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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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Microinjection of titin Z1-Z2 into the cytoplasm of  cardiac myocytes results in disruption of myofibrils: stress fibers  in fibroblasts are unaffected. Cells were microinjected with (a, b,  g, and h) a nonspecific monoclonal antibody (MOPC-21) to identify injected cells and (c–f, i, and j) with the nonspecific antibody  plus purified titin Z1-Z2. 2 h after injection, the cells were fixed  and stained with (a) Cy2-conjugated anti–mouse antibodies and  (b) Texas red–conjugated phalloidin. Note that the control microinjected antibody used to mark the injected cells appears to  localize to some myofibrils in the cardiomyocytes and to some  stress fibers in fibroblasts. Arrows point to the typical striated  and stress fiber staining seen with Texas red–phalloidin (b) in  cardiac myocytes and (h and j) in fibroblasts, respectively; arrowheads point to disrupted myofibrils in cardiac myocytes (d and f).  Bar, 10 μm.
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Figure 10: Microinjection of titin Z1-Z2 into the cytoplasm of cardiac myocytes results in disruption of myofibrils: stress fibers in fibroblasts are unaffected. Cells were microinjected with (a, b, g, and h) a nonspecific monoclonal antibody (MOPC-21) to identify injected cells and (c–f, i, and j) with the nonspecific antibody plus purified titin Z1-Z2. 2 h after injection, the cells were fixed and stained with (a) Cy2-conjugated anti–mouse antibodies and (b) Texas red–conjugated phalloidin. Note that the control microinjected antibody used to mark the injected cells appears to localize to some myofibrils in the cardiomyocytes and to some stress fibers in fibroblasts. Arrows point to the typical striated and stress fiber staining seen with Texas red–phalloidin (b) in cardiac myocytes and (h and j) in fibroblasts, respectively; arrowheads point to disrupted myofibrils in cardiac myocytes (d and f). Bar, 10 μm.

Mentions: As another complementary approach to rule out the possibility that the observed phenotype was due to overexpressing too much protein and to rule out effects due to the fusion proteins used, varying amounts of purified titin Z1-Z2 fragments were microinjected directly into the cytoplasm of the cells; again, a severe disruption of the myofibrils was observed (Fig. 10, cells microinjected with titin Z1-Z2 and double stained for [c and e] the anti–mouse Ig antibodies used to identify injected cells and [d and f] actin; compare with control microinjected cells [a and b]). Approximately, 80% of the cardiac myocytes that were positively identified as being injected expressed this phenotype.


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)

Microinjection of titin Z1-Z2 into the cytoplasm of  cardiac myocytes results in disruption of myofibrils: stress fibers  in fibroblasts are unaffected. Cells were microinjected with (a, b,  g, and h) a nonspecific monoclonal antibody (MOPC-21) to identify injected cells and (c–f, i, and j) with the nonspecific antibody  plus purified titin Z1-Z2. 2 h after injection, the cells were fixed  and stained with (a) Cy2-conjugated anti–mouse antibodies and  (b) Texas red–conjugated phalloidin. Note that the control microinjected antibody used to mark the injected cells appears to  localize to some myofibrils in the cardiomyocytes and to some  stress fibers in fibroblasts. Arrows point to the typical striated  and stress fiber staining seen with Texas red–phalloidin (b) in  cardiac myocytes and (h and j) in fibroblasts, respectively; arrowheads point to disrupted myofibrils in cardiac myocytes (d and f).  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Microinjection of titin Z1-Z2 into the cytoplasm of cardiac myocytes results in disruption of myofibrils: stress fibers in fibroblasts are unaffected. Cells were microinjected with (a, b, g, and h) a nonspecific monoclonal antibody (MOPC-21) to identify injected cells and (c–f, i, and j) with the nonspecific antibody plus purified titin Z1-Z2. 2 h after injection, the cells were fixed and stained with (a) Cy2-conjugated anti–mouse antibodies and (b) Texas red–conjugated phalloidin. Note that the control microinjected antibody used to mark the injected cells appears to localize to some myofibrils in the cardiomyocytes and to some stress fibers in fibroblasts. Arrows point to the typical striated and stress fiber staining seen with Texas red–phalloidin (b) in cardiac myocytes and (h and j) in fibroblasts, respectively; arrowheads point to disrupted myofibrils in cardiac myocytes (d and f). Bar, 10 μm.
Mentions: As another complementary approach to rule out the possibility that the observed phenotype was due to overexpressing too much protein and to rule out effects due to the fusion proteins used, varying amounts of purified titin Z1-Z2 fragments were microinjected directly into the cytoplasm of the cells; again, a severe disruption of the myofibrils was observed (Fig. 10, cells microinjected with titin Z1-Z2 and double stained for [c and e] the anti–mouse Ig antibodies used to identify injected cells and [d and f] actin; compare with control microinjected cells [a and b]). Approximately, 80% of the cardiac myocytes that were positively identified as being injected expressed this phenotype.

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