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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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Overexpression of  titin Z1-Z2 or T-cap results  in marked disruption of  Z-discs. Cardiac myocytes  transfected with a plasmid  encoding the fusion proteins:  (a and b) myc-titin Z1-Z2  and (c and d) myc–T-cap.  Cells were fixed 48 h after  transfection and double  stained with (a and c) monoclonal anti-myc antibodies  followed by FITC-conjugated donkey anti–mouse  antibodies and (b and d)  polyclonal anti–α-actinin  antibodies followed by Texas  red–conjugated F(ab′)2  fragments of donkey anti– rabbit antibodies. * marks  cells that were not transfected. Bar, 10 μm.
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Figure 8: Overexpression of titin Z1-Z2 or T-cap results in marked disruption of Z-discs. Cardiac myocytes transfected with a plasmid encoding the fusion proteins: (a and b) myc-titin Z1-Z2 and (c and d) myc–T-cap. Cells were fixed 48 h after transfection and double stained with (a and c) monoclonal anti-myc antibodies followed by FITC-conjugated donkey anti–mouse antibodies and (b and d) polyclonal anti–α-actinin antibodies followed by Texas red–conjugated F(ab′)2 fragments of donkey anti– rabbit antibodies. * marks cells that were not transfected. Bar, 10 μm.

Mentions: To gain insight into the functional significance of the NH2 terminus of titin in cells, we overexpressed titin Z1-Z2 under the control of a strong viral promoter (CMV) in the hope that the expressed protein would compete with T-cap to generate dominant-negative effects on sarcomere formation. First, we transfected cells with a plasmid encoding a myc-epitope–tagged titin Z1-Z2 fragment. A myc epitope was inserted at the NH2 terminus to distinguish the recombinant protein from endogenous protein. Severe disruption of Z-disc structure was observed in all transfected cells where titin Z1-Z2 was overexpressed (24, 48, and 72 h after transfection) as seen by a marked loss in α-actinin staining, as compared with nontransfected cells, which demonstrate a strong periodic Z-line staining with anti–α-actinin antibodies (Fig. 8, identical cells stained for [a] myc and [b] α-actinin; compare staining patterns in transfected cells that stain positively with anti-myc antibodies with nontransfected cells marked with *). As a complementary approach, to support the idea that the effects we were observing were a result of disrupting the interaction between titin and T-cap, cardiac myocytes were also transfected with a plasmid encoding a myc–T-cap fusion protein. Overexpression of T-cap resulted in an identical phenotype: severe disruption of Z-lines was discerned by staining for α-actinin (Fig. 8, staining for [c] myc and [d] for α-actinin; compare staining patterns in transfected cells with nontransfected cells marked with *). The severe phenotype observed suggested that other sarcomeric components might also be disrupted by the overexpression of these peptides. To study this, the distribution of the major thin filament protein, actin; the major thick filament protein, myosin; and different epitopes of titin along the length of the molecule were also observed in both pCMVmyc-titin Z1-Z2 and pCMVmyc–T-cap–transfected cells. Results from this experiment revealed that overexpression of either titin Z1-Z2 or T-cap resulted in disruption of all sarcomeric proteins studied. (See Fig. 9 for cardiac myocytes transfected with pCMVmyc titin Z1-Z2 and double stained for [a] myc and [b] actin, and [f] myc and [g] myosin; compare staining patterns in transfected cells that stain positively with anti-myc antibodies with nontransfected cells marked with * in b and e. Staining for other contractile protein constituents studied is not shown.) Similarly, when the T-cap–GFP COOH-terminal fusion protein was overexpressed, a similar disruption was observed, while overexpression of GFP alone did not lead to the disruption of myofibrils in the transfected cells (data not shown). This suggests that the observed phenotype was not due to overexpressing too much protein in this cell type (i.e., by using such a strong promoter; others have also observed no effect in the same cell type for example, Turnacioglu et al., 1997).


