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The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems.

Pieperhoff S, Rickelt S, Heid H, Claycomb WC, Zimbelmann R, Kuhn C, Winter-Simanowski S, Kuhn C, Frey N, Franke WW - J. Cell. Mol. Med. (2012)

Bottom Line: Using a set of novel, highly sensitive and specific antibodies we now report that myozap is also a major constituent of the cytoplasmic plaques of the adherens junctions (AJs) connecting the endothelial cells of the mammalian blood and lymph vascular systems, including the desmoplakin-containing complexus adhaerentes of the virgultar cells of lymph node sinus.We conclude that myozap is a general component of cell-cell junctions not only in the myocardium but also in diverse endothelia of the blood and lymph vascular systems of adult mammals, suggesting that this protein not only serves a specific role in the heart but also a broader set of functions in the vessel systems.We also propose to use myozap as an endothelial cell type marker in diagnoses.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Group Cell Biology, German Cancer Research Center, DKFZ, Heidelberg, Germany.

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Immunofluorescence microscopy of protein myozap in the composite junctions of mammalian cardiomyocytes. (A) Immunofluorescence micrograph of a cryostat section through bovine myocardium using myozap mAb (clone 517.67, red), in combination with nuclear DAPI staining (blue) and phase contrast microscopy. Note the intense positive reaction of the intercalated disks (IDS). (B) Double-label immunofluorescence microscopy of primary cardiomyocyte cultures of neonatal rat hearts, using murine mAbs to α-cardiac actin (green) and polyclonal guinea pig antibodies to myozap (red), in comparison with DAPI staining (blue). Note the specific reaction of the composite junctions of the IDS. (C–F) Cardiomyocyte-derived HL-1 mouse cells grown in dense culture monolayers and labelled with myozap mAb (red), in a differential interference contrast (DIC) image (C) or in comparison with the actin microfilament-associated junctional protein ZO-1 (D and E; green) and the major adherens junction plaque protein β-catenin (F; green). Note the intense, specific reaction of the composite junctions of the intercalated disk-derived structures (A, B). Note also the colocalization of myozap with parts of the structures positive for further adherens junction-associated plaque proteins (yellow merged colour in D–F). Bars: 100 μm (A–C); 20 μm (D–F).
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fig02: Immunofluorescence microscopy of protein myozap in the composite junctions of mammalian cardiomyocytes. (A) Immunofluorescence micrograph of a cryostat section through bovine myocardium using myozap mAb (clone 517.67, red), in combination with nuclear DAPI staining (blue) and phase contrast microscopy. Note the intense positive reaction of the intercalated disks (IDS). (B) Double-label immunofluorescence microscopy of primary cardiomyocyte cultures of neonatal rat hearts, using murine mAbs to α-cardiac actin (green) and polyclonal guinea pig antibodies to myozap (red), in comparison with DAPI staining (blue). Note the specific reaction of the composite junctions of the IDS. (C–F) Cardiomyocyte-derived HL-1 mouse cells grown in dense culture monolayers and labelled with myozap mAb (red), in a differential interference contrast (DIC) image (C) or in comparison with the actin microfilament-associated junctional protein ZO-1 (D and E; green) and the major adherens junction plaque protein β-catenin (F; green). Note the intense, specific reaction of the composite junctions of the intercalated disk-derived structures (A, B). Note also the colocalization of myozap with parts of the structures positive for further adherens junction-associated plaque proteins (yellow merged colour in D–F). Bars: 100 μm (A–C); 20 μm (D–F).

Mentions: Using Northern and Western blot tests, protein myozap (Mr 54 kD) had been detected not only in heart but also in placenta and lung tissue [18]. Immunoblot analyses using the novel very sensitive antibodies have now allowed us to identify this protein not only in myocardial tissues of the diverse mammals examined but also in cultures of murine cardiomyocyte-derived, permanently growing HL-1 cells [22] as well as in lung tissue and, in lesser amounts, in certain other tissues such as kidney and liver (Fig. 1, upper panel). Fractionation experiments have further shown that protein myozap is enriched in the Triton-insoluble, cytoskeletal residue fractions containing cell–cell junction structures (Fig. 1, lower panel). Remarkably, in these fractions relatively more protein myozap has been recovered than some of the ‘classic’ cell junction constituents such as N-cadherin and plakoglobin (Fig. 1, lower panel). Two-dimensional gel electrophoresis of the polypeptides present in HL-1 cell lysates, followed by immunoblotting with the new myozap antibodies, further allowed us to distinguish two major variants of this polypeptide with isoelectric points of ca. 6.02 and 6.20. The novel murine monoclonal and guinea pig polyclonal antibodies also specifically and intensely decorated composite junction structures, whether they occurred in frozen myocardial tissue, in primary cultures of neonatal rat cardiomyocytes, or in HL-1 cardiomyocyte cultures (Fig. 2A–C). In all comparative localization experiments using myocardial tissue or HL-1 transformed cardiomyocyte cultures (Fig. 2C–F) protein myozap showed partial colocalization with several other junction plaque proteins, including actin filament-binding ones (for an example, see protein ZO-1; Fig. 2D and E) and members of the armadillo protein family (Fig. 2F presents β-catenin for example).


The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems.

