The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.     

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.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates - IV: coalescence and amalgamation of desmosomal and adhaerens junction components - late processes in mammalian heart development

In the adult mammalian heart, the cardiomyocytes and thus their terminally anchored myofibrillar bundles are connected by large arrays of closely spaced or even fused adhering junctions (AJs), termed "intercalated disks" (IDs). In recent years, the ID complex has attracted special attention as it has become clear that several human hereditary cardiomyopathies are caused by mutations of genes encoding ID marker proteins, in particular some that are also known as constituents of epithelial desmosomes. Previously, we have shown that in the mature myocardial ID the compositional differences between desmosome-like and adhaerens junctions are, by and large, lost and a composite hybrid structure, the area composita, is formed. We now report results from immunofluorescence and (immuno-)electron microscopic studies of heart formation during mouse embryogenesis and postnatal growth and show that the formation of the IDs with extended area composita structures is a late, primarily postnatal process. While up to birth small distinct desmosomes and AJs are resolved as predominant ID structures, areae compositae of increasing sizes and merged marker protein patterns occupy most of the IDs in the mature heart. Differences in the patterns of ID formation and amalgamation of the two ensembles of junction proteins in time and space are also demonstrated. Together with corresponding observations during rat and human heart development our results indicate that ID topogenesis and area composita formation are also late developmental processes in other mammals. We discuss the importance of the ID and the areae compositae in cardiac functions and, consequently, in cardiomyopathies and possible myocardial regeneration processes.     

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.     

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell-cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell-cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell-cell junctions as well as medical consequences are discussed.     

Plakophilins—hard work in the desmosome,recreation in the nucleus?

The linkage of the different types of cytoskeletal proteins to cell adhesion structures at the cytoplasmic membrane and the connection of these contact sites to corresponding sites of adjacent cells is a prerequisite for integrity and stability of cells and tissues. The structurally most prominent types of such cell-cell adhesion complexes are the desmosomes (maculae adhaerentes), which are found in all epithelia and certain non-epithelial tissues. As an element of the cytoskeleton, intermediate filaments are connected to the adhesive desmosomal transmembrane proteins by the cytoplasmic desmosomal plaque proteins. At least three different types of proteins are found in the desmosomal plaque, one of which is represented by the plakophilins, a recently described sub-family of sequence-related armadillo-repeat proteins. Consisting of three isoforms, plakophilins (plakophilin 1 to 3, PKP 1 to 3) are located in all desmosomes in a differentiation-dependent manner. While PKP 2 and PKP 3 are part of almost all desmosome-bearing cell types (PKP 2 except for differentiated cells of stratified epithelia and PKP 3 for hepatocytes and cardiomyocytes), PKP 1 is restricted to desmosomes of cells of stratified and complex epithelia. Besides the architectural function that plakophilins seem to fulfill in the desmosomes, at least PKP 1 and 2 are also localized in the nucleus independently of any differentiation-related processes and with an up to now enigmatic function in this compartment. In the following article we want to summarize the current knowledge concerning structure, function and regulation of the plakophilins that has been achieved during the last decade.     

Cardiac telocytes — their junctions and functional implications

Telocytes (TCs) form a cardiac network of interstitial cells. Our previous studies have shown that TCs are involved in heterocellular contacts with cardiomyocytes and cardiac stem/progenitor cells. In addition, TCs frequently establish 'stromal synapses' with several types of immunoreactive cells in various organs ( www.telocytes.com ). Using electron microscopy (EM) and electron microscope tomography (ET), we further investigated the interstitial cell network of TCs and found that TCs form 'atypical' junctions with virtually all types of cells in the human heart. EM and ET showed different junction types connecting TCs in a network (puncta adhaerentia minima, processus adhaerentes and manubria adhaerentia). The connections between TCs and cardiomyocytes are 'dot' junctions with nanocontacts or asymmetric junctions. Junctions between stem cells and TCs are either 'stromal synapses' or adhaerens junctions. An unexpected finding was that TCs have direct cell-cell (nano)contacts with Schwann cells, endothelial cells and pericytes. Therefore, ultrastructural analysis proved that the cardiac TC network could integrate the overall 'information' from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells/progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and planar contacts) and the mean intermembrane distance is within the macromolecular interaction range (10-30 nm). In conclusion, TCs make a network in the myocardial interstitium, which is involved in the long-distance intercellular signaling coordination. This integrated interstitial system appears to be composed of large homotropic zones (TC-TC junctions) and limited (distinct) heterotropic zones (heterocellular junctions of TCs).     