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)

Overexpression of  titin Z1-Z2 or T-cap results  in marked disruption of  Z-discs. Cardiac myocytes  transfected with a plasmid  encoding the fusion proteins:  (a and b) myc-titin Z1-Z2  and (c and d) myc–T-cap.  Cells were fixed 48 h after  transfection and double  stained with (a and c) monoclonal anti-myc antibodies  followed by FITC-conjugated donkey anti–mouse  antibodies and (b and d)  polyclonal anti–α-actinin  antibodies followed by Texas  red–conjugated F(ab′)2  fragments of donkey anti– rabbit antibodies. * marks  cells that were not transfected. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132961&req=5

Figure 8: Overexpression of titin Z1-Z2 or T-cap results in marked disruption of Z-discs. Cardiac myocytes transfected with a plasmid encoding the fusion proteins: (a and b) myc-titin Z1-Z2 and (c and d) myc–T-cap. Cells were fixed 48 h after transfection and double stained with (a and c) monoclonal anti-myc antibodies followed by FITC-conjugated donkey anti–mouse antibodies and (b and d) polyclonal anti–α-actinin antibodies followed by Texas red–conjugated F(ab′)2 fragments of donkey anti– rabbit antibodies. * marks cells that were not transfected. Bar, 10 μm.
Mentions: To gain insight into the functional significance of the NH2 terminus of titin in cells, we overexpressed titin Z1-Z2 under the control of a strong viral promoter (CMV) in the hope that the expressed protein would compete with T-cap to generate dominant-negative effects on sarcomere formation. First, we transfected cells with a plasmid encoding a myc-epitope–tagged titin Z1-Z2 fragment. A myc epitope was inserted at the NH2 terminus to distinguish the recombinant protein from endogenous protein. Severe disruption of Z-disc structure was observed in all transfected cells where titin Z1-Z2 was overexpressed (24, 48, and 72 h after transfection) as seen by a marked loss in α-actinin staining, as compared with nontransfected cells, which demonstrate a strong periodic Z-line staining with anti–α-actinin antibodies (Fig. 8, identical cells stained for [a] myc and [b] α-actinin; compare staining patterns in transfected cells that stain positively with anti-myc antibodies with nontransfected cells marked with *). As a complementary approach, to support the idea that the effects we were observing were a result of disrupting the interaction between titin and T-cap, cardiac myocytes were also transfected with a plasmid encoding a myc–T-cap fusion protein. Overexpression of T-cap resulted in an identical phenotype: severe disruption of Z-lines was discerned by staining for α-actinin (Fig. 8, staining for [c] myc and [d] for α-actinin; compare staining patterns in transfected cells with nontransfected cells marked with *). The severe phenotype observed suggested that other sarcomeric components might also be disrupted by the overexpression of these peptides. To study this, the distribution of the major thin filament protein, actin; the major thick filament protein, myosin; and different epitopes of titin along the length of the molecule were also observed in both pCMVmyc-titin Z1-Z2 and pCMVmyc–T-cap–transfected cells. Results from this experiment revealed that overexpression of either titin Z1-Z2 or T-cap resulted in disruption of all sarcomeric proteins studied. (See Fig. 9 for cardiac myocytes transfected with pCMVmyc titin Z1-Z2 and double stained for [a] myc and [b] actin, and [f] myc and [g] myosin; compare staining patterns in transfected cells that stain positively with anti-myc antibodies with nontransfected cells marked with * in b and e. Staining for other contractile protein constituents studied is not shown.) Similarly, when the T-cap–GFP COOH-terminal fusion protein was overexpressed, a similar disruption was observed, while overexpression of GFP alone did not lead to the disruption of myofibrils in the transfected cells (data not shown). This suggests that the observed phenotype was not due to overexpressing too much protein in this cell type (i.e., by using such a strong promoter; others have also observed no effect in the same cell type for example, Turnacioglu et al., 1997).

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