosin
Terminal α-actinin?
EM mapped epitope
Overview of the layout of titin, nebulin and thin filaments in Z-discs from skeletal muscles. Titin: the amino-terminal region spans the Z-disc, so that titin filaments from neighboring sarcomeres fully overlap within the Z-lines [10••]. Within the Z-disc, titin filaments connect to α actinin Z-filaments at
multiple sites which then cross-link the titin and thin filaments [10••,21••,22••,23••]. Nebulin: filaments insert ~30 nm from the center of the Z-disc [14••]. Arrows indicate the location of the titin and nebulin epitopes that were mapped by immuno-electron microscopy.
to seven copies of a 45-residue repeat, each of which is referred to as a titin Z-repeat [20]. In vitro studies indicate that the Z-repeats bind the extreme carboxy-terminal 10 kDa of α-actinin — a major component of Z-lines that links the antiparallel actin filaments from adjacent sarcomeres [10••,21••,22••]. Additionally, the Z-line titin residues 750–826 (which are distinct and just carboxy-terminal to the Z-repeats) may contain another binding site for α-actinin but in this case the interaction may occur with α-actinin’s spectrin-like repeat domains (R2–R3) [23••] in the central rod.
might directly or indirectly involve T-cap, while the center region of α-actinin directly connects — via its spectrin-like domains R2-R3 — to titin Z/I junction residues. Inside the Z-line, α-actinin connects titin filaments from opposite sarcomeres by directly cross-linking Z-repeats. Unknown linker molecules may also be involved. A schema of the layout of the titin, nebulin and thin filaments in the Z-line is shown in Figure 3.
From what is known about the in vitro binding properties of Z-line titin, the following speculative model emerges. Residues 1–200 (Z1 and Z2) reside in the periphery of the Z-line of the sarcomere adjacent to the one in which the titin filament is extended, where they mark the edge of the Z-line region (‘Z-line titin edge residues’). Titin residues 200–750 (‘Z-line titin integrative residues’) span the Z-disk, whereas residues 750–826 are located at the Z-line junction with the I-band (‘titin Z/I junction residues’). Z-line titin edge and Z/I junction residues of overlapping titin filaments from neighboring sarcomeres may be cross-linked via unique Z-filaments. Cross-linking of the titin edge residues
Interactions of titin’s I-band
Near the Z-line, titin filaments associate with actin (thin) filaments, as indicated by increased titin extensibility after removal of actin by gelsolin extraction [24•–26•]. It is unclear which titin motifs mediate this interaction. To date, no splicing variants have been identified in this region, suggesting that the titin–actin interaction is required in all muscle types. The lateral association of titin with the thin filaments may straighten the otherwise folded titin molecule and thread it correctly into the Zdisc where it is organized into a highly ordered tetragonal lattice, cross-linked by α-actinin. Whether titin–actin interactions occur elsewhere in the I-band is unresolved, as a weak interaction that affects Ig-domain unfolding in
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cardiac muscle is suggested by some studies [26•], but not by others [24•]. Within the central I-band region (and also within the M-line region) of titin, binding sites are found for the muscle-specific calpain protease p94 [27]. The p94 binding motifs are excluded in some titin isoforms by exon-skipping [5,28]. The biological significance of the binding of p94 to titin and differential expression of the p94 binding sites is unknown. A-band titin interactions
The eleven-domain super-repeats contain multiple domains that bind to the tail portion of myosin, as well as to the thick-filament-associated protein, C-protein (MyBP-C) [29,30] which is organized transversely in A-bands. Phosphorylation of this protein may modulate contraction. Ig domains within each titin super-repeat have been identified that bind in vitro to the carboxy-terminal region of C-protein [31]. This interaction may explain why carboxy-protein associates with the thick filament at up to 11 positions in situ. The carboxyl terminus of C-protein also binds to the myosin tail [32]; therefore, within the C-zone of the A-band, titin may specify the number and location of myosin and C-protein molecules that are incorporated into thick filaments. Titin’s FNIII repeats are restricted to the A-band, whereas the Ig repeats are present throughout the molecule; therefore, titin’s FNIII repeats may represent a family of myosin-tail-binding motifs. The three-dimensional structure of a titin FNIII domain reveals the presence of two polyproline type II helices on one surface of the FNIII domain [8••]. In this regard, it may be relevant that titin’s A-band FNIII domains are generally proline-rich and that the positions of the proline residues are conserved. Future studies are needed to address whether the polyproline type II helix motifs act as weak myosin-binding motifs functioning to tether the titin to thick filaments. It is likely that titin interacts with myosin along the whole of the thick filament and not only in the C-zone, as at a wide range of sarcomere lengths, A-band titin epitopes remain at the same location relative to the M-line (e.g. [1,9,33,34••]). M-line titin interactions
Within the M-line region of the A-band, the titin Ig-repeat domain M5 has been found to bind myomesin in vitro [13••]. M5 is adjacent to four tandem copies of Lys–Ser–Pro (KSP motifs in the single letter code for amino acids) that are potential substrates of an unknown KSP-directed serine/threonine kinase [35]. Possibly, KSP phosphorylation may regulate titin–myomesin interactions [13••]. Together, C-protein and myomesin might function in the lateral registration of thick filaments. A caveat of many of the above studies is that they are mostly based upon in vitro binding data. Future studies will be required to address the functional role of titin’s interactions using in vivo models. So far, only the functional role of Z-line titin has been investigated by
dominant-negative transient expression studies in cultured myocytes. Overexpression of the amino-terminal titin Z1-Z2 domain, its ligand T-cap [10••,19], the first 362 residues of titin [36], the entire Z-disc region of titin [37] or a carboxy-terminal truncated fragment of αactinin (missing it’s binding site for titin) [38••] all result in the disruption of myofibrils. Thus, the association of titin filaments with Z-discs, via their interaction with Tcap and α-actinin, appears to be critical for the assembly and maintenance of myofibril structure. It is warranted to extend this approach to other regions of titin. In addition, molecular genetic approaches will be invaluable to study the roles of single titin domains and their specific ligands in transgenic models.
Differential expression of titin Only a single copy of the titin gene has been identified in vertebrate genomes, but many distinct isoforms are generated by differential splicing (Figure 2). Z-disc titin isoforms
The family of 45-residue titin Z-repeats are differentially expressed in different striated muscle tissues. Seven Z-repeats are expressed in rabbit heart muscle, a mixture of either six or four Z-repeats in rabbit soleus muscle, and four Z-repeats in rabbit psoas muscle [22••]. The titin Z-repeats have been shown to bind to the carboxyl terminus of αactinin [21••,22••]. We speculate that the differential expression of the Z-repeats leads to variable numbers of actin–titin cross-links within the Z-line resulting in different mechanical properties of the Z-lines. This organization could be important to fine-tune the strength of the Z-discs and the transmission of force between adjacent sarcomeres. Titin isoforms
The extensible portion of titin in the sarcomere is restricted to the I-band region of the molecule. Within this region, both the proximal tandem Ig segments and the PEVK segment are expressed in muscle-type-specific length isoforms. As a result of this differential expression, the I-band region of titins from different striated muscles range from 0.7 MDa in the cardiac isoform to 1.5 MDa in human soleus muscle (see Figure 2) and therefore, it was speculated that expression of different titin isoforms could adjust the elastic properties of the titin filament system to the physiological demands placed on the sarcomere [5]. Indeed, studies of single myofibrils have suggested that both the tandem Ig and the PEVK segments behave as extensible springs, which together may act as a two-spring system [33,34••]. Recent single molecule mechanical studies [39••–41••] are consistent with this model for titin elasticity in which the tandem Ig segments remain permanently folded whereas the PEVK segment behaves as a potentially unfolding polypeptide. Consequently, two distinct springs with different entropic properties and bending rigidities are serially linked. Consistent with the idea that the bending rigidity of the native tandem Ig segments are likely to be much higher than that of the PEVK segment, tandem Ig and PEVK segments
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extend under low force and high force, respectively [42••,43••]. This property explains their observed sequential extension in the skeletal muscle sarcomere; furthermore, by serially linking the mechanically distinct tandem Ig and PEVK segments, a unique passive force response is achieved which is not attainable with a single elastic segment.
