The cell-cell junctions of mammalian testes. III. Absence of an endothelial cell layer covering the peritubular wall of the seminiferous tubules-an immunocytochemical correction of a 50-year-old error in the literature.

In the molecular biological and ultrastructural studies of the peritubular wall cells encasing the seminiferous tubules of mammalian testes, we found it necessary to characterize the outermost cell layer bordering on the interstitial space in detail. For half a century, the extremely thin cells of this monolayer have in the literature been regarded as part of a lymphatic endothelium, in particular in rodents. However, our double-label immunofluorescence microscopical results have shown that in all six mammalian species examined, including three rodent ones (rat, mouse, guinea pig), this classification is not correct: the very attenuated cells of this monolayer are not of lymphatic endothelial nature as they do not contain established endothelial marker molecules. In particular, they do not contain claudin-5-positive tight junctions, VE-cadherin-positive adherens junctions, "lymph vessel endothelium hyaluronan receptor 1" (LYVE-1), podoplanin, protein myozap and "von Willebrand Factor" (vWF). By contrast and as controls, all these established marker molecules for the lymphatic endothelial cell type are found in the endothelia of the lymph and-partly also-blood vessels located nearby in the interstitial space. Thus, our results provide evidence that the monolayer cells covering the peritubular wall do not contain endothelial marker molecules and hence are not endothelial cells. We discuss possible methodological reasons for the maintenance of this incorrect cell type classification in the literature and emphasize the value of molecular analyses using multiple cell type-specific markers, also with respect to physiology and medical sciences.

Striatin is a novel modulator of cell adhesion.

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.

The cell-cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions-a novel architectonic cell-cell junction system.

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral "lamellar smooth muscle cells" (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing ß-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in "dense bodies," "focal adhesions," and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.

Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells.

Proteins of the striatin family (striatins 1-4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E-N-cadherin heterodimers and with the plaque proteins α- and ß-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the "multimodulator" scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs.

Mice with cardiac-restricted overexpression of Myozap are sensitized to biomechanical stress and develop a protein-aggregate-associated cardiomyopathy.

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes and has been recognized as a hot spot for cardiomyopathy. We have previously identified Myozap as a novel cardiac-enriched ID protein, which interacts with several other ID proteins and is involved in RhoA/SRF signaling in vitro. To now study its potential role in vivo we generated a mouse model with cardiac overexpression of Myozap. Transgenic (Tg) mice developed cardiomyopathy with hypertrophy and LV dilation. Consistently, these mice displayed upregulation of the hypertrophy-associated and SRF-dependent gene expression. Pressure overload (transverse aortic constriction, TAC) caused exaggerated cardiac hypertrophy, further loss of contractility and LV dilation. Similarly, a physiological stimulus (voluntary running) also led to significant LV dysfunction. On the ultrastructural level, Myozap-Tg mouse hearts exhibited massive protein aggregates composed of Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the alterations observed in desmin-related cardiomyopathy. Interestingly, desmin was not detectable in the aggregates, yet was largely displaced from the ID. Molecular analyses revealed induction of autophagy and dysregulation of the unfolded protein response (UPR), associated with apoptosis. Taken together, cardiac overexpression of Myozap leads to cardiomyopathy, mediated, at least in part by induction of Rho-dependent SRF signaling in vivo. Surprisingly, this phenotype was also accompanied by protein aggregates in cardiomyocytes, UPR alteration, accelerated autophagy and apoptosis. Thus, this mouse model may also offer additional insight into the pathogenesis of protein-aggregate-associated cardiomyopathies and represents a new candidate gene itself.

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 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.

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 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.

Myozap, a novel intercalated disc protein, activates serum response factor-dependent signaling and is required to maintain cardiac function in vivo.

