A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.     

Evolution: structural and functional diversity of cadherin at the adherens junction

Adhesion between cells is essential to the evolution of multicellularity. Indeed, morphogenesis in animals requires firm but flexible intercellular adhesions that are mediated by subcellular structures like the adherens junction (AJ). A key component of AJs is classical cadherins, a group of transmembrane proteins that maintain dynamic cell-cell associations in many animal species. An evolutionary reconstruction of cadherin structure and function provides a comprehensive framework with which to appreciate the diversity of morphogenetic mechanisms in animals.     

Cadherin-actin interactions at adherens junctions

The adherens junction (AJ) is a major cell-cell junction that mediates cell recognition, adhesion, morphogenesis, and tissue integrity. Although AJs transmit forces generated by actomyosin from one cell to another, AJs have long been considered as an area where signal transduction from cadherin ligation takes place through cell adhesion. Through the efforts to understand embryonic or cellular morphogenesis, dynamic interactions between the AJ and actin filaments have become crucial issues to be addressed since actin association is essential for AJ development, remodeling and function. Here, I provide an overview of cadherin-actin interaction from morphological aspects and of possible molecular mechanisms revealed by recent studies.     

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.     

E-N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells

Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this "cadherin switch" hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them. We show that the cadherins in a major portion of AJs in these cells can be chemically cross-linked in E-N heterodimers. We also show that cells possessing E-N heterodimer AJs can form semistable hemihomotypic AJs with purely N-cadherin-based AJs of mesenchymally derived cells, including stroma cells. We conclude that these heterodimers are the major AJ constituents of several endoderm-derived tissues and tumors and that the prevailing concept of antagonistic roles of these two cadherins in developmental and tumor biology has to be reconsidered.     

Modulation of Cell–Cell Adherens Junctions by Surface Clustering of the N-Cadherin Cytoplasmic Tail

Cadherins mediate the formation of cell–cell adherens junctions (AJ) by homophilic interactions through their extracellular domains as well as by interacting with the actin cytoskeleton via their cytoplasmic portions. Cadherin clustering initiates cytoplasmic signaling that results in the assembly of structural components into cell–cell AJ. To elucidate the function of the cytoplasmic tail of cadherins in initiating the assembly signal, we generated and characterized a chimeric cadherin tail fused to an inert transmembrane anchor. The chimera enabled us to cluster the cadherin cytoplasmic tail in the absence of extracellular portions of the molecule. The transfected cadherin tail chimera localized to cell–cell AJ of epithelial cells, indicating that the submembrane junctional plaque has the capacity to recruit additional cadherins, with no involvement of their extracellular domains. Expression of the chimera in cells of mesenchymal origin resulted in dominant negative effects on the formation of cell–cell AJ. Surface clustering of cadherin cytoplasmic tails induced the recruitment of components and structural assembly of cell–cell AJ, thereby reversing the initial dominant–negative effects. We conclude that the cadherin cytoplasmic tail contains information required to direct the molecule to cell–cell AJ. Its function as modulator of cell–cell AJ depends on cell type and on whether the tail is clustered.     

A Common Clathrin‐Mediated Machinery Co‐ordinates Cell–Cell Adhesion and Bacterial Internalization

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E‐cadherin, hijack the host adherens junction (AJ) machinery and invade non‐phagocytic cells by a clathrin‐dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell–cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E‐cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E‐cadherin. We demonstrate that non‐adherent cells expressing the InlA chimera, as bacteria, can be internalized by E‐cadherin‐expressing adherent cells. Together these results reveal that a common clathrin‐mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.     

Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension.     