Titin as a molecular template in myofibril assembly One of the challenges of muscle research is to identify the sequence of events involved in assembling thick and thin filaments and Z-lines to form sarcomeres. The direct interaction of titin with various sarcomeric proteins and the presence of many titin isoforms are consistent with its proposed role as a molecular template during myofibril assembly. Interestingly, studies have suggested that thick and thin filaments, assemble independently (for recent discussion, see [44]). The coordinated integration of the thick and thin filament systems into sarcomeres would require a structure interacting with both filament systems. In vertebrates, the titin-based third filament system has emerged as an excellent candidate to fulfill this role because it spans half-sarcomeres and interacts with many thick and thin filaments components at multiple sites. Finally, titin’s catalytic serine/threonine kinase domain from its M-line end has recently been shown to phosphorylate the T-cap/telethonin protein in vitro [45••]. T-cap(telethonin) is a Z-disc protein that binds to titin’s amino-terminus [10••,19], and this interaction is required for sarcomere formation [10••]; therefore, the Z-disc and the M-line ends of the titin molecule may communicate with each other during myofibril assembly by a T-cap(telethonin)-dependent mechanism. Such a mechanism could coordinate the integration of thick and thin filaments, which appear to originally assemble independently into sarcomeres [47]. Titin is one of the first myofibrillar proteins that assembles into nascent sarcomeres in in vivo studies [46–48] and in most, but not all, studies using primary striated muscle cell cultures as model systems (for in vitro studies see for example [2,49•,50] and references therein). In this regard, one would predict that tissue-specific titin splice pathways would emerge early during myogenesis to allow for the interpretation of titin’s sarcomeric blueprint. Sarcomeres in different muscle types could therefore be specified with different Z-line and I-band ultrastructures, as well as elastic properties. It is unclear whether this finely-tuned regulation by titin of myofibril assembly, and the fiber-type specificity which is dependent on the myosin isoforms that are expressed are coordinated, or whether both levels of regulation are controlled independently. One enigma that requires resolution is that because of the elastic properties of the titin molecule it is difficult to understand how nascent titin could remained sprawled out in a linear fashion as a blueprint for molecules to bind to it. One proposal is that the synthesis and linearizing of titin might be assisted by the periodic organization of titin’s mRNA with a significant fraction of newly synthesized titin being assembled during translation [50]. It is also possible
23
that ligand binding straightens the otherwise coiled protein at specific binding sites and possibly at some distance beyond (due to the high bending rigidity of the molecule), allowing titin to function as a ruler and as a ‘fishing line’ connecting different components of the sarcomere. Finally, specific molecules such as, titin-cap within the Z-lines may act as ‘bolts’ to anchor the long molecule, providing a framework upon which myofibrils could be constructed. In the invertebrate nematode C. elegans, unique concepts for regulation of myofibril assembly have emerged. Although longer than in vertebrates, thick filament lengths in C. elegans are also precisely determined at 7.4 µm. They appear to be assembled onto polymeric paramyosin molecules which serve as a core template (for review, see [51•]). The characterization of mutants with perturbed myofibril assembly have identified additional thick-filament-associated proteins that — apart from structural linker molecule functions — may have regulatory ‘assemblase’-like functions [52••,53••]. Interestingly, in vertebrates, both the titin and nebulin filaments contain motifs that are predicted to participate in regulatory signal transduction pathways. Most notably are the SH3 domains at the carboxyl terminus of nebulin/nebulette, and the kinase domain near titin’s carboxyl terminus whose three-dimensional structure recently was determined [45••]. Identification of the titin kinase and nebulin SH3 interacting ligands might also reveal vertebrate assemblaselike factors. The dynamic interaction of ‘assemblases’ with precise structural templates — paramyosin in C. elegans, and titin in vertebrates — may explain why throughout the animal kingdom, myofibrils are precisely assembled with almost crystalline order, yet are very dynamic structures whose components can be readily exchanged [54••]. In summary, although many unanswered questions remain, substantial progress has been made during the past few years in elucidating the mechanisms involved in myofibrillogenesis. This quest will be further aided by identifying specific ligands and functional domains of the third filament system. It is certain that the study of myofibrillogensis will continue to cede fundamental insights into the nature of macromolecular assembly processes.
Acknowledgements We especially thank Thomas Centner for the illustrations. We also thank the many members of our (past and present) laboratories especially Bernhard Kolmerer, Karoly Trombitás, and Thomas Centner who have made critical contributions, both scientifically and intellectually, to the development of this review. Work in our laboratories is supported by grants from the Human Frontier Science Program (Carol Gregorio, Hiroyuki Sorimachi, Siegfried Labeit), National Institutes of Health HL57461 (Carol Gregorio), National Institutes of Health HL61497 (Henk Granzier), the Deutsche Forschungsgemeinschaft (La668/2-3 and La668/3-3) and the Forschungsfond für Klinische Medizin Mannheim (Siegfried Labeit).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Trinick J: Cytoskeleton: titin as a scaffold and spring. Curr Biol 1996, 6:258-260.
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Epstein HF, Fischman DA: Molecular analysis of protein assembly in muscle development. Science 1991, 251:1039-1044.
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Gregorio CC, Fowler VM: Tropomodulin function and thin filament assembly in cardiac myocytes. Trends Cardiovasc Med 1996, 6:136-141.
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Schiaffino S, Reggiani C: Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev 1996, 76:371-423.
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Labeit S, Kolmerer B: Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science 1995, 270:293-296.
6. •
Pelin K, Ridanpää M, Donner K, Laing N, Kolmerer B, Millevoi S, Labeit S, Chapelle A, Wallgren-Pettersson C: Refined localization of the genes for nebulin and titin genes on chromosome 2q allows the assignment of nebulin as a candidate gene for autosomal recessive nemaline myopathy. Eur J Hum Genet 1997, 5:229-234. High resolution radiation hybrid mapping of the titin and nebulin gene loci in humans demonstrates that both genes are located on the long arm of chromosome 2, a few megabases apart. Nebulin, but not titin, is linked to the most common form of nemaline myopathy (the autosomal recessive ‘main stream’ form). Affected patients have pronounced muscle weakness and inclusion bodies in their myocytes which represent abnormal Z-disc protein assemblies. 7. •
Improta S, Krueger JK, Gautel M, Atkinson RA, Lefevre JF, Moulton S, Trewhella J, Pastore A: The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity. J Mol Biol 1998, 284:761-777. A structural study of expressed tandem Ig repeats from I-band titin using nuclear magnetic resonance and small angle X-ray scattering techniques. Shortening of the tandem Ig segments may result from the bending of interdomain linker regions. 8. ••
Muhle-Goll C, Pastore A, Nilges M: The three-dimensional structure of a type I module from titin: a prototype of intracellular fibronectin type III domains. Structure 1998, 6:1291-1302. The three-dimensional structure of an A-band FNIII domain was determined by nuclear magnetic resonance. This is the first solved structure of an intracellular member of the FNIII superfamily. Interestingly, the prolines are found to be clustered on one surface of the domain and constitute two polyproline helix II conformations. Since the proline residues are conserved within the titin FNIII family, they might attach titin to the thick filaments in a conserved fashion. 9.
Fürst DO, Osborn M, Nave R, Weber K: The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J Cell Biol 1988, 106:1563-1572.
10. Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, •• Suzuki K, Obermayr F, Herrmann B, Granzier H et al.: The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19 kD ligand (T-cap) is required for sarcomeric integrity. J Cell Biol 1998, 143:1013-1027. This study focuses on the molecular interactions of Z-line titin. Results demonstrate the molecular layout of titin within the Z-line: mammalian titins have at least four potential binding sites for α-actinin, and the amino-terminal titin Z1 and Z2 repeats bind to a novel 19 kDa protein, referred to as titincap (T-cap). Using dominant-negative approaches in cardiac myocytes, the interaction of titin Z1 and Z2 domains with T-cap is shown to be required for the structural integrity of sarcomeres. 11. Witt CC, Olivieri N, Centner T, Kolmerer B, Millevoi S, Labeit D, • Jockusch H, Pastore A, Labeit S: A survey of the primary structure and the interspecies conservation of I-band titin’s elastic elements in vertebrates. J Struct Biol 1998, 122:1-10. This study shows that the differentially expressed tandem Ig repeats in I-band titin’s elastic region form a specific subgroup of the titin Ig repeat family and differ structurally from those expressed elsewhere in the molecule. 12. Obermann WM, Gautel M, Steiner F, van der Ven PF, Weber K, Fürst DO: The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy. J Cell Biol 1996, 134:1441-1453. 13. Obermann WM, Gautel M, Weber K, Fürst DO: Molecular structure •• of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin. EMBO J 1997, 16:211-220. A thorough study of the layout and molecular interactions of M-line titin. The carboxy-terminal 200 kDa of the titin filament spans the entire M-line region; Ig repeats inside the M-line region may provide attachment sites for the M-line protein myomesin.
