The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Role of the Crumbs proteins in ciliogenesis, cell migration and actin organization.

Epithelial cell organization relies on a set of proteins that interact in an intricate way and which are called polarity complexes. These complexes are involved in the determination of the apico-basal axis and in the positioning and stability of the cell-cell junctions called adherens junctions at the apico-lateral border in invertebrates. Among the polarity complexes, two are present at the apical side of epithelial cells. These are the Par complex including aPKC, PAR3 and PAR6 and the Crumbs complex including, CRUMBS, PALS1 and PATJ/MUPP1. These two complexes interact directly and in addition to their already well described functions, they play a role in other cellular processes such as ciliogenesis and polarized cell migration. In this review, we will focus on these aspects that involve the apical Crumbs polarity complex and its relation with the cortical actin cytoskeleton which might provide a more comprehensive hypothesis to explain the many facets of Crumbs cell and tissue properties.

The localisation of the apical Par/Cdc42 polarity module is specifically affected in microvillus inclusion disease.

BACKGROUND INFORMATION: Microvillus inclusion disease (MVID) is a genetic disorder affecting intestinal absorption. It is caused by mutations in MYO5B or syntaxin 3 (STX3) affecting apical membrane trafficking. Morphologically, MVID is characterised by a depletion of apical microvilli and the formation of microvillus inclusions inside the cells, suggesting a loss of polarity. To investigate this hypothesis, we examined the location of essential apical polarity determinants in five MVID patients. RESULTS: We found that the polarity determinants Cdc42, Par6B, PKCζ/ι and the structural proteins ezrin and phospho-ezrin were lost from the apical membrane and accumulated either in the cytoplasm or on the basal side of enterocytes in patients, which suggests an inversion of cell polarity. Moreover, microvilli-like structures were observed at the basal side as per electron microscopy analysis. We next performed Myo5B depletion in three dimensional grown human Caco2 cells forming cysts and found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore, we observed that a majority of cysts displayed an inverted polarity phenotype as seen in some patients. Finally, we found that this loss of polarity was specific for MVID: tissue samples of patients with Myo5B-independent absorption disorders showed normal polarity but we identified Cdc42 as a potentially essential biomarker for trichohepatoenteric syndrome. CONCLUSION: Our findings indicate that the loss of Myo5B induces a strong loss of enterocyte polarity, potentially leading to polarity inversion. SIGNIFICANCE: Our results show that polarity determinants could be useful markers to help establishing a diagnosis in patients. Furthermore, they could be used to characterise other rare intestinal absorption diseases.

Drebrin E depletion in human intestinal epithelial cells mimics Rab8a loss of function.

Intestinal epithelial cells are highly polarized and exhibit a complex architecture with a columnar shape and a specialized apical surface supporting microvilli organized in a brush border. These microvilli are rooted in a dense meshwork of acto-myosin called the terminal web. We have shown recently that Drebrin E, an F-actin-binding protein, is a key protein for the organization of the terminal web and the brush border. Drebrin E is also required for the columnar cell shape of Caco2 cells (human colonic cells). Here, we found that the subcellular localization of several apical markers including dipeptidyl peptidase IV (DPPIV) was strikingly modified in Drebrin E-depleted Caco2 cells. Instead of being mostly present at the apical surface, these proteins are accumulated in an enlarged subapical compartment. Using known intracellular markers, we show by both confocal and electron microscopy that this compartment is related to lysosomes. We also demonstrate that the enrichment of DPPIV in this compartment originates from apical endocytosis and that depletion of Rab8a induces an accumulation of apical proteins in a similar compartment. Consistent with this, the phenotype observed in Drebrin E knock-down Caco2 cells shares some features with a pathology called microvillar inclusion disease (MVID) involving both Myosin Vb and Rab8a. Taken together, these results suggest that Drebrin E redirects the apical recycling pathway in intestinal epithelial cells to the lysosomes, demonstrating that Drebrin E is a key regulator in apical trafficking in Caco2 cells.

Apico-basal elongation requires a drebrin-E-EB3 complex in columnar human epithelial cells.

Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB-ßII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and ßII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilizing the actin-based terminal web.

The multi-PDZ domain protein-1 (MUPP-1) expression regulates cellular levels of the PALS-1/PATJ polarity complex.