N-cadherin-mediated cell adhesion restricts cell proliferation in the dorsal neural tube

Neural progenitors are organized as a pseudostratified epithelium held together by adherens junctions (AJs), multiprotein complexes composed of cadherins and α- and β-catenin. Catenins are known to control neural progenitor division; however, it is not known whether they function in this capacity as cadherin binding partners, as there is little evidence that cadherins themselves regulate neural proliferation. We show here that zebrafish N-cadherin (N-cad) restricts cell proliferation in the dorsal region of the neural tube by regulating cell-cycle length. We further reveal that N-cad couples cell-cycle exit and differentiation, as a fraction of neurons are mitotic in N-cad mutants. Enhanced proliferation in N-cad mutants is mediated by ligand-independent activation of Hedgehog (Hh) signaling, possibly caused by defective ciliogenesis. Furthermore, depletion of Hh signaling results in the loss of junctional markers. We therefore propose that N-cad restricts the response of dorsal neural progenitors to Hh and that Hh signaling limits the range of its own activity by promoting AJ assembly. Taken together, these observations emphasize a key role for N-cad-mediated adhesion in controlling neural progenitor proliferation. In addition, these findings are the first to demonstrate a requirement for cadherins in synchronizing cell-cycle exit and differentiation and a reciprocal interaction between AJs and Hh signaling.     

Adherens Junction: Molecular Architecture and Regulation

The adherens junction (AJ) is an element of the cell–cell junction in which cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions. Cadherins associate with cytoplasmic proteins, called catenins, which in turn bind to cytoskeletal components, such as actin filaments and microtubules. These molecular complexes further interact with other proteins, including signaling molecules, rendering the AJs into highly dynamic and regulatable structures. The AJs of such nature contribute to the physical linking of cells, as well as to the regulation of cell–cell contacts, which is essential for morphogenesis and remodeling of tissues and organs. Thus, elucidating the molecular architecture of the AJs and their regulatory mechanisms are crucial for understanding how the multicellular system is organized.The adherens junction (AJ) is a form of cell–cell adhesion structure observed in a variety of cell types, as well as in different animal species. It is characterized by a pair of plasma membranes apposed with a distance of 10–20 nm between them, whose intercellular space is occupied by rod-shaped molecules bridging the membranes (Hirokawa and Heuser 1981; Miyaguchi 2000), and the cytoplasmic side of the AJ is associated with condensed actin filaments. In polarized epithelia of vertebrates, the AJ is part of the tripartite junctional complex localized at the juxta-luminal region, which comprises the tight junction (zonula occludens), AJ, and desmosome (macula adherens) aligned in this order from the apical end of the junction (Farquhar and Palade 1963). In this type of epithelia, the AJ is specifically termed the “zonula adherens” or “adhesion belt,” as it completely encloses the cells along with the F-actin lining, called the circumferential actin belt (Fig. 1). The AJs in other cell types assume different morphologies: For example, the AJs in fibroblastic cells are spotty and discontinuous (Yonemura et al. 1995), and those in neurons are organized into tiny puncta as a constituent of the synaptic junctions (Uchida et al. 1996).Open in a separate windowFigure 1.Morphological variations of the adherens junction. In Caco2 cells (colonic carcinoma line), E-cadherin is localized along the actin circumferential belt to organize the zonula adherens (arrow). At the lateral portions of cell junction (arrowheads), E-cadherin signals are punctate, only occasionally overlapping with actin signals in this specific sample. The lateral patterns of cadherin and actin distribution, however, vary with cellular conditions. In MCF10A cells (mammary epithelial line), spotty adherens junctions are seen, where actin filaments perpendicularly terminate at E-cadherin puncta.A major function of AJs is to maintain the physical association between cells, as disruption of them causes loosening of cell–cell contacts, leading to disorganization of tissue architecture. Calcium chelators such as EDTA and EGTA are widely used as a reagent to promote the dissociation of cells in tissues or monolayer cultures. A major target of these chelators is the AJ, as this is a calcium-sensitive structure; although, calcium removal is generally insufficient for the complete dispersion of cells because of the presence of calcium-independent cell–cell adhesion mechanisms (Takeichi et al. 1977). Early studies to search for the molecules responsible for the calcium-dependent junctions resulted in the identification of a group of type-I transmembrane proteins, and its founding member was termed cadherin (Yoshida and Takeichi 1982; Yoshida-Noro et al. 1984). Related molecules identified were also called by various names, such as uvomorulin (Peyrieras et al. 1983), LCAM (Gallin et al. 1983), and ACAM (Volk and Geiger 1984). Later studies revealed that the cadherins form a superfamily, and therefore, the original cadherins are now called “classic” cadherins.Another series of studies have identified nectins, a family of immunoglobulin-like transmembrane proteins, as an AJ component. Nectins function in a calcium-independent way to promote cell–cell adhesion (Nakanishi and Takai 2004). In this article, we overview the molecular organization of the AJs constructed with these membrane proteins, as well as the regulatory mechanisms that operate to sustain or remodel these junctions, paying much attention to the linkages between the AJ and cytoskeletal or signaling proteins.     

