The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures.

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the "blood-testis barrier", formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or "desmosome-like" junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and "desmosome-like" junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and ß-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the "areae adhaerentes", i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5-7 nm internal diameter ("cribelliform junctions"). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.

Protein LUMA is a cytoplasmic plaque constituent of various epithelial adherens junctions and composite junctions of myocardial intercalated disks: a unifying finding for cell biology and cardiology.

In a series of recent reports, mutations in the gene encoding a protein called LUMA (or TMEM43), widely speculated to be a tetraspan transmembrane protein of the nuclear envelope, have been associated with a specific subtype of cardiomyopathy (arrhythmogenic cardiomyopathies) and cases of sudden death. However, using antibodies of high specificity in immunolocalization experiments, we have discovered that, in mammals, LUMA is a component of zonula adhaerens and punctum adhaerens plaques of diverse epithelia and epithelial cell cultures and is also located in (or in some species associated with) the plaques of composite junctions (CJs) in myocardiac intercalated disks (IDs). In CJs, LUMA often colocalizes with several other CJ marker proteins. In all these cells, LUMA has not been detected in the nuclear envelope. Surprisingly, under certain conditions, similar CJ localizations have also been seen with some antibodies commercially available for some time. The identification of LUMA as a plaque component of myocardiac CJs leads to reconsiderations of the molecular composition and architecture, the development, the functions, and the pathogenic states of CJs and IDs. These findings now also allow the general conclusion that LUMA has to be added to the list of mutations of cardiomyocyte junction proteins that may be involved in cardiomyopathies.

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells.

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions ("tessellate junctions"), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.

The adhering junctions of valvular interstitial cells: molecular composition in fetal and adult hearts and the comings and goings of plakophilin-2 in situ, in cell culture and upon re-association with scaffolds.

The interstitial cells of cardiac valves represent one of the most frequent cell types in the mammalian heart. In order to provide a cell and molecular biological basis for the growth of isolated valvular interstitial cells (VICs) in cell culture and for the use in re-implantation surgery we have examined VICs in situ and in culture, in fetal, postnatal and adult hearts, in re-associations with scaffolds of extracellular matrix (ECM) material and decellularized heart valves. In all four mammalian species examined (human, bovine, porcine and ovine), the typical mesenchymal-type cell-cell adherens junctions (AJs) connecting VICs appear as normal N-cadherin based puncta adhaerentia. Their molecular ensemble, however, changes under various growth conditions insofar as plakophilin-2 (Pkp2), known as a major cytoplasmic plaque component of epithelial desmosomes, is recruited to and integrated in the plaques of VIC-AJs as a major component under growth conditions characterized by enhanced proliferation, i.e., in fetal heart valves and in cell cultures. Upon re-seeding onto decellularized heart valves or in stages of growth in association with artificial scaffolds, Pkp2 is - for the most part - lost from the AJs. As Pkp2 has recently also been detected in AJs of cardiac myxomata and diverse other mesenchymal tumors, the demonstrated return to the normal Pkp2-negative state upon re-association with ECM scaffolds and decellularized heart valves may now provide a safe basis for the use of cultured VICs in valve replacement surgery. Even more surprising, this type of transient acquisition of Pkp2 has also been observed in distinct groups of endothelial cells of the endocardium, where it seems to correspond to the cell type ready for endothelial-mesenchymal transition (EMT).

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

Recently the protein myozap, a 54-kD polypeptide which is not a member of any of the known cytoskeletal and junctional protein multigene families, has been identified as a constituent of the plaques of the composite junctions in the intercalated disks connecting the cardiomyocytes of mammalian hearts. 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. In light and electron microscopic immunolocalization experiments we show that myozap colocalizes with several proteins of desmosomal plaques as well as with AJ-specific transmembrane molecules, including VE-cadherin. In biochemical analyses, rigorous immunoprecipitation experiments have revealed N-cadherin, desmoplakin, desmoglein-2, plakophilin-2, plakoglobin and plectin as very stably bound complex partners. 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.

Protein myozap--a late addition to the molecular ensembles of various kinds of adherens junctions.

