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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 β-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.

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.

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 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 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.