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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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T-cap assembles at  the Z-disc in cardiac myocytes. Fluorescent confocal  image of a cardiac myocyte  expressing (a) T-cap–GFP  that was fixed and stained  with (b) anti–titin T12 antibodies followed by Texas  red–conjugated donkey anti– mouse antibodies. (c) Merged  image of a and b demonstrating overlap of the two fluorochromes. Bar, 10 μm.
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Figure 7: T-cap assembles at the Z-disc in cardiac myocytes. Fluorescent confocal image of a cardiac myocyte expressing (a) T-cap–GFP that was fixed and stained with (b) anti–titin T12 antibodies followed by Texas red–conjugated donkey anti– mouse antibodies. (c) Merged image of a and b demonstrating overlap of the two fluorochromes. Bar, 10 μm.

Mentions: For functional studies on the assembly of T-cap, primary cultures of chick cardiac myocytes were chosen as a model system. Owing to their flat, well-spread morphology, these cells are ideally suited for detailed immunolocalization observations of the spatial relationships among assembling myofibrillar proteins. To determine the cellular localization of assembled T-cap, we transfected cardiac myocytes with a fusion construct of T-cap and GFP. Confocal analysis of T-cap–GFP–transfected cells, which were costained with the well-characterized monoclonal titin T12 antibody, which stains an epitope of titin in close proximity to the Z-line (Fürst et al., 1988), revealed overlap of the two fluorochromes at the level of resolution of the light microscope (identical results were obtained when T-cap–GFP– transfected cells were costained with anti–α-actinin antibodies; data not shown) (Fig. 7, a–c). This result indicates that newly synthesized T-cap is targeted to Z-lines in live cardiac muscle cells, which is consistent with the ultrastructural localization of this protein (Fig. 6) and its binding partner, titin Z1-Z2 (Fig. 1).


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)

T-cap assembles at  the Z-disc in cardiac myocytes. Fluorescent confocal  image of a cardiac myocyte  expressing (a) T-cap–GFP  that was fixed and stained  with (b) anti–titin T12 antibodies followed by Texas  red–conjugated donkey anti– mouse antibodies. (c) Merged  image of a and b demonstrating overlap of the two fluorochromes. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132961&req=5

Figure 7: T-cap assembles at the Z-disc in cardiac myocytes. Fluorescent confocal image of a cardiac myocyte expressing (a) T-cap–GFP that was fixed and stained with (b) anti–titin T12 antibodies followed by Texas red–conjugated donkey anti– mouse antibodies. (c) Merged image of a and b demonstrating overlap of the two fluorochromes. Bar, 10 μm.
Mentions: For functional studies on the assembly of T-cap, primary cultures of chick cardiac myocytes were chosen as a model system. Owing to their flat, well-spread morphology, these cells are ideally suited for detailed immunolocalization observations of the spatial relationships among assembling myofibrillar proteins. To determine the cellular localization of assembled T-cap, we transfected cardiac myocytes with a fusion construct of T-cap and GFP. Confocal analysis of T-cap–GFP–transfected cells, which were costained with the well-characterized monoclonal titin T12 antibody, which stains an epitope of titin in close proximity to the Z-line (Fürst et al., 1988), revealed overlap of the two fluorochromes at the level of resolution of the light microscope (identical results were obtained when T-cap–GFP– transfected cells were costained with anti–α-actinin antibodies; data not shown) (Fig. 7, a–c). This result indicates that newly synthesized T-cap is targeted to Z-lines in live cardiac muscle cells, which is consistent with the ultrastructural localization of this protein (Fig. 6) and its binding partner, titin Z1-Z2 (Fig. 1).

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