Vertebrate Z-lines link actin filaments in one sarcomere to actin filaments with opposite polarity, in the adjacent sarcomere. Although there are substantial variations in Z-lines from different vertebrate muscles and fiber types, all vertebrate Z-lines share the packaging of the antiparallel thin filaments from opposite half-sarcomeres into a tetragonal lattice, which is cross-linked by α-actinin (otherwise referred to as Z-filaments: Yamaguchi et al., 1985
; Vigoreaux, 1994
; Luther et al., 1995
; Schroeter et al., 1996
; Squire, 1997
). Other less characterized components of Z-lines, including titin, are likely to be important in the regulation of Z-line assembly. Thus, a detailed knowledge of titin's layout and of its molecular interactions in Z-lines is necessary.
The Z1 and Z2 Ig domains (residues 1–200) are expressed in all striated muscles, whereas the subsequent residues 200–700, including the Z-repeats, are differentially expressed (Sorimachi et al., 1997
). We sought to define the titin Z-disc layout more precisely by generating antibodies to the constitutively expressed titin Z1-Z2 domains, and to the differentially expressed residues 560– 650. Immunolocalization at the ultrastructural level revealed that the NH2
-terminal Z1-Z2 domains localized to the periphery of the Z-disc; this finding suggests that titin molecules from opposite sarcomeres fully cross each other within the Z-disc (Fig. ). A possible explanation for the conflicting Z-line layout models that have been proposed by others (Gautel et al., 1996
; Young et al., 1998
) is that the antibodies used in these studies were generated against titin repeats which may cross-react with other Z repeats (e.g., Fürst et al., 1989b
; Gautel et al., 1996
; anti– Zr5-6 antibodies used in this study). Here, we also used immunoelectron microscopy to localize the nonrepetitive T-cap. As expected from our localization of titin Z1-Z2, T-cap epitopes were also detected at the edges of the Z-disc (Fig. ). Furthermore, the Z-repeat epitopes were present throughout the Z-line (Fig. ). This may suggest that the Z-repeat family longitudinally extends within the Z-disc lattice (Fig. ). Taken together, the immunolocalization studies with the anti–Z1-Z2, anti-Zr5/6, and anti– T-cap antibodies demonstrate that the titin NH2
-terminal region spans the entire Z-line with the titin Z1-Z2 (residues 1–200)/T-cap protein complex extending to the I-Z junction of the adjacent sarcomere. In combination with other immunoelectron microscopy data (Yajima et al., 1996
; Gautel et al., 1996
; Young et al., 1998
), it can be concluded that ~200–700 residues of titin correspond to titin's Z-line–spanning region. Therefore, we suggest the term “Z-disc integrative domain” for this segment of titin.
In order for the ~500 residues to span the 110-nm-wide Z-line of rabbit soleus muscle, each residue of Z-disc titin would have a mean axial extension of ~2.2 Å per residue. This figure is intermediate between the values predicted for an extended α-helix (1.5 Å per residue) or an extended β-sheet structure (3.2–3.4 Å per residue). Sequence analysis of the titin residues 200–700 reveals segments with both predicted β-sheet and α-helical secondary structural folds. Therefore, we speculate that titin in the interior of the Z-line is composed of extended β-sheet and α-helical secondary structure elements connected in series. More highly folded domains in Z-disc titin, such as globular folds, appear unlikely. Based on our model, we would predict that residues 200–700 of the titin gene contain differentially expressed exons that are skipped in tissues and fibers that have narrower Z-lines. In support of this hypothesis, many differential splicing events have been observed to occur in this region (Gautel et al., 1996
; Sorimachi et al., 1997
). Furthermore, studies on chick breast muscles that have narrow Z-lines revealed that the differentially expressed Z-line segment of titin is very short and contains only ~450 residues (Yajima et al., 1996
). It will be interesting to also determine Z-disc titin sequences from teleost bony fish muscles, which have ~50-nm-wide Z-lines (Luther et al., 1995
), in contrast to the ~70–110-nm-wide Z-lines of mammalian muscles.
Rabbit cardiac Z-disc titin contains seven copies of the 45-residue Z-repeats, soleus muscle expresses isoforms of four and six repeats, and rabbit psoas muscle contains four repeats (Sorimachi et al., 1997
). Previously the Z-repeat, Zr7, was shown to interact with α-actinin (Ohtsuka et al., 1997
; Sorimachi et al., 1997
). Here, in vitro binding studies show that although Zr1, 2, 3, and 7 (repeats expressed in psoas muscle) together bind to the COOH terminus of α-actinin, each of these single Z-repeats are alone sufficient for this interaction. In contrast, the single repeats Zr4, 5, and 6 from soleus and heart muscles failed to bind in our assay; however, the triple repeat construct Zr4, 5, 6 interacted with α-actinin. This may suggest that the individual repeats Zr4, 5, and 6 also bind, although possibly more weakly, to the COOH terminus of α-actinin. Earlier studies using the yeast two-hybrid system detected titin/ α-actinin interactions only within the Z-repeat region (Ohtsuku et al., 1997; Sorimachi et al., 1997
). However, after the original submission of this manuscript, it was reported that specific titin Z-repeats also bind to the central spectrin repeats within α-actinin, and that another non– Z-repeat α-actinin binding site (residues 760–826 of the human cardiac titin) is present in titin (Young et al., 1998
). It was concluded from these findings that two distinct types of titin interactions lead to an asymmetrical sorting of α-actinin. Further studies will be required to resolve the range of possible in vivo titin/α-actinin interactions. At present, we speculate, based upon our data, that the titin Z-repeats may regulate the number and distribution of Z-filaments in Z-lines by their binding to the COOH-terminal α-actinin domains. In cardiac titin, Z-repeats may provide up to seven attachment sites for Z-filaments per titin molecule, whereas soleus and psoas muscle titins may provide six and four, respectively (Sorimachi et al., 1997
). The differential expression of the titin Z-repeats may explain the presence of different numbers of Z-filament layers in the Z-lines (Rowe, 1973
). As for the functional significance, we hypothesize that the differential expression of Z-repeats modulates the α-actinin/titin/actin network of the Z-lines and that this allows the mechanical strength of the Z-line to be varied.