14. Millevoi S, Trombitas K, Kostin S, Schaper J, Pelin K, Kolmerer B, •• Granzier H, Labeit S: Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs. J Mol Biol 1998, 282:111-123. Millevoi and colleagues report the cloning, full-length sequence and gene mapping of nebulette, a nebulin-related protein from cardiac muscle. Both nebulin and nebulette are filamentous proteins that insert into the periphery of the Z-line. Similar to titin’s 45-residue Z-repeats, nebulin/nebulette also contain a family of differentially expressed Z-repeats. It is likely that the differential expression of titin and nebulin/nebulette accounts for Z-line tissue diversity. 15. Moncman CL, Wang K: Nebulette: a 107 kD nebulin-like protein in cardiac muscle. Cell Motil Cytoskeleton 1995, 32:205-225. 16. Goll DE, Suzuki, A, Temple J, Holmes GR: Studies on purified α-actinin. I. Effect of temperature and tropomyosin on the α-actinin/F-actin interaction. J Mol Biol 1972, 67:469-488. 17. Squire JM: Architecture and function in the muscle sarcomere. • Curr Opin Struct Biol 1997, 7:247-257. A very stimulating review on recent progress on the structural biology of the sarcomere. 18. Vigoreaux JO: The muscle Z band: lessons in stress management. J Muscle Res Cell Motil 1994, 15:237-255. 19. Mues A, van der Ven PFM, Young P, Fürst DO, Gautel M: Two immunoglobulin-like domains of the Z-disc portion of titin interact in a conformation-dependent way with telethonin. FEBS Lett 1998, 428:111-114. 20. Gautel M, Goulding D, Bullard B, Weber K, Fürst DO: The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers. J Cell Sci 1996, 109:2747-2754. 21. Ohtsuka H, Yajima H, Kimura S, Maruyama K: Binding of the N •• terminal fragment of connectin/titin to alpha-actinin as revealed by yeast two-hybrid systems. FEBS Lett 1997, 401:65-67. See annotation [22••]. 22. Sorimachi H, Freiburg A, Kolmerer B, Ishiura S, Stier G, Gregorio CC, •• Labeit D, Linke WA, Suzuki K, Labeit S: Tissue-specific expression and alpha-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs. J Mol Biol 1998, 270:688-695. Both studies [21••,22••] show that the 10 kDa carboxy-terminal domain of α-actinin binds to titin’s 45-residue Z-repeats. Thus, antiparallel α-actinin dimers can attach at both ends to titin filaments, whereas their amino termini can cross-link them to thin filaments. The Z-repeats are shown to be expressed in different copy numbers in different tissues, possibly accounting for the variability of Z-filaments in different muscle types. 23. Young P, Ferguson C, Bañuelos S, Gautel M: Molecular structure of •• the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of α-actinin. EMBO J 1998, 17:1614-1624. Besides confirming the interaction of titin’s Z-repeats with α-actinin, Young and colleagues identified that a single site at the periphery of Z-disc titin also may bind to α−actinin. This site just flanks the Z-repeats and, in contrast to them, does not bind to the carboxy-terminal end of α-actinin, but to the central rod domains (R2-R3) containing spectrin-like repeats. Thus, titin may contain two distinct α-actinin binding motifs. 24. Linke WA, Ivemeyer M, Labeit S, Hinssen H, Ruegg JC, Gautel M: • Actin–titin interaction in cardiac myofibrils: probing a physiological role. Biophys J 1997, 73:905-919. See annotation [26•]. 25. Trombitas K, Granzier H: Actin removal from cardiac myocytes • shows that near the Z-line titin attaches to actin while under tension. Amer J Physiol 1997, 273:C662-C670. See annotation [26•]. 26. Granzier H, Kellermayer M, Trombitas K: Titin elasticity and • mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction. Biophys J 1997, 73:2043-2053. These studies [24•,25•] report that the portion of I-band titin near the Z-disc resists stretch when thin filaments are intact, and that following thin-filament removal titin near the Z-disk collapses in short sarcomeres and extends upon sarcomere elongation [26•]. Therefore, this part of I-band titin is predicted to interact with actin. 27.
Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishiura S, Sasagawa N, Sorimachi N, Shimada H, Tagawa K, Maruyama K, Suzuki K: Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J Biol Chem 1995, 270:31158-31162.
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28. Kolmerer B, Olivieri N, Witt CC, Herrmann BG, Labeit S: Genomic organization of the M line titin and its tissue-specific expression in two distinct isoforms. J Mol Biol 1996, 256:556-563. 29. Labeit S, Gautel M, Lakey A, Trinick J: Towards a molecular understanding of titin. EMBO J 1992, 11:1711-1716. 30. Houmeida A, Holt J, Tskhovrebova L, Trinick J: Studies of the interaction between titin and myosin. J Cell Biol 1995, 131:1471-1481. 31. Freiburg A, Gautel M: A molecular map of the interaction between titin and myosin-binding protein C. Implications for sarcomeric assembly in familiar hypertrophic cardiomyopathy. Eur J Biochem 1996, 235:317-323. 32. Okagaki T, Weber FE, Fischman DA, Vaughan KT, Mikawa T, Reinach FC: The major myosin-binding domain of skeletal muscle MyBP-C (Cprotein) resides in the COOH-terminal, immunoglobulin C2 motif. J Cell Biol 1993, 123:619-626. 33. Linke WA, Ivemeyer M, Olivieri N, Kolmerer B, Rüegg JC, Labeit S: Towards a molecular understanding of the elasticity of titin. J Mol Biol 1996, 261:62-71. 34. Linke WA, Stockmeier MR, Ivemeyer M, Hosser H, Mundel P: • Characterizing titin’s I-band Ig domain region as an entropic spring. J Cell Sci 1998, 111:1567-1574. Both studies [33,34•] use intact myofibrils to characterize the behavior of the tandem Ig and PEVK titin segments, and conclude that they may act as a two-spring system. 35. Gautel M, Leonard K, Labeit S: Phosphorylation of KSP-motifs in the C-terminal region of titin in differentiating myoblasts. EMBO J 1993, 12:3827-3834. 36. Turnacioglu KK, Mittal B, Sanger JM, Sanger JW: Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin. Cell Motil Cytoskeleton 1996, 34:108-121. 37.
Peckham M, Young P, Gautel M: Constitutive and variable regions of Z-disk titin/connectin in myofibril formation: a dominantnegative screen. Cell Struct Funct 1997, 22:95-101.
38. Lin Z, Kijikata T, Zhang Z, Choi J, Holtzer S, Sweeney HS, Holtzer H: •• Dispensability of the actin-binding site and spectrin repeats for targeting sarcomeric α-actinin into maturing Z bands in vivo: implications for in vitro binding studies. Development 1998, 199:291-308. An elegant study investigating the roles of specific domains of α-actinin in the initial targeting and maintenance of striated myofibrils. This study indicates the limited value of predicting the behavior of sarcomeric components based on in vitro binding studies. 39. Kellermayer MS, Smith SB, Granzier HL, Bustamante C: Folding•• unfolding transitions in single titin molecules characterized with laser tweezers. Science 1997, 276:1112-1116. See annotation [41••]. 40. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE: Reversible •• unfolding of individual titin immunoglobulin domains by AFM. Science 1997, 276:1109-1112. See annotation [41••]. 41. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM: Elasticity and •• unfolding of single molecules of the giant muscle protein titin. Nature 1997, 387:308-312. The papers [39••,40••,41••] are pioneering studies in which single titin molecules were manipulated and titin’s force-extension properties were characterized at the molecular level. The results obtained reveal that titin behaves as an entropic spring. Unfolding takes place at high force during stretching of the molecule, and refolding at low force during release of the molecule. 42. Trombitas K, Greaser M, Labeit S, Jin J-P, Kellermayer M, Helmes M, •• Granzier H: Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments. J Cell Biol 1998, 140:853-859. See annotation [43••].
25
43. Linke WA, Ivemeyer M, Mundel P, Stockmeyer MR, Kolmerer B. •• Nature of PEVK-titin elasticity in skeletal muscle. Proc Natl Acad Sci USA 1998, 95:8052-8057. Both Trombitas et al. [42••] and Linke et al. [43••] use novel epitope-mapped antibodies to monitor the mechanical behavior of central I-band titin during myofibrillar stretch. They prove that the PEVK (in the single letter code for amino acids) region of titin acts as a highly extensible spring element which is responsible for the passive tension response of the titin filament. 44. Holtzer H, Hijikata T, Lin ZX, Zhang ZQ, Holtzer S, Protasi F, Frazini-Armstrong C, Sweeney HL: Independent assembly of 1.6 µm long bipolar MHC filaments and I-Z-I bodies. Cell Struct Funct 1997, 22:83-93. 45. Mayans O, van der Ven P, Wilm M, Mues A, Young P, Fürst DO, •• Wilmanns M, Gautel M: Structural basis for activation of the titin kinase domain during myofibrillogenesis. Nature 1998, 395:863-869. A pioneering study which reports the three-dimensional structure of the titin kinase domain and may provide a molecular understanding of its mode of activation. The Z-disc protein and titin ligand T-cap(telethonin) has been identified as a potential substrate of the titin kinase domain using in vitro phosphorylation assays. Thereby, M-line and Z-disc assembly might be coordinated during myofibrilogenesis. 46. Tokuyasu KT, Maher PA: Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alphaactinin dots within titin spots at the time of the first myofibril formation. J Cell Biol 1987, 105:2795-2801. 47.
