Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization

The molecular mechanisms of how primordial adherens junctions (AJs) evolve into spatially separated belt-like AJs and tight junctions (TJs) during epithelial polarization are not well understood. Previously, we reported the establishment of ZO-1/ZO-2-deficient cultured epithelial cells (1[ko]/2[kd] cells), which lacked TJs completely. In the present study, we found that the formation of belt-like AJs was significantly delayed in 1(ko)/2(kd) cells during epithelial polarization. The activation of Rac1 upon primordial AJ formation is severely impaired in 1(ko)/2(kd) cells. Our data indicate that ZO-1 plays crucial roles not only in TJ formation, but also in the conversion from "fibroblastic" AJs to belt-like "polarized epithelial" AJs through Rac1 activation. Furthermore, to examine whether ZO-1 itself mediate belt-like AJ and TJ formation, respectively, we performed a mutational analysis of ZO-1. The requirement for ZO-1 differs between belt-like AJ and TJ formation. We propose that ZO-1 is directly involved in the establishment of two distinct junctional domains, belt-like AJs and TJs, during epithelial polarization.     

Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.     

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.     

Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles

The development of an ovarian follicle requires a complex set of reciprocal interactions between the oocyte and granulosa cells in order for both types of cells to develop properly. These interactions are largely orchestrated by the oocyte via paracrine factors such as growth differentiation factor 9 (GDF9). To examine these interactions further, a study was conducted of the effects of oocytes at different stages of development on proteins synthesized by mouse granulosa cells during the transition of granulosa cells (GCs) from preantral, secondary (2 degrees ) follicles (2 degrees GCs) to mural granulosa cells (3 degrees GCs) of antral tertiary (3 degrees ) follicles. The ability of recombinant GDF9 to mimic the effects of oocytes was also determined. Effects were evaluated by high- resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis. Coculture of the 2 degrees GCs with growing oocytes (GOs) from 2 degrees follicles brought about many of the changes in granulosa cell phenotype associated with the 2 degrees to 3 degrees follicle transition. GDF9 likewise brought about many of these changes, but only a subset of GDF9-affected protein spots were also affected by coculture with GOs. Coculture of 2 degrees GCs with the nearly fully grown oocytes (FGOs) from 3 degrees follicles had a reduced effect on 2 degrees GC phenotype, in comparison with coculture with GOs. For some proteins, oocyte coculture or GDF9 treatment appeared to have opposite effects on 2 degrees GCs and 3 degrees GCs. Additional effects of GDF9 and oocytes were seen in cultures of 2 degrees GCs for proteins other than those that differed between untreated control 2 degrees and 3 degrees GCs. These results indicate that GOs and GDF9 can each induce 2 degrees GCs to shift their phenotype toward that of 3 degrees GCs. The ability of the oocyte to produce this effect is diminished with oocyte development. The transition in the GC phenotype promoted by oocytes appears stable because differences in 2 degrees GCs promoted by oocytes and GDF9 were observed in untreated 3 degrees GCs. We conclude that the influence of the oocyte on GCs changes with the progression of their development, and so too does the response of the GCs to the oocyte. Moreover, by acting on the 2 degrees GCs, GOs are able to influence stably the phenotype of 3 degrees GCs. Thus, at or near the 2 degrees to 3 degrees follicle transition, signals from the growing oocyte contribute to the development of the mural GC phenotype.     

Interaction of integrin alpha(v)beta3 with nectin. Implication in cross-talk between cell-matrix and cell-cell junctions

Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.     

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.     

Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.     

Involvement of nectin in inactivation of integrin alpha(v)beta(3) after the establishment of cell-cell adhesion

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.     

Role of nectin in formation of E-cadherin-based adherens junctions in keratinocytes: analysis with the N-cadherin dominant negative mutant

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin-based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin-based AJs in keratinocytes.     

ADIP,a novel Afadin- and alpha-actinin-binding protein localized at cell-cell adherens junctions

Afadin is an actin filament (F-actin)-binding protein that is associated with the cytoplasmic tail of nectin, a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule. Nectin and afadin strictly localize at cell-cell adherens junctions (AJs) undercoated with F-actin bundles and are involved in the formation of AJs in cooperation with E-cadherin in epithelial cells. In epithelial cells of afadin (-/-) mice and (-/-) embryoid bodies, the proper organization of AJs is markedly impaired. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of AJs has not yet been fully understood. Here we identified a novel afadin-binding protein, named ADIP (afadin DIL domain-interacting protein). ADIP consists of 615 amino acids with a calculated M(r) of 70,954 and has three coiled-coil domains. Northern and Western blot analyses in mouse tissues indicated that ADIP was widely distributed. Immunofluorescence and immunoelectron microscopy revealed that ADIP strictly localized at cell-cell AJs undercoated with F-actin bundles in small intestine absorptive epithelial cells. This localization pattern was the same as those of afadin and nectin. ADIP was undetectable at cell-matrix AJs. ADIP furthermore bound alpha-actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to alpha-catenin. These results indicate that ADIP is an afadin- and alpha-actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through alpha-actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and alpha-actinin.     