RATIONALE: The intercalated disc (ID) is a highly specialized cell-cell contact structure that ensures mechanical and electric coupling of contracting cardiomyocytes. Recently, the ID has been recognized to be a hot spot of cardiac disease, in particular inherited cardiomyopathy. OBJECTIVE: Given its complex structure and function we hypothesized that important molecular constituents of the ID still remain unknown. METHODS AND RESULTS: Using a bioinformatics screen, we discovered and cloned a previously uncharacterized 54 kDa cardiac protein which we termed Myozap (Myocardium-enriched zonula occludens-1-associated protein). Myozap is strongly expressed in the heart and lung. In cardiac tissue it localized to the ID and directly binds to desmoplakin and zonula occludens-1. In a yeast 2-hybrid screen for additional binding partners of Myozap we identified myosin phosphatase-RhoA interacting protein (MRIP), a negative regulator of Rho activity. Myozap, in turn, strongly activates SRF-dependent transcription through its ERM (Ezrin/radixin/moesin)-like domain in a Rho-dependent fashion. Finally, in vivo knockdown of the Myozap ortholog in zebrafish led to severe contractile dysfunction and cardiomyopathy. CONCLUSIONS: Taken together, these findings reveal Myozap as a previously unrecognized component of a Rho-dependent signaling pathway that links the intercalated disc to cardiac gene regulation. Moreover, its subcellular localization and the observation of a severe cardiac phenotype in zebrafish, implicate Myozap in the pathogenesis of cardiomyopathy.

Mesenchymal-epithelial transitions: spontaneous and cumulative syntheses of epithelial marker molecules and their assemblies to novel cell junctions connecting human hematopoietic tumor cells to carcinomatoid tissue structures.

Using biochemical as well as light- and electron-microscopic immunolocalization methods, in cultures of unicellular human blood tumor cells, we have studied the phenomenon of spontaneous and cumulative syntheses of certain epithelial proteins and glycoproteins and their assemblies to two major kinds of novel cell-cell junctions, adhering junctions (AJs) and junctions based on the epithelial cell adhesion molecule (EpCAM). More than two decades, we have selected and characterized clonal sublines of multipotential hematopoietic K562 cells, which are enriched in newly formed AJs based on cis-clusters of desmoglein Dsg2, in some sublines accompanied by desmocollin Dsc2. Both desmosomal cadherins can be anchored in a submembranous plaque containing plakoglobin and plakophilins Pkp2 and Pkp3, with or without other armadillo proteins and desmoplakin. Also, these cells are often connected by an additional, extended junction system, in which the transmembrane epithelial glycoprotein EpCAM is associated with a cytoplasmic plaque rich in several actin-binding proteins such as afadin, α-actinin, ezrin and vinculin. Both kinds of junctions contribute to connections of K562 cells into epithelioid monolayers or even three-dimensional, tissue-like structures, thus markedly changing the cell biological nature and behavior of the resulting tumor subforms (mesenchymal-epithelial transitions). We discuss molecular mechanisms involved in the formation and function of these junctions, also with respect to tumor spread and metastasis, as well as diagnostic and therapeutic consequences.

A novel kind of tumor type-characteristic junction: plakophilin-2 as a major protein of adherens junctions in cardiac myxomata.

Using novel antibodies of high avidity to--and specificity for--the constitutive desmosomal plaque protein, plakophilin-2 (Pkp2), in a systematic study of the molecular composition of junctions connecting the cells of soft tissue tumors, we have discovered with immunocytochemical, biochemical and electron microscopical methods, a novel type of adherens junctions in all 32 cardiac myxomata examined. These junctions contain cadherin-11 as their major transmembrane glycoprotein, which we could repeatedly show in colocalization with N-cadherin, anchored in a cytoplasmic plaque formed by α- and ß-catenin, together with the further armadillo-type proteins plakoglobin, p120, p0071 and ARVCF. Surprisingly, all adherens junctions of these tumors contained, in addition, another major armadillo protein Pkp2, hitherto known as an obligatory and characteristic constituent of desmosomes in epithelium-derived tumors. We have not detected Pkp2 in a series of noncardiac myxomata studied in parallel. Therefore, we conclude that this acquisition of Pkp2, which we have recently also observed in some mesenchymally derived cells growing in culture, can also occur in tumorigenic transformations in situ. We propose to examine the marker value of Pkp2 in clinical diagnoses of cardiac myxomata and to develop Pkp2-targeted therapeutic reagents.

Beyond vessels: occurrence and regional clustering of vascular endothelial (VE-)cadherin-containing junctions in non-endothelial cells.