Shimada Y, Komiyama M, Begum S, Maruyama K: Development of connectin/titin and nebulin in striated muscles of chicken. Adv Biophys 1996, 33:223-234.
48. Fürst DO, Osborn M, Weber K: Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J Cell Biol 1989, 109:517-527. 49. Dabiri GA, Turnacioglu KK, Sanger JM, Sanger JW: • Myofibrillogenesis visualized in living embryonic cardiomyocytes. Proc Natl Acad Sci USA 1997, 94:9493-9498. The distribution patterns of green fluorescent protein tagged α-actinin were visualized during myofibril assembly in cultured cardiac myocytes. Nonmuscle myosin IIB is detected at the leading edges of the cells. 50. Fulton AB, Alftine C: Organization of protein and mRNA for titin and other myofibril components during myofibrillogenesis in cultured chicken skeletal muscle. Cell Struct Funct 1997, 22:51-58. 51. Liu F, Barral JM, Bauer CC, Ortiz I, Cook RG, Schmid MF, Epstein HF: • Assemblases and coupling proteins in thick filament assembly. Cell Struct Funct 1997, 22:155-162. A thorough review of thick filament assembly and length regulation. 52. Liu F, Bauer CC, Ortiz I, Cook RG, Schmid MF, Epstein HF: β•• filagenin, a newly identified protein coassembling with myosin and paramyosin in Caenorhabditis elegans. J Cell Biol 1998, 140:347-353. Purification and cloning of a novel protein which coassembles with paramyosin and myosin filaments. β-filagenin may link these two filament systems, comparable to C-protein in vertebrates which links the titin and myosin filaments. 53. Barral JM, Bauer CC, Ortiz I, Epstein HF: Unc-45 mutations in •• Caenorrhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly. J Cell Biol 1998, 143:1215-1225. The UNC-45 locus is proposed to act as a protein machine required for myosin assembly. The UNC-45 gene product contains CRO1/She4p-like domains implicated in signal transduction pathways. Mutations in these regulatory domains interfere with the number and structure of thick filaments assembled. 54. Littlefield R, Fowler VM: Defining actin filament length in striated •• muscle. Annu Rev Dev Cell Biol 1998, 14:487-525. Interesting chapter discussing several hypothetical mechanisms by which muscle cells can generate uniform actin filament length distributions including a mechanism by which titin and its kinase domain may play a role in this process.
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Muscle assembly: a titanic achievement? Carol C Gregorio*, Henk Granzier†, Hiroyuki Sorimachi‡ and Siegfried Labeit§ The formation of perfectly aligned myofibrils in striated muscle represents a dramatic example of supramolecular assembly in eukaryotic cells. Recently, considerable progress has been made in deciphering the roles that titin, the third most abundant protein in muscle, has in this process. An increasing number of sarcomeric proteins (ligands) are being identified that bind to specific titin domains. Titin may serve as a molecular blueprint for sarcomere assembly and turnover by specifying the precise position of its ligands within each halfsarcomere in addition to functioning as a molecular spring that maintains the structural integrity of the contracting myofibrils. Addresses *Departments of Cell Biology and Anatomy, The University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724-5044, USA; e-mail: [email protected] †Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA, 991646520, USA; e-mail: [email protected] ‡Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; e-mail: [email protected] §European Molecular Biology Laboratory, Meyerhofstrasse 1, PO Box 102209, 69012 Heidelberg, Germany; e-mail: [email protected] Current Opinion in Cell Biology 1999, 11:18–25 http://biomednet.com/elecref/0955067401100018 © Elsevier Science Ltd ISSN 0955-0674 Abbreviations FNIII fibronectin type III Ig-domain immunoglobulin-like domain KSP Lys–Ser–Pro PEVK Pro, Glu, Val, Lys SH3 Src-homology-3-like domain
mechanisms may have developed, since the thick filaments in their obliquely striated muscles are much longer than those in vertebrates, and no molecule of titin-like size has yet been identified. Several previous reviews have focused on the general properties, isoforms and order of assembly of the known sarcomeric components [2–4]. Here we emphasize the more recent developments in our knowledge of the possible roles that titin has in myofibril assembly. We first discuss the molecular structure of the titin filament and its detailed layout within the sarcomere. Next, we review progress in identifying specific protein ligands of titin. Finally, we speculate on possible mechanisms involved in orchestrating myofibril assembly and maintenance.
Molecular structure of the titin filament A decade ago, there was much resistance to the idea that a eukaryotic cell could translate megadalton-sized ‘titanic’ polypeptides; however, it is now firmly established that this is possible as a full-length titin cDNA was isolated that encodes a polypeptide of 27,000 residues [5]. A single gene locus for titin was located on the long arm of chromosome 2 in humans (e.g. [6•]). This gene is transcribed to produce a full-length mRNA of up to 100 kilobases, which encodes a titin polypeptide of up to 3.0 MDa.
The sarcomere is the contractile unit of striated muscle. Its formation is a unique example in biology where thousands of molecules assemble into a supramolecular structure with almost crystalline order. As a result of this precise assembly, many different classes of proteins function together to efficiently convert the molecular-level movements produced by actin and myosin into macroscopic movements of muscle.
The titin protein is extensively modular in structure (see Figure 2). It is composed of 132 copies of 100-residue repeats belonging to the fibronectin type III (FNIII) superfamily, and up to 166 copies of 90–95 residue repeats from the immunoglobulin (Ig) superfamily. Together, the Ig and FNIII repeats account for 90% of titin’s mass. Analysis of the three-dimensional structure of selected Ig and FNIII repeats from the I-band, A-band and M-line region (see Figure 1) has revealed that all of the repeats form stable globular domains composed of seven antiparallel β-sheets ([7•,8••], and references therein). The titin filament can therefore be thought of as a microscopic necklace made up of about 300 pearls (the Ig and FNIII domains), which extends across the entire half-sarcomere in both skeletal and cardiac muscle.
It has been speculated that, in vertebrates, the giant filamentous protein titin (also known as connectin), the only known structure that spans the entire half-sarcomere, may act as a molecular blueprint to direct sarcomere assembly (e.g.[1]; see Figure 1). In this role, titin would coordinate the precise assembly of the structural, regulatory and contractile proteins within the sarcomere, a prerequisite for efficient muscle contraction. In the invertebrate Caenorhabditis elegans, different assembly
The Ig and FNIII repeats are arranged in different patterns throughout the molecule (see Figure 1 and below for discussion). About 10% of titin’s mass is organized in nonrepetitive sequences which are situated between the Ig and FNIII repeats. A total of 17 such interdomain insertions are present, 16 of which have no significant homology to each other, or to other known protein sequences. One interdomain insertion that is located close to the carboxy-terminal end of titin is related in
Introduction
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Figure 1 I-band Z-disc Adjacent
A-band
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Current Opinion in Cell Biology
Schema of a skeletal muscle sarcomere (a single contractile unit) with its A-bands that contain myosin thick filaments and I-bands that contain actin thin filaments. In the Z-lines, overlapping thin filaments from opposite sarcomeres are anchored, whereas thick filaments from opposite half-sarcometers are anchored in the M-lines. The layout of the four filament systems (thin, thick, titin and nebulin filaments) are shown. The amino-terminal regions of titin molecules from adjacent
sarcomeres fully overlap in Z-lines [10••]; the carboxy-terminal regions of titin molecules from opposite half-sarcomeres fully overlap in the Mlines [12]. In myofibrils composed of many sarcomeric units, the titin filaments (via their specific ligands) form a continuous filament system. The carboxy-terminal ends of nebulin molecules insert into the Z-disc [14••]; their amino-terminal ends are located at, or in close proximity to, the pointed ends of the thin filaments.
sequence to a serine/threonine kinase domain and is referred to as the ‘titin kinase domain’.
A-band titin
Layout of the titin filament in sarcomeres Initial immuno-electron microscopy studies, with a set of 10 nonrepetitive anti-titin monoclonal antibodies, revealed that a single molecule of titin extends from the Z-line to close to the M-line region [9]. More detailed information on the layout of the titin filament within the sarcomere has been obtained recently by immuno-electron microscopy studies using antibodies raised against specific bacteriallyexpressed domains of titin (see Figures 1 and 2). Z-disc titin
The labeling patterns obtained show that the amino-terminal 80 kDa of titin (or less, depending on the isoform) spans the entire Z-line region [10••]. Titin filaments from opposite sarcomeres therefore overlap within the Z-lines.