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca²⁺ in follicle cells of amphibians. The possible involvements of these Ca²⁺ stores in mediating the hormonal control of meiotic maturation are discussed.     

Development of gap junctions in normal and mutant ovaries of Drosophila melanogaster

An ovarian follicle of Drosophila consists of an oocyte, 15 nurse cells, and hundreds of follicular epithelial cells. A freeze-fracture analysis of the surfaces between glutaraldehyde-fixed ovarian cells showed that all three cell types were interconnected by gap junctions. This is the first report of gap junctions between adjacent nurse cells, between nurse cells and oocytes, and between follicle cells and oocytes in Drosophila. Since we did not observe intramembranous particle clumping into crystalline patterns and since structurally different gap junctions occurred at different times in development and at different cell-cell interfaces, it is unlikely that fixation artifacts influenced particle distribution in our experiments. A computer-assisted morphometric analysis showed that the extent, size, and morphology of gap junctions varied with development and that these junctions can cover up to 9% of the cell surfaces. To test the role of gap junctions in follicular maturation, we studied ovaries from flies homozygous for the female sterile mutation fs(2)A17, in which follicles develop normally until yolk deposition commences. During the development of mutant follicles, gap junctions became abnormal before any other morphological aspect of the follicle. These studies show that gap junctions are available to play an important role in coordinating intercellular activities between all three cell types in ovarian follicles of Drosophila.     

A membrane fusion protein αSNAP is a novel regulator of epithelial apical junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.     

Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions

Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express β-cytoplasmic (β-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic β-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of β-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, β-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.     

The follicle cell-oocyte interaction in ovarian follicles of the stick insect Bacillus rossius (Rossi): (Insecta: Phasmatodea)

Developing ovarian follicles of Bacillus rossius have been examined ultrastructurally in an attempt to understand how inception of vitel-logenesis is controlled. Early vitellogenic follicles are characterized by a thick cuboidal epithelium that is highly interlocked with the oocyte plasma membrane. Gap junctional contacts are present both at the follicle cell/oocyte interface and in between adjacent follicle cells. In addition, microvilli of follicle cells protrude deeply into the cortical ooplasm of these early vitellogenic oocytes. With the onset of vitellogenesis, wide intercellular spaces appear in the follicle cell epithelium and at the follicle cell/oocyte interface. Gap junctions become progressively reduced both on the follicle cell surface and on the oocyte plasma membrane. Microvilli from the two cell types no longer interlock. From a theoretical standpoint each of the two structural differentiations present at the follicle cell/oocyte interface—gap junctions and follicle cell microvilli—could potentially trigger inception of vitellogenesis. Gap junctions might permit the passage of a regulatory molecule, transferring from follicle cells to oocyte, which would control the assembly of coated pits on the oocyte plasma membrane. Alternatively cell interaction via microvilli might induce the appearance of coated pits, thus creating a membrane focus for vitellogenin receptors. Both possibilities are discussed in relation to current literature.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120(ctn), beta-catenin, and alpha-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells.     

Adducins Regulate Remodeling of Apical Junctions in Human Epithelial Cells

Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane–cytoskeleton interface may play a key role in junctional regulation. Spectrin–adducin–ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin–actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and β-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of βII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.     

Suppression of SEMA6C promotes preantral follicles atresia with decreased cell junctions in mice ovaries

Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis. Recent studies revealed that the communication between oocytes and granulosa cells (GCs) is a critical process for female follicle development. In the current study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the cell junctions between oocytes and GCs in mice preantral follicles. The attenuation of SEMA6C expression by siRNA decreased the cell–cell junctions and accelerated follicle atresia in vitro. PI3K-AKT pathway was activated when SEMA6C expression was downregulated. And the LY294002, a PI3K inhibitor, could reverse the effect of low SEMA6C expression on cell junctions in preantral follicles. Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging.