PMC

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.

Immunoelectron microscopy of Z-disc titin. Fibers labeled with anti–titin Z1-Z2 antibodies (A–C). (A) Low-magnification image of a stained section. Darkly stained silver grains reveal epitopes at the edge of the Z-line. (B) High-magnification image of epitopes at edge of Z-line. Notice the absence of clear labeling inside the Z-disc. (C) High-magnification image of stained section. Fibers labeled with anti–titin Zr5-6 antibodies (D–F). (D) Low-magnification image of an unstained section. Darkly stained silver grains reveal epitopes throughout the width of the Z line. (E) High-magnification image. (F) High-magnification image of a stained section. Bar, 1 μm.

Comparison of the T-cap protein sequences from human and mouse predicted by their respective 950-bp full-length cDNAs. The 19-kD T-cap proteins share 90% sequence identity and 95% sequence similarity between species. The human T-cap protein sequence is identical to the recently described telethonin entry (EMBL data library AJ000491) derived from anonymous EST entries (); the mouse sequence data from skeletal muscle are available from GenBank/EMBL/DDBJ under accession number Y14845.

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.

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.

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).

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.

Overexpression of titin Z1-Z2 results in marked disruption of thin and thick filaments in cardiac myocytes: stress fibers in fibroblasts are unaffected. Cardiac myocytes (a, b, and e–g) and fibroblasts (c and d) transfected with a plasmid encoding myc-titin Z1-Z2. 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) Texas red–conjugated phalloidin to stain F-actin, or with (f) polyclonal anti-myc antibodies, followed by FITC-conjugated donkey anti–rabbit antibodies and (e and g) monoclonal antistriated muscle myosin antibodies, followed by Texas red–conjugated donkey anti– mouse antibodies. Staining for myc was not detected in the cell shown in e (data not shown). * marks cells that were not transfected. Bars, 10 μm.

Developmental- and tissue-specific expression of T-cap transcripts. (A) In situ hybridization of whole-mount embryos at day 10.5 pc using probes from the mouse titin-cap cDNA. Transcripts are detected in the heart at 9.5 d of gestation (not shown). At day 10.5 pc, transcription has also commenced in the somites. The transcription pattern is very similar to that of titin (), except that transcripts for the titin-cap protein are also detected in the otic vesicle (B, selected details of A). (C) Presence of titin-cap protein transcripts in human cDNAs from different tissues, as detected by RT-PCR. T-cap protein is expressed exclusively in striated muscles. Lanes 1–3, fetal heart, adult heart, and adult skeletal muscles, respectively; lanes 4–8, normal and pregnant uterus, fetal brain, liver, and spleen, respectively. Controls: lane G, genomic DNA, “no template”; lane M, lambda HindIII size marker.

In vitro interaction of Z-repeats with α-actinin. (A) Binding of four Z-repeat fragments, Zr1, 2, 3, and 7 to an α-actinin fusion protein. A Coomassie blue–stained 15% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing the Z-repeat fragment Zr1, 2, 3, and 7, which corresponds to the splice variant of psoas muscle (lane Lys). These cells were mixed with cells expressing a GST-His double-tagged α-actinin (GST-actn) fusion peptide and lysed together, and lysates were passed over Ni-NTA columns. Interaction of α-actinin with the Z-repeats is indicated by also retaining the nontagged Z-repeats on an Ni-NTA column (arrow Zr1,2,3,7). (B) As in A, but here the Z-repeats were expressed as GST-His₆ double-tagged fusions, and the expressed α-actinin COOH-terminal peptide was tagless. Interaction of the 15-kD from the α-actinin COOH terminus with the Z-repeats is indicated by retaining the α-actinin 15 kD band on the column (arrow actn). Here, Lys indicates a whole-cell lysate of BL21 cells overexpressing the α-actinin COOH terminus; control (C), Zr1-His₆-GST fusion peptide alone loaded on the column, demonstrating that a 15-kD protein from E. coli does not bind nonspecifically on the column. Lanes Zr1–Zr7, interaction of each of the seven Z-repeats alone with the COOH terminus of α-actinin; lane Zr4-6, interaction of the three fused Z-repeats with the COOH terminus of α-actinin. The Z-repeats Zr1, 2, 3, and 7 alone are sufficient for binding to the α-actinin COOH terminus under the conditions used. The Zr4, 5, and 6 alone do not interact with the α-actinin COOH terminus, but as a fusion peptide, they also bind to the COOH terminus of α-actinin. Lane M, molecular mass markers corresponding to 97, 60, 40, 30, 20, and 15 kD.

Interaction of T-cap and the titin Z1-Z2 domains. (a) Titin repeats Z1 and Z2 specifically interact with the 19-kD titin-cap protein by yeast two-hybrid analysis. Numbers above the schematic layout of the NH₂-terminal titin region indicate the nucleotide residue numbers corresponding to the human entry (EMBL data library X90568) (top) and residue numbers of the deduced amino acid sequence (bottom). Two immunoglobulin domains at the NH₂ terminus of titin are indicated as Z1 and Z2, respectively. Bars indicate positions of the cDNAs used for the bait plasmid (pAS2-Z1-Z2-is1), and the truncation constructs, pAS2-Z1-Z2 (containing the Z1 and Z2 domains), pAS2-Z1 (containing Z1 domain only), and pAS2Z2-is1 (containing the Z2 domain fused to residues 200– 257), used for the binding assay to T-cap. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (b) Interaction of the NH₂-terminal 16 kD of T-cap with the titin Z1-Z2 Ig domains in the yeast two-hybrid system. Schematic of T-cap cDNA clone. Numbers above the titin-cap cDNA indicate the nucleotide residues of the human full-length cDNA sequence (sequence data available from EMBL under accession number AJ000491; from ) (top) and residue numbers of the deduced amino acid sequence (bottom). AA...A, a poly(A⁺) tail at the 3′ terminus of the corresponding cDNA clone. Bars indicate positions of cDNA inserts derived from the two-hybrid screening using pAS2-Z1-Z2-is1 (see a) as bait (T-cap-1, -3, -5, -6, and -7), and the truncation constructs of T-cap (T-cap-Δ1 and -Δ2) described in Materials and Methods. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (c) Interaction of expressed T-cap and titin Z1-Z2 peptides. A Coomassie blue– stained 18% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing tagless titin Z1-Z2 (lane C). These cells were mixed with a His₆-tagged 16-kD fragment of T-cap and lysed. Lysates were passed over Ni-NTA agarose columns. Interaction of a stable complex of titin Z1-Z2 and T-cap is indicated by retaining also the nontagged titin Z1-Z2 on the column (arrow marks Z1-Z2 and T-cap in third lane). Lane M, molecular mass markers as in legend for Fig. .