Adherens junctions remain dynamic

One of the four principal categories of cell-cell junctions that hold together and shape distinct tissues and organs in vertebrates, adherens junctions (AJs) form cell-cell contacts that connect transmembrane proteins with cytoskeletal actin filaments to provide architectural strength, aid in morphogenesis, and help to maintain proper tissue homeostasis. The classical organization of AJs, consisting of transmembrane cadherins and cytoplasmically attached β-catenins and α-catenins assembled together into a multiprotein complex, was once thought obligatory to craft a robust and stable connection to actin-based cytoskeletal elements, but this architecture has since been challenged and questioned to exist. In a stimulating paper published in a recent issue of BMC Biology, Millán et al. provide convincing evidence that in confluent vascular endothelial cells a novel dynamic vascular endothelial (VE)-cadherin-based AJ type exists that interacts with and physically connects prominent bundles of tension-mediating actin filaments, stress fibers, between neighboring cells. Stress fibers were known previously to link to integrin-based focal adhesion complexes but not to cell-cell adhesion mediating AJs. These new findings, together with previous results support the concept that different AJ subtypes, sharing the same transmembrane cadherin types, can assemble in various configurations to either increase barrier function and promote physical cell-cell adhesion, or to lessen cell-cell adhesion and promote cell separation and migration.     

Trypanosoma cruzi alters adherens junctions in cardiomyocytes

We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.     

Protein kinases and adherens junction dynamics in the seminiferous epithelium of the rat testis

Earlier studies in multiple epithelia have shown that cell-cell actin-based adherens junction (AJ) dynamics are regulated, at least in part, by the interplay of kinases and phosphatases that determines the intracellular phosphoprotein content. Yet it is virtually unknown regarding the role of protein kinases in Sertoli-germ cell AJ dynamics in the seminiferous epithelium of the testis. To address this issue, an in vitro coculture system utilizing Sertoli and germ cells was used to study the regulation of several protein kinases, including c-Src (the cellular form of the v-src transforming gene of Rous Sarcoma virus, RSV), carboxyl-terminal Src kinase (Csk), and casein kinase 2 (CK2), during AJ assembly. Both Sertoli and germ cells were shown to express c-Src, Csk, and CK2 with a relative Sertoli:germ cell ratio of approximately 1:1, suggesting both cell types contributed equally to the pool of these kinases in the epithelium. c-Src and Csk were shown to be stage-specific proteins during the epithelial cycle, being highest at stages VII-VIII. Studies using immunoprecipitation have illustrated that these kinases were structurally associated with the N-cadherin/beta-catenin, but not the nectin/afadin, protein complex, implicating that the cadherin/catenin protein complex is their likely putative substrate. An induction in c-Src, Csk, and CK2 were detected during Sertoli-germ cell AJ assembly in vitro but not when Sertoli cells were cultured alone. When adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364), a compound known to induce germ cell loss from the seminiferous epithelium, in particular elongating/elongate and round spermatids, by disrupting Sertoli-germ cell AJs, an induction of c-Src and Csk, but not CK2, was detected. Furthermore, a transient increase in the intrinsic kinase activities of c-Src, but not CK2, was also detected. This event was also associated with a loss of protein-protein association of N-cadherin and beta-catenin from the cadherin/catenin/c-Src/Csk/CK2 protein complex. Administration of PP1, a c-Src inhibitor, into adult rats via the jugular vein could induce the loss of spermatocytes and round spermatids, but not elongating/elongate spermatids, from the seminiferous epithelium. This result thus implicates the importance of c-Src in maintaining the integrity of AJs and possibly desmosome-like junctions between Sertoli cells and spermatocytes/round spermatids. In short, the data reported herein have shown that c-Src, Csk, and CK2 are novel protein kinases in AJ dynamics in the testis.     

IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells

The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC‐α phosphorylation, adherens junction (AJ) disassembly (by dislodging β‐catenin from VE‐cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates β‐catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C‐terminal truncated IQGAP1 (IQΔC) that cannot bind β‐catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho‐PKC‐α interacts with the C‐terminal portion of Ecgp96 as well as with VE‐cadherin after IQGAP1‐mediated AJ disassembly. HBMEC overexpressing either C‐terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction ofphospho‐PKC‐α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC‐α phosphorylation and association of phospho‐PKC‐α with Ecgp96, and then signals IQGAP1 to detach β‐catenin from AJs. Subsequently, IQGAP1/β‐catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.     

N‐cadherin regulates the proliferation and differentiation of ventral midbrain dopaminergic progenitors

Adherens junction (AJ) between dopaminergic (DA) progenitors maintains the structure of ventricular zone and polarity of radial glia cells in the ventral midbrain (vMB) during embryonic development. However, it is unclear how loss of N‐cadherin might influence the integrity of the AJ and the process of DA neurogenesis. Here, we used conditional gene targeting approaches to perform the region‐specific removal of N‐cadherin in the neurogenic niche of DA neurons in the vMB. Removal of N‐cadherin in the vMB using Shh‐Cre disrupts the AJs of DA progenitors and radial glia processes in the vMB. Surprisingly, loss of N‐cadherin in the vMB leads to a significant expansion of DA progenitors, including those expressing Sox2, Ngn2, and Otx2. Cell cycle analyses reveal that the cell cycle exit in the progenitor cells is decreased in the mutants from E11.5 to E12.5. In addition, the efficiency of DA progenitors in differentiating into DA neurons is decreased from E10.5 to E12.5, leading to a marked reduction in the number of DA neurons at E11.5, E12.5, and E17.5. Loss of N‐cadherin leads to the diffuse distribution of β‐catenin proteins, which are a critical component of AJ and Wnt signaling, from the AJ throughout the entire cytoplasm in neuroepithelial cells, suggesting that canonical Wnt signaling might be activated in the DA progenitors in vMB. Taken together, these results support the notion that N‐cadherin regulates the proliferation of DA progenitors and the differentiation of DA neurons through canonical Wnt‐β‐catenin signaling in the vMB. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 518–529, 2013     

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators during Drosophila development. We found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent.     

TGF-β3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.     

TGF-beta3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.     

Adherens junction remodeling by the Notch pathway in Drosophila melanogaster oogenesis

Identifying genes involved in the control of adherens junction (AJ) remodeling is essential to understanding epithelial morphogenesis. During follicular epithelium development in Drosophila melanogaster, the main body follicular cells (MBFCs) are displaced toward the oocyte and become columnar. Concomitantly, the stretched cells (StCs) become squamous and flatten around the nurse cells. By monitoring the expression of epithelial cadherin and Armadillo, I have discovered that the rate of AJ disassembly between the StCs is affected in follicles with somatic clones mutant for fringe or Delta and Serrate. This results in abnormal StC flattening and delayed MBFC displacement. Additionally, accumulation of the myosin II heavy chain Zipper is delayed at the AJs that require disassembly. Together, my results demonstrate that the Notch pathway controls AJ remodeling between the StCs and that this role is crucial for the timing of MBFC displacement and StC flattening. This provides new evidence that Notch, besides playing a key role in cell differentiation, also controls cell morphogenesis.