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The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

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Model for the insertion of the NH2-terminal region of  titin into Z-discs. Titin's residues 1–200 are shared by titins from  all muscle tissues and code for two Ig domains (Z1 and Z2). Together, these terminal domains interact with a 19-kD ligand,  referred to as titin-cap (T-cap). The Z1-Z2/T-cap complex is located at the periphery of the Z-line. T-cap contains a 27-residue-long COOH-terminal serine/proline and basic residue-rich domain that codes for potential phosphorylation motifs and is not  required for interaction with titin. Titin residues 200–430 are predicted to extend to the center of the Z-line. Titin residues 430–700  contain the Z-repeats that potentially bind to the COOH terminus of α-actinin. Titins from opposite half-sarcomeres are predicted to share full overlap in Z-lines (about 100 nm in soleus and  cardiac muscle Z-lines).
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Figure 11: Model for the insertion of the NH2-terminal region of titin into Z-discs. Titin's residues 1–200 are shared by titins from all muscle tissues and code for two Ig domains (Z1 and Z2). Together, these terminal domains interact with a 19-kD ligand, referred to as titin-cap (T-cap). The Z1-Z2/T-cap complex is located at the periphery of the Z-line. T-cap contains a 27-residue-long COOH-terminal serine/proline and basic residue-rich domain that codes for potential phosphorylation motifs and is not required for interaction with titin. Titin residues 200–430 are predicted to extend to the center of the Z-line. Titin residues 430–700 contain the Z-repeats that potentially bind to the COOH terminus of α-actinin. Titins from opposite half-sarcomeres are predicted to share full overlap in Z-lines (about 100 nm in soleus and cardiac muscle Z-lines).

Mentions: To ascertain the molecular layout of titin within the Z-line, we generated novel polyclonal antibodies against (a) the first two NH2-terminal Ig domains of titin (anti–Z1-Z2 antibodies), which are expressed in all striated muscles, and (b) the Zr5 and 6 titin repeats (anti–titin Zr5-6 antibodies) (Labeit and Kolmerer, 1995; Sorimachi et al., 1997). Under standard labeling and strong blocking (60 min with 2% BSA) conditions, antibodies that are specific to the titin Z1-Z2 domains stained the borders of the Z-lines only (Fig. 1, A–C). This result was surprising based on previous reports that predicted that the NH2 terminus of titin is found in the middle of the Z-line (Gautel et al., 1996). These new observations indicate that either the NH2 terminus of titin completely spans the Z-line to the adjacent sarcomere or that the NH2-terminal end of the titin molecule does not penetrate the Z-line. Next, to distinguish between these possibilities and to rule out that the absence of integral Z-line labeling was a result of the failure of the first antibody to penetrate the Z-line, labeling studies were also performed with the anti–titin Zr5-6 antibodies. Although these polyclonal antibodies were generated against the Zr5 and 6 repeats which are differentially expressed in striated muscle, they most likely recognize all Zr repeats because of the very high sequence similarity between them (Sorimachi et al., 1997); this is supported by the observation that the anti–titin Zr5-6 antibodies label the Z-lines in myofibrils from rat psoas muscle (data not shown) which only express the Z-repeats 1, 2, 3 and 7 (Sorimachi et al., 1997). Immunolabeling with the anti–titin Zr5-6 antibodies resulted in strong labeling throughout the Z-line, indicating that this structure can be penetrated by antibodies (Fig. 1, D–F). The labeling pattern for the Z-repeats, together with the labeling for the Z1-Z2 Ig domains, suggests that titin molecules from neighboring sarcomeres fully overlap and extend to the opposite ends of the Z-disc (see model: Fig. 11).


The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

Model for the insertion of the NH2-terminal region of  titin into Z-discs. Titin's residues 1–200 are shared by titins from  all muscle tissues and code for two Ig domains (Z1 and Z2). Together, these terminal domains interact with a 19-kD ligand,  referred to as titin-cap (T-cap). The Z1-Z2/T-cap complex is located at the periphery of the Z-line. T-cap contains a 27-residue-long COOH-terminal serine/proline and basic residue-rich domain that codes for potential phosphorylation motifs and is not  required for interaction with titin. Titin residues 200–430 are predicted to extend to the center of the Z-line. Titin residues 430–700  contain the Z-repeats that potentially bind to the COOH terminus of α-actinin. Titins from opposite half-sarcomeres are predicted to share full overlap in Z-lines (about 100 nm in soleus and  cardiac muscle Z-lines).
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Related In: Results  -  Collection

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Figure 11: Model for the insertion of the NH2-terminal region of titin into Z-discs. Titin's residues 1–200 are shared by titins from all muscle tissues and code for two Ig domains (Z1 and Z2). Together, these terminal domains interact with a 19-kD ligand, referred to as titin-cap (T-cap). The Z1-Z2/T-cap complex is located at the periphery of the Z-line. T-cap contains a 27-residue-long COOH-terminal serine/proline and basic residue-rich domain that codes for potential phosphorylation motifs and is not required for interaction with titin. Titin residues 200–430 are predicted to extend to the center of the Z-line. Titin residues 430–700 contain the Z-repeats that potentially bind to the COOH terminus of α-actinin. Titins from opposite half-sarcomeres are predicted to share full overlap in Z-lines (about 100 nm in soleus and cardiac muscle Z-lines).
Mentions: To ascertain the molecular layout of titin within the Z-line, we generated novel polyclonal antibodies against (a) the first two NH2-terminal Ig domains of titin (anti–Z1-Z2 antibodies), which are expressed in all striated muscles, and (b) the Zr5 and 6 titin repeats (anti–titin Zr5-6 antibodies) (Labeit and Kolmerer, 1995; Sorimachi et al., 1997). Under standard labeling and strong blocking (60 min with 2% BSA) conditions, antibodies that are specific to the titin Z1-Z2 domains stained the borders of the Z-lines only (Fig. 1, A–C). This result was surprising based on previous reports that predicted that the NH2 terminus of titin is found in the middle of the Z-line (Gautel et al., 1996). These new observations indicate that either the NH2 terminus of titin completely spans the Z-line to the adjacent sarcomere or that the NH2-terminal end of the titin molecule does not penetrate the Z-line. Next, to distinguish between these possibilities and to rule out that the absence of integral Z-line labeling was a result of the failure of the first antibody to penetrate the Z-line, labeling studies were also performed with the anti–titin Zr5-6 antibodies. Although these polyclonal antibodies were generated against the Zr5 and 6 repeats which are differentially expressed in striated muscle, they most likely recognize all Zr repeats because of the very high sequence similarity between them (Sorimachi et al., 1997); this is supported by the observation that the anti–titin Zr5-6 antibodies label the Z-lines in myofibrils from rat psoas muscle (data not shown) which only express the Z-repeats 1, 2, 3 and 7 (Sorimachi et al., 1997). Immunolabeling with the anti–titin Zr5-6 antibodies resulted in strong labeling throughout the Z-line, indicating that this structure can be penetrated by antibodies (Fig. 1, D–F). The labeling pattern for the Z-repeats, together with the labeling for the Z1-Z2 Ig domains, suggests that titin molecules from neighboring sarcomeres fully overlap and extend to the opposite ends of the Z-disc (see model: Fig. 11).

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Show MeSH