Pieperhoff S, Rickelt S, Heid H, Claycomb WC, Zimbelmann R, Kuhn C, Winter-Simanowski S, Kuhn C, Frey N, Franke WW - J. Cell. Mol. Med. (2012)

Immunofluorescence microscopy of protein myozap in the composite junctions of mammalian cardiomyocytes. (A) Immunofluorescence micrograph of a cryostat section through bovine myocardium using myozap mAb (clone 517.67, red), in combination with nuclear DAPI staining (blue) and phase contrast microscopy. Note the intense positive reaction of the intercalated disks (IDS). (B) Double-label immunofluorescence microscopy of primary cardiomyocyte cultures of neonatal rat hearts, using murine mAbs to α-cardiac actin (green) and polyclonal guinea pig antibodies to myozap (red), in comparison with DAPI staining (blue). Note the specific reaction of the composite junctions of the IDS. (C–F) Cardiomyocyte-derived HL-1 mouse cells grown in dense culture monolayers and labelled with myozap mAb (red), in a differential interference contrast (DIC) image (C) or in comparison with the actin microfilament-associated junctional protein ZO-1 (D and E; green) and the major adherens junction plaque protein β-catenin (F; green). Note the intense, specific reaction of the composite junctions of the intercalated disk-derived structures (A, B). Note also the colocalization of myozap with parts of the structures positive for further adherens junction-associated plaque proteins (yellow merged colour in D–F). Bars: 100 μm (A–C); 20 μm (D–F).
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Related In: Results  -  Collection

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fig02: Immunofluorescence microscopy of protein myozap in the composite junctions of mammalian cardiomyocytes. (A) Immunofluorescence micrograph of a cryostat section through bovine myocardium using myozap mAb (clone 517.67, red), in combination with nuclear DAPI staining (blue) and phase contrast microscopy. Note the intense positive reaction of the intercalated disks (IDS). (B) Double-label immunofluorescence microscopy of primary cardiomyocyte cultures of neonatal rat hearts, using murine mAbs to α-cardiac actin (green) and polyclonal guinea pig antibodies to myozap (red), in comparison with DAPI staining (blue). Note the specific reaction of the composite junctions of the IDS. (C–F) Cardiomyocyte-derived HL-1 mouse cells grown in dense culture monolayers and labelled with myozap mAb (red), in a differential interference contrast (DIC) image (C) or in comparison with the actin microfilament-associated junctional protein ZO-1 (D and E; green) and the major adherens junction plaque protein β-catenin (F; green). Note the intense, specific reaction of the composite junctions of the intercalated disk-derived structures (A, B). Note also the colocalization of myozap with parts of the structures positive for further adherens junction-associated plaque proteins (yellow merged colour in D–F). Bars: 100 μm (A–C); 20 μm (D–F).
Mentions: Using Northern and Western blot tests, protein myozap (Mr 54 kD) had been detected not only in heart but also in placenta and lung tissue [18]. Immunoblot analyses using the novel very sensitive antibodies have now allowed us to identify this protein not only in myocardial tissues of the diverse mammals examined but also in cultures of murine cardiomyocyte-derived, permanently growing HL-1 cells [22] as well as in lung tissue and, in lesser amounts, in certain other tissues such as kidney and liver (Fig. 1, upper panel). Fractionation experiments have further shown that protein myozap is enriched in the Triton-insoluble, cytoskeletal residue fractions containing cell–cell junction structures (Fig. 1, lower panel). Remarkably, in these fractions relatively more protein myozap has been recovered than some of the ‘classic’ cell junction constituents such as N-cadherin and plakoglobin (Fig. 1, lower panel). Two-dimensional gel electrophoresis of the polypeptides present in HL-1 cell lysates, followed by immunoblotting with the new myozap antibodies, further allowed us to distinguish two major variants of this polypeptide with isoelectric points of ca. 6.02 and 6.20. The novel murine monoclonal and guinea pig polyclonal antibodies also specifically and intensely decorated composite junction structures, whether they occurred in frozen myocardial tissue, in primary cultures of neonatal rat cardiomyocytes, or in HL-1 cardiomyocyte cultures (Fig. 2A–C). In all comparative localization experiments using myocardial tissue or HL-1 transformed cardiomyocyte cultures (Fig. 2C–F) protein myozap showed partial colocalization with several other junction plaque proteins, including actin filament-binding ones (for an example, see protein ZO-1; Fig. 2D and E) and members of the armadillo protein family (Fig. 2F presents β-catenin for example).

Bottom Line: Using a set of novel, highly sensitive and specific antibodies we now report that myozap is also a major constituent of the cytoplasmic plaques of the adherens junctions (AJs) connecting the endothelial cells of the mammalian blood and lymph vascular systems, including the desmoplakin-containing complexus adhaerentes of the virgultar cells of lymph node sinus.We conclude that myozap is a general component of cell-cell junctions not only in the myocardium but also in diverse endothelia of the blood and lymph vascular systems of adult mammals, suggesting that this protein not only serves a specific role in the heart but also a broader set of functions in the vessel systems.We also propose to use myozap as an endothelial cell type marker in diagnoses.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Group Cell Biology, German Cancer Research Center, DKFZ, Heidelberg, Germany.

Show MeSH
Related in: MedlinePlus