MUPP-1 (multi-PDZ domain protein-1) and PATJ (PALS-1-associated tight junction protein) proteins are closely related scaffold proteins and bind to many common interactors including PALS-1 (protein associated with Lin seven) a member of the Crumbs complex. Our goal is to understand how MUPP-1 and PATJ and their interaction with PALS-1 are regulated in the same cells. We have shown that in MCF10A cells there are at least two different and co-existing complexes, PALS-1/MUPP-1 and PALS-1/PATJ. Surprisingly, MUPP-1 levels inversely correlated with PATJ protein levels by acting on the stabilization of the PATJ/PALS-1 complex. Upon MUPP-1 depletion, the increased amounts of PATJ are in part localized at the migrating front of MCF10A cells and are able to recruit more PAR3 (partition defective 3). All together these data indicate that a precise balance between MUPP-1 and PATJ is achieved in epithelial cells by regulating their association with PALS-1.

Super-resolution imaging uncovers the nanoscopic segregation of polarity proteins in epithelia.

Epithelial tissues acquire their integrity and function through the apico-basal polarization of their constituent cells. Proteins of the PAR and Crumbs complexes are pivotal to epithelial polarization, but the mechanistic understanding of polarization is challenging to reach, largely because numerous potential interactions between these proteins and others have been found, without a clear hierarchy in importance. We identify the regionalized and segregated organization of members of the PAR and Crumbs complexes at epithelial apical junctions by imaging endogenous proteins using stimulated-emission-depletion microscopy on Caco-2 cells, and human and murine intestinal samples. Proteins organize in submicrometric clusters, with PAR3 overlapping with the tight junction (TJ) while PALS1-PATJ and aPKC-PAR6ß form segregated clusters that are apical of the TJ and present in an alternated pattern related to actin organization. CRB3A is also apical of the TJ and partially overlaps with other polarity proteins. Of the numerous potential interactions identified between polarity proteins, only PALS1-PATJ and aPKC-PAR6ß are spatially relevant in the junctional area of mature epithelial cells, simplifying our view of how polarity proteins could cooperate to drive and maintain cell polarity.

Many of our organs, including the lungs and the intestine, are lined with a single layer of cells that separate the inside of the organ from the surrounding environment inside the body. These so-called epithelial cells form a tightly packed barrier and have a very characteristic organization. The apical surface faces the outside world, while the basal surface faces the inner tissues. These different interfaces are reflected in the organization of the cells themselves. The shape, composition, and role of the apical cell surface are distinct from those of the basal surface, and they also contain different proteins. In some epithelial cells, the apical surface specializes and forms protruding structures called microvilli. Thus, epithelial cells are said to be polarized along this apical­basal axis. Over the last 30 years, many labs have identified and studied which proteins help epithelial cells become and stay polarized. Previous biochemical experiments showed that these so-called polarity proteins interact with each other in many different ways. But it remains unclear whether some of these interactions are more important than others, and where exactly in the apical or basal membranes these interactions take place. Mangeol et al. used super-resolution microscopy to observe the polarity of proteins at the apical membranes of both human and mouse cells from the small intestine to answer these questions. They focused on areas called tight junctions, where the intestinal cells connect with each other to form the barrier between the outside and the inside. First, all the polarity proteins clustered together in various formations, they were not distributed uniformly. For example, one protein called PAR3 was at the level of the tight junctions, whereas other proteins were closer to the apical surface and the outside world. Only two pairs of proteins ­ PAR6 and aPKC, and PALS1 and PATJ ­ formed stable clusters with each other. This finding was unexpected because previous biochemical experiments had predicted multiple interactions. Third, the PALS1/PATJ complexes stayed at the bottom of the microvilli protrusions, whereas PAR6/aPKC were inside the protrusions. Taken together, these experiments reveal a detailed snapshot of how the polarity proteins themselves are organized at the apical surface of epithelial cells. Future work will be able to address how these protein complexes behave over time.

Evolution of mechanisms controlling epithelial morphogenesis across animals: new insights from dissociation-reaggregation experiments in the sponge Oscarella lobularis.

