Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus)

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.     

Keap1 in adhesion complexes

Cell adhesion complexes are sensors that interact with the extracellular environment and allow for the transmission of signals found outside the cell across the plasma membrane to the cell interior. Keap1 is a newly identified component of cell adhesion complexes. We investigated Keap1's association with these complexes in diverse tissues and cell types. Keap1 is present in focal adhesion (FA)-like assemblies in kidney proximal tubule cells where it colocates with actin. In liver, Keap1 is found in the adherens junctions (AJ) and at the base of the bile canaliculi. To study Keap1's involvement in both the integrin-based FA and the cadherin-based AJ, we induced formation of these complexes in fibroblasts, using a serum starvation followed by a serum supplementation method. When compared with vinculin, a component of all FA, we found that Keap1 assembles only in the peripheral FA. Within the peripheral FA, Keap1 was present in distinct foci along the length of the FA and these foci were different from vinculin, talin, paxillin, and phospho-tyrosine rich regions of the FA. Unlike most FA components, Keap1 was also recruited to the newly formed AJ. As Keap1 homologues are actin-bundling proteins, we hypothesize that Keap1's function is to bundle F-actin within these diverse types of cell adhesion components.     

Multiple Neuronal Connexins in the Mammalian Retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Multiple neuronal connexins in the mammalian retina

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.     

Fate of annular gap junctions in the papillary cells of the enamel organ in the rat incisor

Summary To investigate the mechanisms whereby annular gap junctions in the papillary cells of the enamel organ are degraded intracellularly, continuously growing rat incisors were examined by electron microscopy of routine thin sections as well as for the cytochemical localization of inorganic trimetaphosphatase activity. Routine thin-section analysis revealed small flat or undulated gap junctions, hemi-annular gap junctions between an invaginated cell process and a cell body, and fully internalized cytoplasmic annular gap junctions. Both hemi-annular and annular gap junctions usually contain various organelles and/or inclusions, such as mitochondria, endoplasmic reticulum, ribosomes, vesicles, and lysosomes in the cytoplasm confined by the junctional membranes. Annular gap junctions are sometimes fused with vesicular or tubulovesicular structures. Cytochemistry of inorganic trimetaphosphatase activity revealed an intense enzymatic reaction within a system of tubular structures and round or oval dense bodies. Both structures are believed to correspond to primary lysosomes. A part of the Golgi apparatus also shows a weak reaction. Although hemi-annular gap junctions never show enzymatic reaction, annular gap junctions sometimes contain reaction products throughout their interior cytoplasm and inclusions. Fusion of annular gap-junctional membranes with reaction-positive tubular structures is also observed. In one instance, revealed in serial sections, an annular gap junction was encircled entirely by a reaction-positive structure. These results suggest that cytoplasmic annular gap junctions are formed by endocytosis of hemi-annular gap junctional membranes from the cell surface and then degraded intracellularly by lysosomal enzymes.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Molecular basis for pacemaker cells in epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.     

Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells.     

Regulation of vascular endothelial junction stability and remodeling through Rap1-Rasip1 signaling

The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell–cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions.     

Differences in claudin synthesis in primary cultures of acinar cells from rat salivary gland are correlated with the specific three-dimensional organization of the cells