The genes encoding transmembrane glycoproteins of the cadherin family, i.e., the Ca(2+)-dependent cell-cell adhesion molecules, are typically expressed in cell-type- or cell-lineage-specific patterns. One of them, vascular endothelial (VE)-cadherin, is widely considered to be specific for vascular endothelia in which it is either the sole or the predominant cadherin, often co-existing with N-cadherin. This specificity of VE-cadherin for vascular endothelial cells is important not only in blood and lymph vessel biology and medicine, but also for cell-type-based diagnoses, notably those of metastatic tumors. Surprisingly, however, we have recently noted the frequent synthesis, surface exposure, and junction assembly of VE-cadherin in certain other cells, in which this glycoprotein is clustered into adherens junctions (AJs), either alone or in combination with N-cadherin and/or cadherin-11. Such cells include mammalian astrocytes and glioma, probably mostly astrocytoma cells growing in culture, and a specific subtype of astrocytoma in situ. Moreover, VE-cadherin synthesis and AJ assembly, plus the regional clustering of such AJs in certain domains, are not clonally fixed but can appear again and again in cells of the progeny of cloned homogeneous-appearing individual cells, thus resulting in clonal cell colonies that are often heterogeneous in their cadherin junction patterns. We discuss the constitutive presence of VE-cadherin in some non-endothelial cells with respect to certain architectural features and possible physiological and pathogenic functions of the cells, and in comparison with recent reports of VE-cadherin-positive melanomas.

The junctions that don't fit the scheme: special symmetrical cell-cell junctions of their own kind.

Immunocytochemical, electron-, and immunoelectron-microscopical studies have revealed that, in addition to the four major "textbook categories" of cell-cell junctions (gap junctions, tight junctions, adherens junctions, and desmosomes), a broad range of other junctions exists, such as the tiny puncta adhaerentia minima, the taproot junctions (manubria adhaerentia), the plakophilin-2-containing adherens junctions of mesenchymal or mesenchymally derived cell types including malignantly transformed cells, the composite junctions (areae compositae) of the mature mammalian myocardium, the cortex adhaerens of the eye lens, the interdesmosomal "sandwich" or "stud" junctions in the subapical layers of stratified epithelia and the tumors derived therefrom, and the complexus adhaerentes of the endothelial and virgultar cells of the lymph node sinus. On the basis of their sizes and shapes, other morphological criteria, and their specific molecular ensembles, these junctions and the genes that encode them cannot be subsumed under one of the major categories mentioned above but represent special structures in their own right, appear to serve special functions, and can give rise to specific pathological disorders.

Cordial connections: molecular ensembles and structures of adhering junctions connecting interstitial cells of cardiac valves in situ and in cell culture.

Remarkable efforts have recently been made in the tissue engineering of heart valves to improve the results of valve transplantations and replacements, including the design of artificial valves. However, knowledge of the cell and molecular biology of valves and, specifically, of valvular interstitial cells (VICs) remains limited. Therefore, our aim has been to determine and localize the molecules forming the adhering junctions (AJs) that connect VICs in situ and in cell culture. Using biochemical and immunolocalization methods at the light- and electron-microscopic levels, we have identified, in man, cow, sheep and rat, the components of VIC-connecting AJs in situ and in cell culture. These AJs contain, in addition to the transmembrane glycoproteins N-cadherin and cadherin-11, the typical plaque proteins alpha- and beta-catenin as well as plakoglobin and p120, together with minor amounts of protein p0071, i.e. a total of five plaque proteins of the armadillo family. While we can exclude the occurrence of desmogleins, desmocollins and desmoplakin, we have noted with surprise that AJs of VICs in cell cultures, but not those growing in the valve tissue, contain substantial amounts of the desmosomal plaque protein, plakophilin-2. Clusters of AJs occur not only on the main VIC cell bodies but are also found widely dispersed on their long filopodia thus forming, in the tissue, a meshwork that, together with filopodial attachments to paracrystalline collagen fiber bundles, establishes a three-dimensional suprastructure, the role of which is discussed with respect to valve formation, regeneration and function.

Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (complexus adhaerens).