BACKGROUND: The ancestral presence of epithelia in Metazoa is no longer debated. Porifera seem to be one of the best candidates to be the sister group to all other Metazoa. This makes them a key taxon to explore cell-adhesion evolution on animals. For this reason, several transcriptomic, genomic, histological, physiological and biochemical studies focused on sponge epithelia. Nevertheless, the complete and precise protein composition of cell-cell junctions and mechanisms that regulate epithelial morphogenetic processes still remain at the center of attention. RESULTS: To get insights into the early evolution of epithelial morphogenesis, we focused on morphogenic characteristics of the homoscleromorph sponge Oscarella lobularis. Homoscleromorpha are a sponge class with a typical basement membrane and adhaerens-like junctions unknown in other sponge classes. We took advantage of the dynamic context provided by cell dissociation-reaggregation experiments to explore morphogenetic processes in epithelial cells in a non-bilaterian lineage by combining fluorescent and electron microscopy observations and RNA sequencing approaches at key time-points of the dissociation and reaggregation processes. CONCLUSIONS: Our results show that part of the molecular toolkit involved in the loss and restoration of epithelial features such as cell-cell and cell-matrix adhesion is conserved between Homoscleromorpha and Bilateria, suggesting their common role in the last common ancestor of animals. In addition, sponge-specific genes are differently expressed during the dissociation and reaggregation processes, calling for future functional characterization of these genes.

Polarity complex proteins.

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.

Hook2, a microtubule-binding protein, interacts with Par6α and controls centrosome orientation during polarized cell migration.

Polarity protein complexes function during polarized cell migration and a subset of these proteins localizes to the reoriented centrosome during this process. Despite these observations, the mechanisms behind the recruitment of these polarity complexes such as the aPKC/PAR6α complex to the centrosome are not well understood. Here we identify Hook2 as an interactor for the aPKC/PAR6α complex that functions to localize this complex at the centrosome. We first demonstrate that Hook2 is essential for the polarized Golgi re-orientation towards the migration front. Depletion of Hook2 results in a decrease of PAR6α at the centrosome during cell migration, while overexpression of Hook2 in cells induced the formation of aggresomes with the recruitment of PAR6α, aPKC and PAR3. In addition, we demonstrate that the interaction between the C-terminal domain of Hook2 and the aPKC-binding domain of PAR6α localizes PAR6α to the centrosome during cell migration. Our data suggests that Hook2, a microtubule binding protein, plays an important role in the regulation of PAR6α recruitment to the centrosome to bridge microtubules and the aPKC/PAR complex. This data reveals how some of the polarity protein complexes are recruited to the centrosome and might regulate pericentriolar and microtubule organization and potentially impact on polarized migration.

Hook2 is involved in the morphogenesis of the primary cilium.

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation.

Crumbs proteins in epithelial morphogenesis.

Cell polarity is an essential feature of most eukaryotic cells, especially epithelial cells in multicellular animals. Polarity protein complexes that regulate epithelial organization have been identified. In this review, it is proposed to describe how the Crumbs complex acts in the process of cell polarity and epithelial organization. During the last decade, several partners of Crumbs, an apical transmembrane protein, have been identified and their direct or indirect associations with the cytoplasmic domain of Crumbs have been dissected. In addition, mutants of several of the genes encoding proteins belonging to the Crumbs network have been obtained in animals ranging from flies to mouse, which have led to a better understanding of their functions in vivo. These functions include polarity axis formation, stabilization of epithelial apico-lateral junctions, photoreceptor organization and ciliogenesis. Since human CRUMBS1 mutations are associated with retina degeneration, it has become essential to define Crumbs network and to understand exactly how this network acts in polarized cells, with a view to developing suitable therapeutic approaches for treating this severe degenerative disease.

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex.

In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. On the other hand, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. Here we report, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2.

PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells.

The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Dpatj gene and its role in epithelial morphogenesis and polarity remains unknown. We have produced downregulated PATJ stable lines of Caco2 to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains.

Enterocytin: A new specific enterocyte marker bearing a B30.2-like domain.

Enterocyte differentiation is correlated to the expression of specific proteins which only a few of them are identified. In this study, we characterize a new marker of enterocyte differentiation using monoclonal antibodies. We showed that small intestinal enterocytes specifically express a new 47 kDa protein named Enterocytin. Expression of this protein increase along the crypt-villus axis and it is concentrated in the terminal web, lateral plasma membrane domain, and nucleus membrane of mature enterocytes. A 1.8-kb cDNA of Enterocytin was isolated by expression cloning from a cDNA library of rabbit small intestine. The amino acid sequence obtained shows an N-terminal region with a coiled-coil structure and a B30.2-like domain in the C-terminus region. By co-transfection and immunoprecipitation procedures on Cos cells, it was observed that the coiled-coil domain is involved in the homodimerization of Enterocytin. In the human intestine, a similar 47 kDa protein was detected, exclusively in the small intestinal enterocytes. In addition, expression of this protein in Caco2 cells is correlated with the state of differentiation of these cells. The restricted expression of Enterocytin in the intestine and its localization in mature cells suggest that it may contribute to the differentiation processes and maintenance of the enterocytic polarity.