The cardiac ultrastructure of Chimaera monstrosa L. (Elasmobranchii: Holocephali)

Summary The ultrastructure of the heart in Chimaera monstrosa L. is described. The endocardial and the epicardial cells are similar in the three cardiac regions. Myocardial cells show small variations.The myofibre, 4–6 m thick, contains one or a few myofibrils. Each myosin filament is surrounded by six actin filaments. The sarcomere banding pattern includes the Z-, A-, I-, M-, N-, and H-band. End-to-end attachments between myofibres are composed of alternating desmosomes and fasciae adhaerentes. Desmosomes and nexuses occur between longitudinally oriented cell surfaces. The sarcoplasmic reticulum is poorly developed but well defined. Peripheral coupling-like structures are common, T-tubules are absent. Membrane bound dense bodies occur in all regions. Areas with ribosomes and single myosin filaments are often seen.The epicardial cells have a regular hexagonal surface and are much thicker than the endocardial cells. Numerous short and a few longer cytoplasmic extensions face the pericardial cavity.The fiat endocardial cells contain a large nucleus and small amounts of cytoplasm.     

Apoptosis in the developing human heart resembles apoptosis in epithelial tissues

It is widely accepted that apoptosis plays an important role in the development of the heart as well as in different heart diseases. Despite extensive research efforts, many issues regarding apoptosis in the heart remain unsolved, including the detection of apoptotic cardiomyocytes, their morphological features, the mechanisms of their removal and the clinical significance of apoptosis in the heart. It has been suggested that fetal cardiomyocytes resemble epithelial tissues. To test this hypothesis, we analyzed the expression of an epithelial marker cytokeratin 18 (CK18) and caspase-cleaved-CK18, recognized by antibody M30, as well as the expression of cleaved caspase-3 and desmosomal and classical cadherins, major components of desmosomes and adherens junctions in fetal hearts in comparison to infant and adult human hearts. We found that, in fetal hearts, cardiomyocytes expressed CK18 and that apoptotic cardiomyocytes expressed caspase-cleaved CK18, being recognized by antibody M30. Furthermore, desmosomal and classical cadherins exhibited a membraneous reaction similar to epithelial tissues. In adults and children after the age of 6 months, cadherins were localized in the intercalated disks, cardiomyocytes lost CK18 expression and apoptotic cardiomyocytes were no longer recognized by M30. We conclude that apoptosis in the developing human heart resembles apoptosis in epithelial tissues, exhibiting different characteristics than in the adult human heart.     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intercellular adhering junctions with an asymmetric molecular composition: desmosomes connecting Merkel cells and keratinocytes

Merkel cells (MCs) are special neuroendocrine epithelial cells that occur as individual cells or as cell groups within the confinements of a major epithelium formed and dominated by other epithelial cells. In the epidermis and some of its appendages MCs are mostly located in the basal cell layer, occasionally also in suprabasal layers and generally occur in linear arrays in outer root sheath cell layers of hair follicles. As MCs are connected to the adjacent keratinocytes by a series of adhering junctions (AJs), of which the desmosomes are the most prominent, these junctions represent heterotypic cell–cell connections, i.e. a kind of structure not yet elucidated in molecular terms. Therefore, we have studied these AJs in order to examine the molecular composition of the desmosomal halves. Using light- and electron-microscopic immunolocalization and keratin 20 as the MC-specific cell type marker we show that the plaques of the MC half of the desmosomes specifically and constitutively contain plakophilin Pkp2. This protein, however, is absent in the keratinocyte half of such heterotypic desmosomes which instead contains Pkp1 and/or Pkp3. We discuss the developmental, tissue-architectonic and functional importance of such asymmetric junctions in normal physiology as well as in diseases, in particular in the formation of distant tumor cell metastasis.     