The carboxy-terminal 2 MDa of titin are located in the Aband; this region of titin is rendered inextensible by its tight association with the myosin thick filament. It contains two types of super-repeats, both consisting of FNIII and Ig repeats arranged into distinct patterns [5]. Within the central portion of the thick filament, seven FNIII and four Ig domains form a pattern, which is repeated 11 times and referred to as the 11-domain super-repeat (Figure 2). This super-repeat structure appears to correspond with the presence of the 11–43 nm crossbridge repeats of the thick filaments within the C-zone of the A-band; therefore, the 11 titin super-repeats are also referred to as ‘C-zone A-band titin’. More towards the amino-terminus within the A-band, five FNIII and two Ig repeats are arranged into a pattern that is repeated seven times. This part of the titin filament is referred to as ‘D-zone A-band titin’ (see Figure 2). M-line titin
I-band titin
Between 800 kDa and 1.5 MDa of titin are located within the I-band, depending on the particular isoform (see below). The central section of I-band titin — corresponding to its elastic region — is formed by tandemly arranged Ig repeats which are contained in two separate blocks, the proximal and the distal tandem Ig segments [11•]. Both of the tandem Ig groups are separated by up to 2200 residues that are unusually rich in P (proline), E (glutamate), V (valine), and K (lysine). This segment from the central I-band titin is referred to as the ‘PEVK domain’ [5].
The M-line region of titin corresponds to ~200 kDa of the final carboxy-terminal region of the protein. The kinase domain of titin is located at the periphery of the M-line region [12]. As in Z-lines, where titin filaments from opposite sarcomeres fully overlap [10••], titin filaments from opposite half-sarcomeres fully overlap within the M-line region [13••]. The overlapping ends are interconnected by their binding to other sarcomeric components (see below). Titin filaments with opposite polarity overlap in both the Z-lines and M-lines, forming a contiguous system within myofibrils (Figure 1).
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Figure 2
Half I-band Z-disc Tandem Ig region
PEVK-segment
NH 2 N2-A
P1
Half A-band A/I junction
P-zone
C-zone 11domain super-repeat
D-zone 7domain super-repeat
M-line P1
COOH Kinase domain Current Opinion in Cell Biology
Titin domains Ig-module
Titin ligands : :(by yeast two-hybrid) PEVK-segment
FN-module
Z-repeats
Unique sequence
Splice site
α-actinin
Calmodulin
p94
Titin-cap/ telethonin
Titin ligands: (by protein biochemical studies) C-protein P1
Myomesin Myosin
Phosphorylation site
Molecular structure of the titin filament, and location of differential splicing sites that result in different titin isoforms. Above the corresponding titin domains, blocks indicate ligands that have been
identified by yeast two-hybrid studies. Below, blocks indicate putative titin ligands identified by in vitro studies using expressed domains (see text).
Comparison of the titin and nebulin filament layouts: functional implications
Z-lines vary in width in different tissue types (for reviews see [17•,18]). Interestingly, both Z-disc titin and nebulin contain differentially expressed repeat families, referred to as Z-repeats. We speculate that together, the differential expression of nebulin’s carboxy-terminal and titin’s amino-terminal Z-repeats may account for the variability of vertebrate Z-lines. Regulating the amount of titin and actin filament overlap in the Z-line and the degree of Z-line insertion of nebulin and tropomyosin is likely to be physiologically important as the width of the Z-line is likely to be correlated to the mechanical properties of muscle; fast and slow-twitch fibers have narrow and wide Z-lines, respectively.
A fourth structural filament system of striated muscle is formed by nebulin, an 800 kDa thin-filament-binding protein specific to skeletal muscle. This protein is sugegsted to function as a molecular ruler to specify the length of thin filaments. The SH3-containing carboxy-terminal domain of a single molecule of nebulin inserts into the Zdisc, associate with the thin filament along its length. The amino-terminus whereas the central regions of nebulin locates in close proximity to the pointed (free) ends of the thin filaments (see Figure 1). In cardiac muscle, nebulette — a 109 kDa nebulin-related protein, sharing extensive structural homology with nebulin’s Z-disc (carboxy-terminal) region — appears to substitute for the Z-disc integrative function of nebulin (see below), but not to share a role in thin filament specification, as it is predicted to extend along only ~25% of the thin filament [14••,15]. In contrast to actin and titin, which span the entire Z-line, nebulin filaments appear to insert only within the periphery of the Z-line [14••]. Similarly, polymers of tropomyosin, which coil around and stabilize actin filaments, are predicted to be absent from the central Z-disc region of the thin filament, as α-actinin, a major structural component of the Z-line, inhibits tropomyosin binding to actin filaments [16]. It is possible that the amino-terminal region of titin may specify the principal amount of thin filament overlap, while the carboxy-terminal region of nebulin (or nebulette) may specify where tropomyosin binding on the actin filament terminates.
Interaction of titin with other sarcomeric components Here, we describe the proteins that interact with titin — starting from its amino-terminal Z-disc to its carboxy-terminal M-line end — and identify their potential binding sites in human titin sequences (see Figure 2). Interactions of titin’s Z-disc region
Using the yeast two-hybrid system to search for binding partners of the amino-terminal Z-line portion (~80 kDa) of titin, three distinct interactions have been identified. First, the extreme amino-terminal titin Ig repeats Z1 and Z2 (residues 1–200) bind to a novel Z-line protein [10••,19], which we suggest calling the ‘titin cap’ (T-cap) [10••]. Both the Z1 and Z2 repeats are required for binding. Further away from the amino terminus, the Z-disc titin residues 450–750 contain up
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Figure 3
Z-disc 50nm
50nm *
*
*
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Zr7
Z-repeats COOH
Zr7
*
*
Zr1
*
* Zr1
*
To M-line
* Zr7
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NH2
Current Opinion in Cell Biology
Nebulin: SH3-domain
Titin: Ig-domains
Z1/Z2
Z-repeats *(differentially expressed)
Nebulin repeats *(differentially expressed)
Unique sequence
Ser-rich domain
Titin-cap protein
Actin (thin) filament decorated with tropomyosin
Terminal α-actinin?
EM mapped epitope
Overview of the layout of titin, nebulin and thin filaments in Z-discs from skeletal muscles. Titin: the amino-terminal region spans the Z-disc, so that titin filaments from neighboring sarcomeres fully overlap within the Z-lines [10••]. Within the Z-disc, titin filaments connect to α actinin Z-filaments at
multiple sites which then cross-link the titin and thin filaments [10••,21••,22••,23••]. Nebulin: filaments insert ~30 nm from the center of the Z-disc [14••]. Arrows indicate the location of the titin and nebulin epitopes that were mapped by immuno-electron microscopy.
to seven copies of a 45-residue repeat, each of which is referred to as a titin Z-repeat [20]. In vitro studies indicate that the Z-repeats bind the extreme carboxy-terminal 10 kDa of α-actinin — a major component of Z-lines that links the antiparallel actin filaments from adjacent sarcomeres [10••,21••,22••]. Additionally, the Z-line titin residues 750–826 (which are distinct and just carboxy-terminal to the Z-repeats) may contain another binding site for α-actinin but in this case the interaction may occur with α-actinin’s spectrin-like repeat domains (R2–R3) [23••] in the central rod.
might directly or indirectly involve T-cap, while the center region of α-actinin directly connects — via its spectrin-like domains R2-R3 — to titin Z/I junction residues. Inside the Z-line, α-actinin connects titin filaments from opposite sarcomeres by directly cross-linking Z-repeats. Unknown linker molecules may also be involved. A schema of the layout of the titin, nebulin and thin filaments in the Z-line is shown in Figure 3.