-terminal immunoglobulin domains Z1 and Z2 are shared by all titins. We searched for a potential function(s) of these domains. Using the yeast two-hybrid approach and by subsequent studies with expressed recombinant proteins, we found that titin Z1 and Z2 domains bind to a 19-kD sarcomeric protein. Deletion studies indicated that both the Z1 and Z2 domains are required for this interaction, suggesting that the binding site for the 19-kD protein contains residues from both domains. Surprisingly, the identified sequence of the human 19-kD protein is identical to a recently determined cDNA sequence from a putative thick filament–associated protein, telethonin (Valle et al., 1997
). Note that thick filament association in this study was determined by a single immunofluorescence study. To clarify this, we analyzed further if the cloned 19-kD protein is indeed a Z-disc protein interacting with titin, or rather a thick filament–associated protein. Initially, we investigated the targeting of the 19-kD titin ligand into myofibrils by expressing it in cardiac myocytes as a green fluorescent fusion protein. This experiment demonstrated that the 19-kD protein in cardiac myocytes is targeted to the Z-line, but not to the A band. Moreover, localization studies using a polyclonal antibody that specifically recognizes the 19-kD protein demonstrated staining at the Z-line, but not the A band. These data are consistent with the finding that the 19-kD protein is a ligand of the Z1-Z2 titin domains. Based on the 19-kD protein's localization, assembly, and interaction with the extreme NH2
-terminal domains of titin, we propose the name “titin-cap” (T-cap) for the 19-kD protein, rather than the initial suggestion, Telethonin, which referred to a putative thick filament protein (Valle et al., 1997
On the transcriptional level, T-cap transcripts are abundantly expressed in heart and skeletal muscles (Valle et al., 1997
). At day 9.5 pc, the distribution of T-cap transcripts in somites and in the developing heart closely resembles that of titin (Fig. for T-cap; Kolmerer et al., 1996
for titin). Future studies will be needed to address the significance of T-cap transcripts in the otic vesicle during early development, and if the initial appearance of titin transcripts at 8.0 pc (Kolmerer et al., 1996
) precedes that of T-cap.
To elucidate further the functional significance of titin's association with the Z-line components, we overexpressed the NH2-terminal domains Z1 and Z2 of titin and their binding partner, T-cap, in primary cultures of cardiac myocytes. Using either microinjection or transfection techniques, overexpression of either molecule resulted in severe myofibril disruption. One explanation for this phenomenon is that a dominant-negative phenotype occurred. That is, overexpressed titin Z1-Z2 fragments competed for the T-cap binding site of endogenous titin, and overexpressed T-cap competed for the titin binding site on endogenous T-cap. These events might result in inhibiting endogenous titin and/or T-cap from assembling and stabilizing Z-discs.
The results from our titin Z1-Z2 and T-cap overexpression studies suggest that the association of titin filaments with Z-discs is critical for the assembly and maintenance of myofibril structure. Disruption of myofibrils has also been reported as a result of overexpression of (a
) the first 362 amino acids of titin (zeugmatin) in cardiac and skeletal muscle cultures (Turnacioglu et al., 1997
) the entire Z-disc region of titin in the myogenic cell line Hsk btsA58 (Peckham et al., 1997
); and (c
) a COOH-terminal truncated fragment of α-actinin (thus, missing it's binding site for titin) in skeletal muscle cultures (Schultheiss et al., 1992
). Thus, the association of titin filaments with Z-discs (i.e., via its interaction with T-cap and α-actinin) is critical for the assembly and maintenance of myofibril structure.
It is not difficult to understand why disrupting the interaction of titin with α-actinin would result in Z-line disruption, since it is likely that the interaction of these two proteins (together with the thin filaments) contributes greatly to the structural continuity of the sarcomere. It is more difficult to understand why overexpression of the 19-kD T-cap, which interacts with the most NH2-terminal region of titin, located at the periphery of the Z-line, is also required for Z-line/sarcomere maintenance. A highly speculative idea is that perhaps T-cap acts as a “bolt,” functioning to anchor the giant titin filament at its NH2 terminus. Future studies will be focused on exploring further the interaction of titin with T-cap, e.g., determining how the interaction of T-cap with titin is regulated.
In summary, we report here that the NH2
terminus of titin spans the Z-line. So far, using different regions of Z-disc titin as baits in yeast two-hybrid studies, two ligands for this region of titin have been identified: α-actinin (Ohtsuka et al., 1997
; Sorimachi et al., 1997
; Young et al., 1998
) and T-cap (this study). Studies in live myocytes demonstrate that all components of this network, including the NH2
terminus of titin, COOH terminus of α-actinin, and T-cap are required for Z-line structure. Since the amount of titin filament overlap coincides with Z-line width, and many studies have demonstrated that α-actinin and Z-disc titin are important in myofibril formation, it appears likely that the NH2
-terminal region of titin is intimately involved in the assembly of Z-lines during myogenesis.