The ultrastructure of the epithelium of the ciliary body

Summary The fine structure of the human and rabbit ciliary body epithelium has been studied with the electron microscope, both under normal conditions and after paracentesis of the anterior chamber. The disposition of the junctional complexes in the two layers of the ciliary epithelium is described in detail. Junctional complexes appear particularly developed between the apical surfaces of the cells of the two layers, but are present, as in other monolayered epithelia, also between the lateral surfaces of adjacent cells of each layer. The junctional complexes connecting the apical surfaces of the cells of the two layers are represented by zonulae occludentes, zonulae adhaerentes and desmosomes following each other irregularly, with interposition of rounded dilatations of the intercellular space called ciliary channels. The zonulae occludentes and adhaerentes found along the lateral surfaces of the epithelial cells probably form discontinuous and overlapping fasciae. Moreover, the existence of a peculiar dove-tail arched junction called macula occludens is suggested. Few differences were encountered when comparing the arrangement of junctional complexes in the ciliary epithelium of man with that of the rabbit. Many desmosomes connecting the basal portion of lateral surfaces of the non-pigmented cells were found only in human ciliary bodies.The study of the modifications of the junctional apparatus of the ciliary epithelium following paracentesis of the anterior chamber, confirms the functional hypotheses on junctional complexes previously suggested: in particular only zonulae occludentes cause a real block of the intercellular spaces. On the basis of the present work, the close relationships between the number, kind and disposition of junctional complexes in epithelia and the functional possibilities of these epithelia are stressed.Dedicated to Professor W. Bargmann on his 60th birthday.Dr. Orzalesi is the recipient of a research grant from Ministero della Pubblica Istruzione for 1964.     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. II. Colocalizations of desmosomal and fascia adhaerens molecules in the intercalated disk

Using immunofluorescence histochemistry and immunoelectron microscopy on sections through myocardiac tissues of diverse mammalian (human, cow, rat, mouse) and fish species we show that both desmosomal and fascia adhaerens proteins identified by gel electrophoresis and immunoblot occur in the area composita, the by far major type of plaque-bearing junctions of the intercalated disks (IDs) connecting cardiomyocytes. Specifically, we demonstrate that desmoplakin and the other desmosomal proteins occur in these junctions, together with N-cadherin, cadherin-11, alpha- and beta-catenin as well as vinculin, afadin and proteins p120(ctn), ARVCF, p0071, and ZO-1, suggestive of colocalization. We conclude that the predominant type of adhering junction present in IDs is a junction sui generis, termed area composita, that is characterized by an unusually high molecular complexity and an intimate association of molecules of both ensembles, the desmosomal one and the fascia adhaerens category. We discuss possible myocardium-specific, complex-forming interactions between members of the two ensembles and the relevance of our findings for the formation and functioning of the heart and for the understanding of hereditary and other cardiomyopathies. We further propose to use this highly characteristic area composita ensemble of molecules as cardiomyocyte markers for the monitoring of cardiomyogenesis, cardiomyocyte regeneration and possible cardiomyocyte differentiation from mesenchymal stem cells.     

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. We show that atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, we find a balance of microtubule and actin interactions controls AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost.     

Establishment of cardiac cytoarchitecture in the developing mouse heart

Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.     

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 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.     

High resolution scanning electron microscopy of the intracellular surface of intercalated disks in human heart

The three-dimensional architecture of human cardiac intercalated disks was examined by high resolution scanning electron microscopy of osmium-macerated specimens. This methodology permits viewing of in situ intercalated disks from a vantage point inside individual cardiomyocytes. The erose nature of these structures was rendered in stark relief. Areas covered with clusters of particles were present on some membranous projections--these may represent a combination of desmosomes and fasciae adherentes. On the other hand, areas devoid of particles may correspond to gap junctions.     

Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning

The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPs) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs.     

CELLULAR MECHANISM OF INTESTINAL PERMEABILITY ALTERATIONS PRODUCED BY CHELATION DEPLETION

The absorption of phenolsulfonphthalein (phenol red) was used as a measure in vivo of intestinal permeability in anesthetized rats. A chelating agent, sodium ethylenediaminetetraacetate (NaEDTA), placed in the lumen evoked a fivefold increase in membrane permeability; at the same time the mucosal content of magnesium and calcium decreased significantly. Making either magnesium or calcium available to the luminal surface of the membrane in isotonic solution restored normal permeability and brought the cation contents above the original levels. Electron micrographs of tissues treated in vivo with NaEDTA revealed (a) rounded swellings on the microvilli in the area of the junctional complexes between adjacent epithelial cells, (b) widening of intercellular channels particularly in the region of the intermediate junctions (zonulae adhaerentes), and (c) loss of architectural detail in the region of the desmosomes (maculae adhaerentes) with separation of their dense borders. All of these alterations in fine structure could be reversed by in vivo cation replacements which reinstated normal permeability. The implications of these findings on mechanisms of fluid transport across epithelial membranes are discussed, and a working hypothesis for the role of divalent cations in membrane permeability regulation is presented.