From what is known about the in vitro binding properties of Z-line titin, the following speculative model emerges. Residues 1–200 (Z1 and Z2) reside in the periphery of the Z-line of the sarcomere adjacent to the one in which the titin filament is extended, where they mark the edge of the Z-line region (‘Z-line titin edge residues’). Titin residues 200–750 (‘Z-line titin integrative residues’) span the Z-disk, whereas residues 750–826 are located at the Z-line junction with the I-band (‘titin Z/I junction residues’). Z-line titin edge and Z/I junction residues of overlapping titin filaments from neighboring sarcomeres may be cross-linked via unique Z-filaments. Cross-linking of the titin edge residues
Interactions of titin’s I-band
Near the Z-line, titin filaments associate with actin (thin) filaments, as indicated by increased titin extensibility after removal of actin by gelsolin extraction [24•–26•]. It is unclear which titin motifs mediate this interaction. To date, no splicing variants have been identified in this region, suggesting that the titin–actin interaction is required in all muscle types. The lateral association of titin with the thin filaments may straighten the otherwise folded titin molecule and thread it correctly into the Zdisc where it is organized into a highly ordered tetragonal lattice, cross-linked by α-actinin. Whether titin–actin interactions occur elsewhere in the I-band is unresolved, as a weak interaction that affects Ig-domain unfolding in
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cardiac muscle is suggested by some studies [26•], but not by others [24•]. Within the central I-band region (and also within the M-line region) of titin, binding sites are found for the muscle-specific calpain protease p94 [27]. The p94 binding motifs are excluded in some titin isoforms by exon-skipping [5,28]. The biological significance of the binding of p94 to titin and differential expression of the p94 binding sites is unknown. A-band titin interactions
The eleven-domain super-repeats contain multiple domains that bind to the tail portion of myosin, as well as to the thick-filament-associated protein, C-protein (MyBP-C) [29,30] which is organized transversely in A-bands. Phosphorylation of this protein may modulate contraction. Ig domains within each titin super-repeat have been identified that bind in vitro to the carboxy-terminal region of C-protein [31]. This interaction may explain why carboxy-protein associates with the thick filament at up to 11 positions in situ. The carboxyl terminus of C-protein also binds to the myosin tail [32]; therefore, within the C-zone of the A-band, titin may specify the number and location of myosin and C-protein molecules that are incorporated into thick filaments. Titin’s FNIII repeats are restricted to the A-band, whereas the Ig repeats are present throughout the molecule; therefore, titin’s FNIII repeats may represent a family of myosin-tail-binding motifs. The three-dimensional structure of a titin FNIII domain reveals the presence of two polyproline type II helices on one surface of the FNIII domain [8••]. In this regard, it may be relevant that titin’s A-band FNIII domains are generally proline-rich and that the positions of the proline residues are conserved. Future studies are needed to address whether the polyproline type II helix motifs act as weak myosin-binding motifs functioning to tether the titin to thick filaments. It is likely that titin interacts with myosin along the whole of the thick filament and not only in the C-zone, as at a wide range of sarcomere lengths, A-band titin epitopes remain at the same location relative to the M-line (e.g. [1,9,33,34••]). M-line titin interactions
Within the M-line region of the A-band, the titin Ig-repeat domain M5 has been found to bind myomesin in vitro [13••]. M5 is adjacent to four tandem copies of Lys–Ser–Pro (KSP motifs in the single letter code for amino acids) that are potential substrates of an unknown KSP-directed serine/threonine kinase [35]. Possibly, KSP phosphorylation may regulate titin–myomesin interactions [13••]. Together, C-protein and myomesin might function in the lateral registration of thick filaments. A caveat of many of the above studies is that they are mostly based upon in vitro binding data. Future studies will be required to address the functional role of titin’s interactions using in vivo models. So far, only the functional role of Z-line titin has been investigated by
dominant-negative transient expression studies in cultured myocytes. Overexpression of the amino-terminal titin Z1-Z2 domain, its ligand T-cap [10••,19], the first 362 residues of titin [36], the entire Z-disc region of titin [37] or a carboxy-terminal truncated fragment of αactinin (missing it’s binding site for titin) [38••] all result in the disruption of myofibrils. Thus, the association of titin filaments with Z-discs, via their interaction with Tcap and α-actinin, appears to be critical for the assembly and maintenance of myofibril structure. It is warranted to extend this approach to other regions of titin. In addition, molecular genetic approaches will be invaluable to study the roles of single titin domains and their specific ligands in transgenic models.
Differential expression of titin Only a single copy of the titin gene has been identified in vertebrate genomes, but many distinct isoforms are generated by differential splicing (Figure 2). Z-disc titin isoforms
The family of 45-residue titin Z-repeats are differentially expressed in different striated muscle tissues. Seven Z-repeats are expressed in rabbit heart muscle, a mixture of either six or four Z-repeats in rabbit soleus muscle, and four Z-repeats in rabbit psoas muscle [22••]. The titin Z-repeats have been shown to bind to the carboxyl terminus of αactinin [21••,22••]. We speculate that the differential expression of the Z-repeats leads to variable numbers of actin–titin cross-links within the Z-line resulting in different mechanical properties of the Z-lines. This organization could be important to fine-tune the strength of the Z-discs and the transmission of force between adjacent sarcomeres. Titin isoforms
The extensible portion of titin in the sarcomere is restricted to the I-band region of the molecule. Within this region, both the proximal tandem Ig segments and the PEVK segment are expressed in muscle-type-specific length isoforms. As a result of this differential expression, the I-band region of titins from different striated muscles range from 0.7 MDa in the cardiac isoform to 1.5 MDa in human soleus muscle (see Figure 2) and therefore, it was speculated that expression of different titin isoforms could adjust the elastic properties of the titin filament system to the physiological demands placed on the sarcomere [5]. Indeed, studies of single myofibrils have suggested that both the tandem Ig and the PEVK segments behave as extensible springs, which together may act as a two-spring system [33,34••]. Recent single molecule mechanical studies [39••–41••] are consistent with this model for titin elasticity in which the tandem Ig segments remain permanently folded whereas the PEVK segment behaves as a potentially unfolding polypeptide. Consequently, two distinct springs with different entropic properties and bending rigidities are serially linked. Consistent with the idea that the bending rigidity of the native tandem Ig segments are likely to be much higher than that of the PEVK segment, tandem Ig and PEVK segments
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extend under low force and high force, respectively [42••,43••]. This property explains their observed sequential extension in the skeletal muscle sarcomere; furthermore, by serially linking the mechanically distinct tandem Ig and PEVK segments, a unique passive force response is achieved which is not attainable with a single elastic segment.
Titin as a molecular template in myofibril assembly One of the challenges of muscle research is to identify the sequence of events involved in assembling thick and thin filaments and Z-lines to form sarcomeres. The direct interaction of titin with various sarcomeric proteins and the presence of many titin isoforms are consistent with its proposed role as a molecular template during myofibril assembly. Interestingly, studies have suggested that thick and thin filaments, assemble independently (for recent discussion, see [44]). The coordinated integration of the thick and thin filament systems into sarcomeres would require a structure interacting with both filament systems. In vertebrates, the titin-based third filament system has emerged as an excellent candidate to fulfill this role because it spans half-sarcomeres and interacts with many thick and thin filaments components at multiple sites. Finally, titin’s catalytic serine/threonine kinase domain from its M-line end has recently been shown to phosphorylate the T-cap/telethonin protein in vitro [45••]. T-cap(telethonin) is a Z-disc protein that binds to titin’s amino-terminus [10••,19], and this interaction is required for sarcomere formation [10••]; therefore, the Z-disc and the M-line ends of the titin molecule may communicate with each other during myofibril assembly by a T-cap(telethonin)-dependent mechanism. Such a mechanism could coordinate the integration of thick and thin filaments, which appear to originally assemble independently into sarcomeres [47]. Titin is one of the first myofibrillar proteins that assembles into nascent sarcomeres in in vivo studies [46–48] and in most, but not all, studies using primary striated muscle cell cultures as model systems (for in vitro studies see for example [2,49•,50] and references therein). In this regard, one would predict that tissue-specific titin splice pathways would emerge early during myogenesis to allow for the interpretation of titin’s sarcomeric blueprint. Sarcomeres in different muscle types could therefore be specified with different Z-line and I-band ultrastructures, as well as elastic properties. It is unclear whether this finely-tuned regulation by titin of myofibril assembly, and the fiber-type specificity which is dependent on the myosin isoforms that are expressed are coordinated, or whether both levels of regulation are controlled independently. One enigma that requires resolution is that because of the elastic properties of the titin molecule it is difficult to understand how nascent titin could remained sprawled out in a linear fashion as a blueprint for molecules to bind to it. One proposal is that the synthesis and linearizing of titin might be assisted by the periodic organization of titin’s mRNA with a significant fraction of newly synthesized titin being assembled during translation [50]. It is also possible
23
that ligand binding straightens the otherwise coiled protein at specific binding sites and possibly at some distance beyond (due to the high bending rigidity of the molecule), allowing titin to function as a ruler and as a ‘fishing line’ connecting different components of the sarcomere. Finally, specific molecules such as, titin-cap within the Z-lines may act as ‘bolts’ to anchor the long molecule, providing a framework upon which myofibrils could be constructed. In the invertebrate nematode C. elegans, unique concepts for regulation of myofibril assembly have emerged. Although longer than in vertebrates, thick filament lengths in C. elegans are also precisely determined at 7.4 µm. They appear to be assembled onto polymeric paramyosin molecules which serve as a core template (for review, see [51•]). The characterization of mutants with perturbed myofibril assembly have identified additional thick-filament-associated proteins that — apart from structural linker molecule functions — may have regulatory ‘assemblase’-like functions [52••,53••]. Interestingly, in vertebrates, both the titin and nebulin filaments contain motifs that are predicted to participate in regulatory signal transduction pathways. Most notably are the SH3 domains at the carboxyl terminus of nebulin/nebulette, and the kinase domain near titin’s carboxyl terminus whose three-dimensional structure recently was determined [45••]. Identification of the titin kinase and nebulin SH3 interacting ligands might also reveal vertebrate assemblaselike factors. The dynamic interaction of ‘assemblases’ with precise structural templates — paramyosin in C. elegans, and titin in vertebrates — may explain why throughout the animal kingdom, myofibrils are precisely assembled with almost crystalline order, yet are very dynamic structures whose components can be readily exchanged [54••]. In summary, although many unanswered questions remain, substantial progress has been made during the past few years in elucidating the mechanisms involved in myofibrillogenesis. This quest will be further aided by identifying specific ligands and functional domains of the third filament system. It is certain that the study of myofibrillogensis will continue to cede fundamental insights into the nature of macromolecular assembly processes.
Acknowledgements We especially thank Thomas Centner for the illustrations. We also thank the many members of our (past and present) laboratories especially Bernhard Kolmerer, Karoly Trombitás, and Thomas Centner who have made critical contributions, both scientifically and intellectually, to the development of this review. Work in our laboratories is supported by grants from the Human Frontier Science Program (Carol Gregorio, Hiroyuki Sorimachi, Siegfried Labeit), National Institutes of Health HL57461 (Carol Gregorio), National Institutes of Health HL61497 (Henk Granzier), the Deutsche Forschungsgemeinschaft (La668/2-3 and La668/3-3) and the Forschungsfond für Klinische Medizin Mannheim (Siegfried Labeit).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Trinick J: Cytoskeleton: titin as a scaffold and spring. Curr Biol 1996, 6:258-260.
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Labeit S, Kolmerer B: Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science 1995, 270:293-296.
6. •
Pelin K, Ridanpää M, Donner K, Laing N, Kolmerer B, Millevoi S, Labeit S, Chapelle A, Wallgren-Pettersson C: Refined localization of the genes for nebulin and titin genes on chromosome 2q allows the assignment of nebulin as a candidate gene for autosomal recessive nemaline myopathy. Eur J Hum Genet 1997, 5:229-234. High resolution radiation hybrid mapping of the titin and nebulin gene loci in humans demonstrates that both genes are located on the long arm of chromosome 2, a few megabases apart. Nebulin, but not titin, is linked to the most common form of nemaline myopathy (the autosomal recessive ‘main stream’ form). Affected patients have pronounced muscle weakness and inclusion bodies in their myocytes which represent abnormal Z-disc protein assemblies. 7. •
Improta S, Krueger JK, Gautel M, Atkinson RA, Lefevre JF, Moulton S, Trewhella J, Pastore A: The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity. J Mol Biol 1998, 284:761-777. A structural study of expressed tandem Ig repeats from I-band titin using nuclear magnetic resonance and small angle X-ray scattering techniques. Shortening of the tandem Ig segments may result from the bending of interdomain linker regions. 8. ••
Muhle-Goll C, Pastore A, Nilges M: The three-dimensional structure of a type I module from titin: a prototype of intracellular fibronectin type III domains. Structure 1998, 6:1291-1302. The three-dimensional structure of an A-band FNIII domain was determined by nuclear magnetic resonance. This is the first solved structure of an intracellular member of the FNIII superfamily. Interestingly, the prolines are found to be clustered on one surface of the domain and constitute two polyproline helix II conformations. Since the proline residues are conserved within the titin FNIII family, they might attach titin to the thick filaments in a conserved fashion. 9.
Fürst DO, Osborn M, Nave R, Weber K: The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J Cell Biol 1988, 106:1563-1572.
10. Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, •• Suzuki K, Obermayr F, Herrmann B, Granzier H et al.: The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19 kD ligand (T-cap) is required for sarcomeric integrity. J Cell Biol 1998, 143:1013-1027. This study focuses on the molecular interactions of Z-line titin. Results demonstrate the molecular layout of titin within the Z-line: mammalian titins have at least four potential binding sites for α-actinin, and the amino-terminal titin Z1 and Z2 repeats bind to a novel 19 kDa protein, referred to as titincap (T-cap). Using dominant-negative approaches in cardiac myocytes, the interaction of titin Z1 and Z2 domains with T-cap is shown to be required for the structural integrity of sarcomeres. 11. Witt CC, Olivieri N, Centner T, Kolmerer B, Millevoi S, Labeit D, • Jockusch H, Pastore A, Labeit S: A survey of the primary structure and the interspecies conservation of I-band titin’s elastic elements in vertebrates. J Struct Biol 1998, 122:1-10. This study shows that the differentially expressed tandem Ig repeats in I-band titin’s elastic region form a specific subgroup of the titin Ig repeat family and differ structurally from those expressed elsewhere in the molecule. 12. Obermann WM, Gautel M, Steiner F, van der Ven PF, Weber K, Fürst DO: The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy. J Cell Biol 1996, 134:1441-1453. 13. Obermann WM, Gautel M, Weber K, Fürst DO: Molecular structure •• of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin. EMBO J 1997, 16:211-220. A thorough study of the layout and molecular interactions of M-line titin. The carboxy-terminal 200 kDa of the titin filament spans the entire M-line region; Ig repeats inside the M-line region may provide attachment sites for the M-line protein myomesin.
14. Millevoi S, Trombitas K, Kostin S, Schaper J, Pelin K, Kolmerer B, •• Granzier H, Labeit S: Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs. J Mol Biol 1998, 282:111-123. Millevoi and colleagues report the cloning, full-length sequence and gene mapping of nebulette, a nebulin-related protein from cardiac muscle. Both nebulin and nebulette are filamentous proteins that insert into the periphery of the Z-line. Similar to titin’s 45-residue Z-repeats, nebulin/nebulette also contain a family of differentially expressed Z-repeats. It is likely that the differential expression of titin and nebulin/nebulette accounts for Z-line tissue diversity. 15. Moncman CL, Wang K: Nebulette: a 107 kD nebulin-like protein in cardiac muscle. Cell Motil Cytoskeleton 1995, 32:205-225. 16. Goll DE, Suzuki, A, Temple J, Holmes GR: Studies on purified α-actinin. I. Effect of temperature and tropomyosin on the α-actinin/F-actin interaction. J Mol Biol 1972, 67:469-488. 17. Squire JM: Architecture and function in the muscle sarcomere. • Curr Opin Struct Biol 1997, 7:247-257. A very stimulating review on recent progress on the structural biology of the sarcomere. 18. Vigoreaux JO: The muscle Z band: lessons in stress management. J Muscle Res Cell Motil 1994, 15:237-255. 19. Mues A, van der Ven PFM, Young P, Fürst DO, Gautel M: Two immunoglobulin-like domains of the Z-disc portion of titin interact in a conformation-dependent way with telethonin. FEBS Lett 1998, 428:111-114. 20. Gautel M, Goulding D, Bullard B, Weber K, Fürst DO: The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers. J Cell Sci 1996, 109:2747-2754. 21. Ohtsuka H, Yajima H, Kimura S, Maruyama K: Binding of the N •• terminal fragment of connectin/titin to alpha-actinin as revealed by yeast two-hybrid systems. FEBS Lett 1997, 401:65-67. See annotation [22••]. 22. Sorimachi H, Freiburg A, Kolmerer B, Ishiura S, Stier G, Gregorio CC, •• Labeit D, Linke WA, Suzuki K, Labeit S: Tissue-specific expression and alpha-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs. J Mol Biol 1998, 270:688-695. Both studies [21••,22••] show that the 10 kDa carboxy-terminal domain of α-actinin binds to titin’s 45-residue Z-repeats. Thus, antiparallel α-actinin dimers can attach at both ends to titin filaments, whereas their amino termini can cross-link them to thin filaments. The Z-repeats are shown to be expressed in different copy numbers in different tissues, possibly accounting for the variability of Z-filaments in different muscle types. 23. Young P, Ferguson C, Bañuelos S, Gautel M: Molecular structure of •• the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of α-actinin. EMBO J 1998, 17:1614-1624. Besides confirming the interaction of titin’s Z-repeats with α-actinin, Young and colleagues identified that a single site at the periphery of Z-disc titin also may bind to α−actinin. This site just flanks the Z-repeats and, in contrast to them, does not bind to the carboxy-terminal end of α-actinin, but to the central rod domains (R2-R3) containing spectrin-like repeats. Thus, titin may contain two distinct α-actinin binding motifs. 24. Linke WA, Ivemeyer M, Labeit S, Hinssen H, Ruegg JC, Gautel M: • Actin–titin interaction in cardiac myofibrils: probing a physiological role. Biophys J 1997, 73:905-919. See annotation [26•]. 25. Trombitas K, Granzier H: Actin removal from cardiac myocytes • shows that near the Z-line titin attaches to actin while under tension. Amer J Physiol 1997, 273:C662-C670. See annotation [26•]. 26. Granzier H, Kellermayer M, Trombitas K: Titin elasticity and • mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction. Biophys J 1997, 73:2043-2053. These studies [24•,25•] report that the portion of I-band titin near the Z-disc resists stretch when thin filaments are intact, and that following thin-filament removal titin near the Z-disk collapses in short sarcomeres and extends upon sarcomere elongation [26•]. Therefore, this part of I-band titin is predicted to interact with actin. 27.
Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishiura S, Sasagawa N, Sorimachi N, Shimada H, Tagawa K, Maruyama K, Suzuki K: Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J Biol Chem 1995, 270:31158-31162.
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28. Kolmerer B, Olivieri N, Witt CC, Herrmann BG, Labeit S: Genomic organization of the M line titin and its tissue-specific expression in two distinct isoforms. J Mol Biol 1996, 256:556-563. 29. Labeit S, Gautel M, Lakey A, Trinick J: Towards a molecular understanding of titin. EMBO J 1992, 11:1711-1716. 30. Houmeida A, Holt J, Tskhovrebova L, Trinick J: Studies of the interaction between titin and myosin. J Cell Biol 1995, 131:1471-1481. 31. Freiburg A, Gautel M: A molecular map of the interaction between titin and myosin-binding protein C. Implications for sarcomeric assembly in familiar hypertrophic cardiomyopathy. Eur J Biochem 1996, 235:317-323. 32. Okagaki T, Weber FE, Fischman DA, Vaughan KT, Mikawa T, Reinach FC: The major myosin-binding domain of skeletal muscle MyBP-C (Cprotein) resides in the COOH-terminal, immunoglobulin C2 motif. J Cell Biol 1993, 123:619-626. 33. Linke WA, Ivemeyer M, Olivieri N, Kolmerer B, Rüegg JC, Labeit S: Towards a molecular understanding of the elasticity of titin. J Mol Biol 1996, 261:62-71. 34. Linke WA, Stockmeier MR, Ivemeyer M, Hosser H, Mundel P: • Characterizing titin’s I-band Ig domain region as an entropic spring. J Cell Sci 1998, 111:1567-1574. Both studies [33,34•] use intact myofibrils to characterize the behavior of the tandem Ig and PEVK titin segments, and conclude that they may act as a two-spring system. 35. Gautel M, Leonard K, Labeit S: Phosphorylation of KSP-motifs in the C-terminal region of titin in differentiating myoblasts. EMBO J 1993, 12:3827-3834. 36. Turnacioglu KK, Mittal B, Sanger JM, Sanger JW: Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin. Cell Motil Cytoskeleton 1996, 34:108-121. 37.
Peckham M, Young P, Gautel M: Constitutive and variable regions of Z-disk titin/connectin in myofibril formation: a dominantnegative screen. Cell Struct Funct 1997, 22:95-101.
38. Lin Z, Kijikata T, Zhang Z, Choi J, Holtzer S, Sweeney HS, Holtzer H: •• Dispensability of the actin-binding site and spectrin repeats for targeting sarcomeric α-actinin into maturing Z bands in vivo: implications for in vitro binding studies. Development 1998, 199:291-308. An elegant study investigating the roles of specific domains of α-actinin in the initial targeting and maintenance of striated myofibrils. This study indicates the limited value of predicting the behavior of sarcomeric components based on in vitro binding studies. 39. Kellermayer MS, Smith SB, Granzier HL, Bustamante C: Folding•• unfolding transitions in single titin molecules characterized with laser tweezers. Science 1997, 276:1112-1116. See annotation [41••]. 40. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE: Reversible •• unfolding of individual titin immunoglobulin domains by AFM. Science 1997, 276:1109-1112. See annotation [41••]. 41. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM: Elasticity and •• unfolding of single molecules of the giant muscle protein titin. Nature 1997, 387:308-312. The papers [39••,40••,41••] are pioneering studies in which single titin molecules were manipulated and titin’s force-extension properties were characterized at the molecular level. The results obtained reveal that titin behaves as an entropic spring. Unfolding takes place at high force during stretching of the molecule, and refolding at low force during release of the molecule. 42. Trombitas K, Greaser M, Labeit S, Jin J-P, Kellermayer M, Helmes M, •• Granzier H: Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments. J Cell Biol 1998, 140:853-859. See annotation [43••].
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43. Linke WA, Ivemeyer M, Mundel P, Stockmeyer MR, Kolmerer B. •• Nature of PEVK-titin elasticity in skeletal muscle. Proc Natl Acad Sci USA 1998, 95:8052-8057. Both Trombitas et al. [42••] and Linke et al. [43••] use novel epitope-mapped antibodies to monitor the mechanical behavior of central I-band titin during myofibrillar stretch. They prove that the PEVK (in the single letter code for amino acids) region of titin acts as a highly extensible spring element which is responsible for the passive tension response of the titin filament. 44. Holtzer H, Hijikata T, Lin ZX, Zhang ZQ, Holtzer S, Protasi F, Frazini-Armstrong C, Sweeney HL: Independent assembly of 1.6 µm long bipolar MHC filaments and I-Z-I bodies. Cell Struct Funct 1997, 22:83-93. 45. Mayans O, van der Ven P, Wilm M, Mues A, Young P, Fürst DO, •• Wilmanns M, Gautel M: Structural basis for activation of the titin kinase domain during myofibrillogenesis. Nature 1998, 395:863-869. A pioneering study which reports the three-dimensional structure of the titin kinase domain and may provide a molecular understanding of its mode of activation. The Z-disc protein and titin ligand T-cap(telethonin) has been identified as a potential substrate of the titin kinase domain using in vitro phosphorylation assays. Thereby, M-line and Z-disc assembly might be coordinated during myofibrilogenesis. 46. Tokuyasu KT, Maher PA: Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alphaactinin dots within titin spots at the time of the first myofibril formation. J Cell Biol 1987, 105:2795-2801. 47.
Shimada Y, Komiyama M, Begum S, Maruyama K: Development of connectin/titin and nebulin in striated muscles of chicken. Adv Biophys 1996, 33:223-234.
48. Fürst DO, Osborn M, Weber K: Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J Cell Biol 1989, 109:517-527. 49. Dabiri GA, Turnacioglu KK, Sanger JM, Sanger JW: • Myofibrillogenesis visualized in living embryonic cardiomyocytes. Proc Natl Acad Sci USA 1997, 94:9493-9498. The distribution patterns of green fluorescent protein tagged α-actinin were visualized during myofibril assembly in cultured cardiac myocytes. Nonmuscle myosin IIB is detected at the leading edges of the cells. 50. Fulton AB, Alftine C: Organization of protein and mRNA for titin and other myofibril components during myofibrillogenesis in cultured chicken skeletal muscle. Cell Struct Funct 1997, 22:51-58. 51. Liu F, Barral JM, Bauer CC, Ortiz I, Cook RG, Schmid MF, Epstein HF: • Assemblases and coupling proteins in thick filament assembly. Cell Struct Funct 1997, 22:155-162. A thorough review of thick filament assembly and length regulation. 52. Liu F, Bauer CC, Ortiz I, Cook RG, Schmid MF, Epstein HF: β•• filagenin, a newly identified protein coassembling with myosin and paramyosin in Caenorhabditis elegans. J Cell Biol 1998, 140:347-353. Purification and cloning of a novel protein which coassembles with paramyosin and myosin filaments. β-filagenin may link these two filament systems, comparable to C-protein in vertebrates which links the titin and myosin filaments. 53. Barral JM, Bauer CC, Ortiz I, Epstein HF: Unc-45 mutations in •• Caenorrhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly. J Cell Biol 1998, 143:1215-1225. The UNC-45 locus is proposed to act as a protein machine required for myosin assembly. The UNC-45 gene product contains CRO1/She4p-like domains implicated in signal transduction pathways. Mutations in these regulatory domains interfere with the number and structure of thick filaments assembled. 54. Littlefield R, Fowler VM: Defining actin filament length in striated •• muscle. Annu Rev Dev Cell Biol 1998, 14:487-525. Interesting chapter discussing several hypothetical mechanisms by which muscle cells can generate uniform actin filament length distributions including a mechanism by which titin and its kinase domain may play a role in this process.