The protein myozap, a polypeptide of 54 kDa, has recently been identified as a component of the cytoplasmic plaques of the composite junctions (areae compositae) in the myocardiac intercalated disks and of the adherens junctions (AJs) in vascular endothelia. Now we report that using very sensitive new antibodies and drastic localization methods, we have also identified this protein as a component of the AJ plaques in simple and complex epithelia, in the adluminal cell layer of the transitional epithelium of the urinary tract and in certain cell layers of diverse stratified epithelia, including gingiva, tongue, pharynx and esophagus, cervix, vagina and epidermis. Myozap has not been identified in desmosomal and tight junction plaques. We have also detected protein myozap in AJ structures of carcinomas. The discovery of a novel major protein in AJ plaques now calls for re-examinations of molecular interactions in AJ formation and maintenance and also offers a new marker for diagnostic immunocytochemistry. We also discuss the need for progressive unravelling, extractive treatments and buffer rinses of sections and cultured cells to reveal obscured or masked antigens, before definitive negative conclusions in immunohistochemistry can be made.

Upregulation of plakophilin-2 and its acquisition to adherens junctions identifies a novel molecular ensemble of cell-cell-attachment characteristic for transformed mesenchymal cells.

In contrast to the desmosome-containing epithelial and carcinoma cells, normal and malignantly transformed cells derived from mesenchymal tissues and tumors are connected only by adherens junctions (AJs) containing N-cadherins and/or cadherin-11, anchored in a cytoplasmic plaque assembled by alpha- and beta-catenin, plakoglobin, proteins p120 and p0071. Here, we report that the AJs of many malignantly transformed cell lines are characterized by the additional presence of plakophilin-2 (Pkp2), a protein hitherto known only as a major component of desmosomal plaques, i.e., AJs of epithelia and carcinomatous cells. This massive acquisition of Pkp2 and its integration into AJ plaques of a large number of transformed cell lines is demonstrated with biochemical and immunolocalization techniques. Upregulation of Pkp2 and its integration into AJs has also been noted in some soft tissue tumors insitu and some highly proliferative colonies of cultured mesenchymal stem cells. As Pkp2 has recently been identified as a functionally important major regulatory organizer in AJs and related junctions in epithelial cells and cardiomyocytes, we hypothesize that the integration of Pkp2 into AJs of "soft tissue tumor" cells also can serve functions in the upregulation of proliferation, the promotion of malignant growth in general as well as the close-packing of diverse kinds of cells and the metastatic behavior of such tumors. We propose to examine its presence in transformed mesenchymal cells and related tumors and to use it as an additional diagnostic criterion.

The area composita of adhering junctions connecting heart muscle cells of vertebrates - III: assembly and disintegration of intercalated disks in rat cardiomyocytes growing in culture.

For cell and molecular biological studies of heart formation and function cell cultures of embryonal, neonatal or adult hearts of various vertebrates, notably rat and chicken, have been widely used. As the myocardium-specific cell-cell junctions, the intercalated disks (ID), have recently been found to be particularly sensitive to losses of - or mutations in - certain cytoskeletal proteins, resulting in cardiac damages, we have examined the ID organization in primary cultures of cardiomyocytes obtained from neonatal rats. Using immunofluorescence and immunoelectron microscopy, we have studied the major ID components for up to 2 weeks in culture, paying special attention to spontaneously beating, individual cardiomyocytes and myocardial cell colonies. While our results demonstrate the formation of some ID-like cardiomyocyte-connecting junction arrays, they also reveal a variety of structural disorders such as rather extended, junction-free ID regions, sac-like invaginations and endocytotic blebs as well as accumulations of intracytoplasmic structures suggestive of endocytosed forms of junction-derived vesicles or of junction fragments resembling fascia adhaerens elements. Moreover, we have noticed a novel type of small, obviously plaque-free cytoplasmic vesicles containing one or both of the desmosomal cadherins, desmocollin Dsc2 and desmoglein Dsg2. We conclude that cardiomyocyte cultures are useful model systems for studies of certain aspects of myocardiac differentiation and functions but, on the other hand, show progressive disintegration and deterioration. The potential value of molecular markers and reagents in studies of myocardial pathology as well as in the monitoring of myocardial differentiation of so-called stem cells is discussed.