Stefanie Winter-Simanowski

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 α‐ and β‐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 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 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.

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).