The lymph node sinus are channel structures of unquestionable importance in immunology and pathology, specifically in the filtering of the lymph, the transport and processing of antigens, the adhesion and migration of immune cells, and the spread of metastatic cancer cells. Our knowledge of the cell and molecular biology of the sinus-forming cells is still limited, and the origin and biological nature of these cells have long been a matter of debate. Here, we review the relevant literature and present our own experimental results, in particular concerning molecular markers of intercellular junctions and cell differentiation. We show that both the monolayer cells lining the sinus walls and the intraluminal virgultar cell meshwork are indeed different morphotypes of the same basic endothelial cell character, as demonstrated by the presence of a distinct spectrum of general and lymphatic endothelial markers, and we therefore refer to these cells as sinus endothelial/virgultar cells (SEVCs). These cells are connected by unique adhering junctions, termed complexus adhaerentes, characterized by the transmembrane glycoprotein VE-cadherin, combined with the desmosomal plaque protein desmoplakin, several adherens junction plaque proteins including alpha- and beta-catenin and p120 catenin, and components of the tight junction ensemble, specifically claudin-5 and JAM-A, and the plaque protein ZO-1. We show that complexus adhaerentes are involved in the tight three-dimensional integration of the virgultar network of SEVC processes along extracellular guidance structures composed of paracrystalline collagen bundle "stays". Overall, the SEVC system might be considered as a local and specific modification of the general lymphatic vasculature system. Finally, physiological and pathological alterations of the SEVC system will be presented, and the possible value of the molecular markers described in histological diagnoses of autochthonous lymph node tumors will be discussed.

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules.

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. VI. Different precursor structures in non-mammalian species.

Recent studies on the formation and molecular organization of the mammalian heart have emphasized the architectural and functional importance of the adhering junctions (AJs), which are densely clustered in the bipolar end regions (intercalated disks, IDs) connecting the elongated cardiomyocytes of the adult heart. Moreover, we learned from genetic studies of mutated AJ proteins that desmosomal proteins, which for the most part are integral components of ID-specific composite AJs (areae compositae, AC), are essential in heart development and function. Developmental studies have shown that the bipolar concentration of cardiomyocyte AJs in IDs is a rather late process and only completed postnatally. Here we report that in the adult hearts of diverse lower vertebrates (fishes, amphibia, birds) most AJs remain separate and distinct in molecular character, representing either fasciae adhaerentes, maculae adhaerentes (desmosomes) or--less frequently--some form of AC. In the mature hearts of the amphibian and fish species examined a large proportion of the AJs connecting cardiomyocytes is not clustered in the IDs but remains located on the lateral surfaces where they appear either as puncta adhaerentia or as desmosomes. In many places, these puncta connect parallel cardiomyocytes in spectacular ladder-like regular arrays (scalae adhaerentes) correlated with--and connected by--electron-dense plaque-like material to sarcomeric Z-bands. In the avian hearts, on the other hand, most AJs are clustered in the IDs but only a small proportion of the desmosomes appears as AC, compared to the dominance of distinct fasciae adhaerentes. We conclude that the fusion and amalgamation of AJs and desmosomes to ACs is a late process both in ontogenesis and in evolution. The significance and possible functional implications of the specific junctional structures in vertebrate evolution and the class-specific requirements of architectural and molecular assembly adaptation during regeneration processes are discussed.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. V. The importance of plakophilin-2 demonstrated by small interference RNA-mediated knockdown in cultured rat cardiomyocytes.

In the adult mammalian heart, the cardiomyocytes are connected by large polar arrays of closely spaced or even fused composite, plaque-bearing adhering junctions (areae compositae, ACs), in a region usually termed "intercalated disk" (ID). We have recently reported that during late embryogenesis and postnatally these polar assemblies of AC-junction structures are gradually formed as replacements of distinct embryonal junctions representing desmosomes and fasciae adhaerentes which then may amalgamate to the fused AC structures, in some regions occupying more than 90% of the total ID area. Previous gene knockout results as well as mutation analyses of specific human cardiomyopathies have suggested that among the various AC constituents, the desmosomal plaque protein, plakophilin-2, plays a particularly important role in the formation, architectural organization and stability of these junctions interconnecting mature cardiomyocytes. To examine this hypothesis, we have decided to study losses of--or molecular alterations in--such AC proteins with respect to their effects on myocardiac organization and functions. Here we report that plakophilin-2 is indeed of obvious importance for myocardial architecture and cell-cell coupling of rat cardiomyocytes growing in culture. We show that siRNA-mediated reduction of the cardiomyocyte content of plakophilin-2 but not of some other major plaque components such as desmoplakin results in progressive disintegration--and losses--of AC junction structures and that numerous variously sized vesicles appear, which are plaque protein-associated as demonstrable by immunofluorescence and immunoelectron microscopy. The importance of plakophilin-2 as a kind of "organizer" protein in the formation, stabilization and functions of the AC structure and the ID architecture is discussed in relation to other junction proteins and to causes of certain cardiomyopathies.