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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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Immunoelectron microscopy of T-cap. Fibers labeled with anti–T-cap antibodies. (A) Low-magnification image of an unstained section. (B) High-magnification image. (C) High-magnification image of a stained section. The T-cap epitope is localized at the  periphery of the Z-disc. (D–F) Location of epitopes as determined from the distance of silver grains to the center of Z-disc. Histograms of results with anti–titin Z1-Z2 (red) and anti–T-cap antibodies (green) reveal peaks 60 nm from the center of Z-disc, while the  anti–Zr5-6 antibodies (purple) label within the Z-disc: the titin NH2 terminus and T-cap colocalize in the Z-disc periphery. Black lines  mark Z-lines. Bar, 1 μm.
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Figure 6: Immunoelectron microscopy of T-cap. Fibers labeled with anti–T-cap antibodies. (A) Low-magnification image of an unstained section. (B) High-magnification image. (C) High-magnification image of a stained section. The T-cap epitope is localized at the periphery of the Z-disc. (D–F) Location of epitopes as determined from the distance of silver grains to the center of Z-disc. Histograms of results with anti–titin Z1-Z2 (red) and anti–T-cap antibodies (green) reveal peaks 60 nm from the center of Z-disc, while the anti–Zr5-6 antibodies (purple) label within the Z-disc: the titin NH2 terminus and T-cap colocalize in the Z-disc periphery. Black lines mark Z-lines. Bar, 1 μm.

Mentions: Single fibers were dissected from human soleus muscle (biopsies obtained in accordance with protocol “Role of titin in human muscle tissue 2,” Washington State University). Fibers were both chemically and mechanically skinned and stretched in relaxing solution that contained high levels of protease inhibitors (for details see Trombitas et al., 1998). Fibers were used within 48 h and immunolabeled with affinity-purified rabbit anti– titin Z1-Z2, anti–Zr5-6. or anti–T-cap antibodies. In brief, skeletal muscle fibers were fixed in 3% formaldehyde/PBS solution, blocked, and labeled with the primary antibodies (typically 50 μg/ml), followed by washing. To optimize labeling, the concentration of antibody and the duration of the blocking step were varied. Results shown were obtained by blocking for 60 min using 2% bovine serum albumin. Affinity-purified F(ab′) fragments, raised in goat against the whole rabbit IgG molecule were used as secondary antibodies (catalog No. 2004; Nanoprobes, Inc., Stonybrook, NY). These antibodies were conjugated with 1.4-nm gold particles that were intensified with HQ silver developer for 4 min (Trombitas et al., 1995). Labeled fibers were embedded in araldite and then sectioned. Some sections were stained with potassium permanganate and lead citrate (Figs. 1, C and F, and 6 C). To optimally visualize silver grains, some sections were unstained (Figs. 1, A, B, D, and E, and 6, A and B). Sections were visualized using a transmission electron microscope (model JEM 1200 IIE; Jeol, Tokyo, Japan). Z-disc–to–epitope distances were measured from electron micrographs after high-resolution scanning (UC-1260; UMAX Data System, Inc., Fremont, CA) and digital image processing using custom-written macros for the image analysis program NIH Image (v. 1.6, Wayne Rasband, National Institutes of Health, Bethesda, MD). For spatial calibration, the electron microscope's magnification was used.


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)

Immunoelectron microscopy of T-cap. Fibers labeled with anti–T-cap antibodies. (A) Low-magnification image of an unstained section. (B) High-magnification image. (C) High-magnification image of a stained section. The T-cap epitope is localized at the  periphery of the Z-disc. (D–F) Location of epitopes as determined from the distance of silver grains to the center of Z-disc. Histograms of results with anti–titin Z1-Z2 (red) and anti–T-cap antibodies (green) reveal peaks 60 nm from the center of Z-disc, while the  anti–Zr5-6 antibodies (purple) label within the Z-disc: the titin NH2 terminus and T-cap colocalize in the Z-disc periphery. Black lines  mark Z-lines. Bar, 1 μm.
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Figure 6: Immunoelectron microscopy of T-cap. Fibers labeled with anti–T-cap antibodies. (A) Low-magnification image of an unstained section. (B) High-magnification image. (C) High-magnification image of a stained section. The T-cap epitope is localized at the periphery of the Z-disc. (D–F) Location of epitopes as determined from the distance of silver grains to the center of Z-disc. Histograms of results with anti–titin Z1-Z2 (red) and anti–T-cap antibodies (green) reveal peaks 60 nm from the center of Z-disc, while the anti–Zr5-6 antibodies (purple) label within the Z-disc: the titin NH2 terminus and T-cap colocalize in the Z-disc periphery. Black lines mark Z-lines. Bar, 1 μm.
Mentions: Single fibers were dissected from human soleus muscle (biopsies obtained in accordance with protocol “Role of titin in human muscle tissue 2,” Washington State University). Fibers were both chemically and mechanically skinned and stretched in relaxing solution that contained high levels of protease inhibitors (for details see Trombitas et al., 1998). Fibers were used within 48 h and immunolabeled with affinity-purified rabbit anti– titin Z1-Z2, anti–Zr5-6. or anti–T-cap antibodies. In brief, skeletal muscle fibers were fixed in 3% formaldehyde/PBS solution, blocked, and labeled with the primary antibodies (typically 50 μg/ml), followed by washing. To optimize labeling, the concentration of antibody and the duration of the blocking step were varied. Results shown were obtained by blocking for 60 min using 2% bovine serum albumin. Affinity-purified F(ab′) fragments, raised in goat against the whole rabbit IgG molecule were used as secondary antibodies (catalog No. 2004; Nanoprobes, Inc., Stonybrook, NY). These antibodies were conjugated with 1.4-nm gold particles that were intensified with HQ silver developer for 4 min (Trombitas et al., 1995). Labeled fibers were embedded in araldite and then sectioned. Some sections were stained with potassium permanganate and lead citrate (Figs. 1, C and F, and 6 C). To optimally visualize silver grains, some sections were unstained (Figs. 1, A, B, D, and E, and 6, A and B). Sections were visualized using a transmission electron microscope (model JEM 1200 IIE; Jeol, Tokyo, Japan). Z-disc–to–epitope distances were measured from electron micrographs after high-resolution scanning (UC-1260; UMAX Data System, Inc., Fremont, CA) and digital image processing using custom-written macros for the image analysis program NIH Image (v. 1.6, Wayne Rasband, National Institutes of Health, Bethesda, MD). For spatial calibration, the electron microscope's magnification was used.

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