Inactivation of aPKClambda results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.     

Planar cell polarity links axes of spatial dynamics in neural-tube closure

Neural-tube closure is a critical step of embryogenesis, and its failure causes serious birth defects. Coordination of two morphogenetic processes--convergent extension and neural-plate apical constriction--ensures the complete closure of the neural tube. We now provide evidence that planar cell polarity (PCP) signaling directly links these two processes. In the bending neural plates, we find that a PCP-regulating cadherin, Celsr1, is concentrated in adherens junctions (AJs) oriented toward the mediolateral axes of the plates. At these AJs, Celsr1 cooperates with Dishevelled, DAAM1, and the PDZ-RhoGEF to upregulate Rho kinase, causing their actomyosin-dependent contraction in a planar-polarized manner. This planar-polarized contraction promotes simultaneous apical constriction and midline convergence of neuroepithelial cells. Together our findings demonstrate that PCP signals confer anisotropic contractility on the AJs, producing cellular forces that promote the polarized bending of the neural plate.     

Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins

Clostridium sordellii lethal toxin (LT) is a glucosyltransferase which inactivates small GTPases from the Rho and Ras families. In the present work, we studied the effects of two variants, LT82 and LT9048, on the integrity of epithelial cell barrier using polarized MCCD (Mouse Cortical Collecting Duct) and MDCK (Madin-Darby Canine Kidney) cells. Our results demonstrate for the first time that LTs have very limited effects on tight junctions. In contrast, we show that both toxins modified the paracellular permeability within 2-4 h. Concomitantly LT82 and LT9048 induced a disorganization of basolateral actin filaments, without modifying apical actin. Both toxins mainly altered adherens junctions by removing E-cadherin-catenin complexes from the membrane to the cytosol. Similar effects on adherens junctions have been observed with other toxins, which directly or indirectly depolymerize actin. Thereby, Rac, a common substrate of both LTs, might play a central role in LT-dependent adherens junction alteration. Here, we show that adherens junction perturbation induced by LTs results neither from a direct effect of toxins on adherens junction proteins nor from an actin-independent Rac pathway, but rather from a Rac-dependent disorganization of basolateral actin cytoskeleton. This further supports that a dynamic equilibrium of cortical actin filaments is essential for functional E-cadherin organization in epithelia.     

Multifaceted role of Rho, Rac, Cdc42 and Ras in intercellular junctions, lessons from toxins

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly-disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

EphA4 activity causes cell shape change and a loss of cell polarity in Xenopus laevis embryos.

The Eph family of receptor tyrosine kinases and their ephrin ligands are believed to limit cell-cell interactions during embryonic development via a repulsive mechanism. Little is known, however, about the intracellular effects of Eph signaling that lead to cellular repulsion. We have used scanning and transmission electron microscopy to examine the effects of EphA4 catalytic activity on cells in early embryos of Xenopus laevis. We show that ectopic EphA4 catalytic activity in superficial blastula cells leads to a more rounded cellular morphology, a loss of apical microvilli, and a loss of the apical/basolateral boundary, in addition to the previously reported loss of cell adhesion. These effects indicate that these epithelial cells have lost their apical/basolateral polarity. We also show that EphA4 catalytic activity causes a preferential loss of adherens junctions, compared to tight junctions. Furthermore, EphA4 catalytic activity was found to result in a change in filamentous actin levels in blastomeres. These results taken together suggest that the actin cytoskeleton might be a target of EphA4 signaling.     

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.     

A 90-degree rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells

In the neural plate and neural tube in the trunk region of the zebrafish embryo, dividing cells are oriented parallel to the plane of the neuroepithelium, while in neural keel/rod, cells divide perpendicular to it. This change in the orientation of mitosis is brought about by a 90 degrees rotation of the mitotic spindle. As the two halves of the neural primordium in keel/rod stage are in apposition, the perpendicular orientation of mitoses in this stage determines that daughter cells become allocated to both sides of the neural tube. To assess the role played by cell junctions in controlling the orientation of dividing cells, we studied the expression of components of adherens and tight junctions in the neuroepithelial cells. We find that these proteins are distributed irregularly at the neural plate stage and become polarised apically in the cell membrane only during the keel/rod stage. The stereotypic orientation of mitoses is perturbed only weakly upon loss of function of the cell junction components ASIP and aPKClambda, suggesting that mitotic orientation depends in part on the integrity of cell junctions and the polarity of the epithelium as a whole. However, the 90-degree rotation of the spindle does not require perfectly polarised cell junctions between the neuroepithelial cells.     

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6–atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical–basal polarity in forming epithelia, the Cdc42–Par6–aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.     

Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

The epithelial cadherin (E-cadherin)-catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell's apical-basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling.     

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.     

N-cadherin-based adherens junction regulates the maintenance,proliferation, and differentiation of neural progenitor cells during development

This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin.     

Adherens junctions: new insight into assembly,modulation and function

Adherens junctions play pivotal roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling. These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells or between cells and extracellular matrix. The best-known adherens junction is the zonula adherens (ZA) that forms a belt surrounding the apical pole of epithelial cells. Recent studies in Drosophila have further illuminated the structure of adherens junctions. Scaffolding proteins encoded by the stardust gene are novel components of the Crumbs complex, which plays a critical role in ZA assembly.1-3 The small GTPase Rap1 controls the symmetric re-assembly of the ZA after cell division.4 Finally, the asymmetric distribution of adherens junction material regulates spindle orientation during asymmetric cell division in the sensory organ lineage.     

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.     

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.     

A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

Actin-dependent membrane association of a Drosophila epithelial APC protein and its effect on junctional Armadillo

BACKGROUND: The adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colon. It promotes the destabilisation of free cytoplasmic beta-catenin (the vertebrate homologue of the Drosophila protein Armadillo), a critical effector of the Wnt signalling pathway. The beta-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. In Drosophila epithelial cells, the ubiquitous form of APC, known as E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilising Armadillo. RESULTS: Using actin-depolymerising drugs, we established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants, whose actin cytoskeleton is disrupted, we found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalised E-APC from membranes, including a mutation in E-APC itself, caused partial detachment of Armadillo from adhesive membranes. CONCLUSIONS: Actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes.     

p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin.

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.