Tight junctions are essential for the maintenance of epithelial cell polarity. We have previously established a system for the primary culture of salivary parotid acinar cells that retain their ability to generate new secretory granules and to secrete proteins in a signal-dependent manner. Because cell polarity and cell-cell adhesion are prerequisites for the formation of epithelial tissues, we have investigated the structure of the tight junctions in these cultures. We have found two types of cellular organization in the culture: monolayers and semi-spherical clusters. Electron microscopy has revealed tight junctions near the apical region of the lateral membranes between cells in the monolayers and cells at the surface of the clusters. The cells in the interior of the clusters also have tight junctions and are organized around a central lumen. These interior cells retain more secretory granules than the surface or monolayer cells, suggesting that they maintain their original character as acinar cells. The synthesis of claudin-4 increases during culture, although it is not detectable in the cells immediately after isolation from the glands. Immunofluorescence microscopy has shown that claudin-4 is synthesized in the monolayers and at the surface of the clusters, but not inside the clusters. Only claudin-3, which is present in the original acinar cells following isolation and in the intact gland, has been detected inside the clusters. These results suggest that differences in claudin expression are related to the three-dimensional structures of the cell cultures and reflect their ability to function as acinar cells. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (16591868, 16791135), by a Suzuki Memorial Grant of the Nihon University School of Dentistry at Matsudo (Joint Research Grant for 2003), by a Nihon University Multidisciplinary Research Grant for 2005 and 2006, and by a Grant-in-Aid for a 2003 Multidisciplinary Research Project from MEXT.     

Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell.     

Mitochondrial filaments and clusters as intracellular power-transmitting cables

Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.     

Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins in rat testis

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I-VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII-I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII-I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.     

The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors

Nectins are Ca(2+)-independent Ig-like cell adhesion molecules (CAMs) which homophilically and heterophilically interact in trans with nectins and form cell-cell adhesion. This cell-cell adhesion is involved in the formation of many types of cell-cell junctions such as adherens junctions, tight junctions, and synaptic junctions, cooperatively with other CAMs such as cadherins and claudins. Nectins transduce signals cooperatively with integrin alpha(v)beta(3), and regulate formation of cell-cell junctions. In addition, nectin interacts in cis with PDGF receptor and regulates its signaling for anti-apoptosis. Furthermore, nectin interacts in trans with nectin-like molecule-5 (Necl-5) and regulate cell movement and proliferation. We describe cooperative roles of nectins with other CAMs and growth factor receptors.     

Self-organization in and on biological spheres

Three biological settings involving self-organization performed by the Turing-Child field inside a sphere and on its surface are considered. In the first setting the interior of a sphere made up of cells communicating via gap junctions is considered. It is suggested that the Turing-Child self-organization is the cause of radial polarization, the first differentiation of an early mammalian embryo. In the second setting, the Turing example of gastrulation of a hollow cellular sphere is considered. It is shown that Child's experimental patterns are predicted and explained by the Turing-Child theory. The third setting is the interior of a biological cell, and it is suggested that it is the self-organization of the Turing-Child field that causes the formation of the mitotic spindle.     

Cell culture dosimetry for low-frequency magnetic fields

Calculations of the current density and electric field distributions induced in cell cultures by an applied low-frequency magnetic field have assumed that the medium is uniform. This paper calculates these distributions for a more realistic, inhomogeneous, anisotropic model in which the cells are regarded as conducting squares surrounded by insulating membranes. Separate parameters are used to specify the resistivities of the cell interior, the cell membrane parallel to its surface, the cell membrane perpendicular to its surface, and the intercellular junction parallel to the membrane. The presence of gap junctions connecting the interiors of adjacent cells is also considered. For vertical applied magnetic fields, the induced currents and field distributions may deviate considerably from the homogeneous medium model if there is sufficiently tight binding of the cells to each other. The presence of gap junctions can produce relatively large transmembrane electric fields or intracellular current densities. These considerations are generally less important for horizontal applied fields. A simple microscopic model of the cell surface is also discussed. © 1996 Wiley-Liss, Inc.     

Self-organization in and on biological spheres

Three biological settings involving self-organization performed by the Turing-Child field inside a sphere and on its surface are considered. In the first setting the interior of a sphere made up of cells communicating via gap junctions is considered. It is suggested that the Turing-Child self-organization is the cause of radial polarization, the first differentiation of an early mammalian embryo. In the second setting, the Turing example of gastrulation of a hollow cellular sphere is considered. It is shown that Child's experimental patterns are predicted and explained by the Turing-Child theory. The third setting is the interior of a biological cell, and it is suggested that it is the self-organization of the Turing-Child field that causes the formation of the mitotic spindle.