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.

The compact genome of the sponge Oopsacas minuta (Hexactinellida) is lacking key metazoan core genes.

BACKGROUND: Explaining the emergence of the hallmarks of bilaterians is a central focus of evolutionary developmental biology-evodevo-and evolutionary genomics. For this purpose, we must both expand and also refine our knowledge of non-bilaterian genomes, especially by studying early branching animals, in particular those in the metazoan phylum Porifera. RESULTS: We present a comprehensive analysis of the first whole genome of a glass sponge, Oopsacas minuta, a member of the Hexactinellida. Studying this class of sponge is evolutionary relevant because it differs from the three other Porifera classes in terms of development, tissue organization, ecology, and physiology. Although O. minuta does not exhibit drastic body simplifications, its genome is among the smallest of animal genomes sequenced so far, and surprisingly lacks several metazoan core genes (including Wnt and several key transcription factors). Our study also provides the complete genome of a symbiotic Archaea dominating the associated microbial community: a new Thaumarchaeota species. CONCLUSIONS: The genome of the glass sponge O. minuta differs from all other available sponge genomes by its compactness and smaller number of encoded proteins. The unexpected loss of numerous genes previously considered ancestral and pivotal for metazoan morphogenetic processes most likely reflects the peculiar syncytial tissue organization in this group. Our work further documents the importance of convergence during animal evolution, with multiple convergent evolution of septate-like junctions, electrical-signaling and multiciliated cells in metazoans.

The elmo-mbc complex and rhogap19d couple Rho family GTPases during mesenchymal-to-epithelial-like transitions.

Many metazoan developmental processes require cells to transition between migratory mesenchymal- and adherent epithelial-like states. These transitions require Rho GTPase-mediated actin rearrangements downstream of integrin and cadherin pathways. A regulatory toolbox of GEF and GAP proteins precisely coordinates Rho protein activities, yet defining the involvement of specific regulators within a cellular context remains a challenge due to overlapping and coupled activities. Here we demonstrate that Drosophila dorsal closure is a powerful model for Rho GTPase regulation during transitions from leading edges to cadherin contacts. During these transitions a Rac GEF elmo-mbc complex regulates both lamellipodia and Rho1-dependent, actomyosin-mediated tension at initial cadherin contacts. Moreover, the Rho GAP Rhogap19d controls Rac and Rho GTPases during the same processes and genetically regulates the elmo-mbc complex. This study presents a fresh framework to understand the inter-relationship between GEF and GAP proteins that tether Rac and Rho cycles during developmental processes.

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.

New genomic data and analyses challenge the traditional vision of animal epithelium evolution.

BACKGROUND: The emergence of epithelia was the foundation of metazoan expansion. Epithelial tissues are a hallmark of metazoans deeply rooted in the evolution of their complex developmental morphogenesis processes. However, studies on the epithelial features of non-bilaterians are still sparse and it remains unclear whether the last common metazoan ancestor possessed a fully functional epithelial toolkit or if it was acquired later during metazoan evolution. RESULTS: To investigate the early evolution of animal epithelia, we sequenced the genome and transcriptomes of two new sponge species to characterize epithelial markers such as the E-cadherin complex and the polarity complexes for all classes (Calcarea, Demospongiae, Hexactinellida, Homoscleromorpha) of sponges (phylum Porifera) and compare them with their homologues in Placozoa and in Ctenophora. We found that Placozoa and most sponges possess orthologues of all essential genes encoding proteins characteristic of bilaterian epithelial cells, as well as their conserved interaction domains. In stark contrast, we found that ctenophores lack several major polarity complex components such as the Crumbs complex and Scribble. Furthermore, the E-cadherin ctenophore orthologue exhibits a divergent cytoplasmic domain making it unlikely to interact with its canonical cytoplasmic partners. CONCLUSIONS: These unexpected findings challenge the current evolutionary paradigm on the emergence of epithelia. Altogether, our results raise doubt on the homology of protein complexes and structures involved in cell polarity and adhesive-type junctions between Ctenophora and Bilateria epithelia.

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.

CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice.

Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between photoreceptors and Müller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.

Targeted ablation of CRB1 and CRB2 in retinal progenitor cells mimics Leber congenital amaurosis.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene.

In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor 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.

Hyper-accumulation of vanadium in animals: Two sponges compete with urochordates.

Vanadium (V) concentrations in organisms are usually very low. To date, among animals, only some urochordate and annelid species contain very high levels of V in their tissues. A new case of hyper-accumulation of V in a distinct animal phylum (Porifera), namely, the two homoscleromorph sponge species Oscarella lobularis and O. tuberculata is reported. The measured concentrations (up to 30 g/kg dry weight) exceed those reported previously and are not found in all sponge classes. In both Oscarella species, V is mainly accumulated in the surface tissues, and in mesohylar cells, as V(IV), before being partly reduced to V(III) in the deeper tissues. Candidate genes from Bacteria and sponges have been identified as possibly being involved in the metabolism of V. This finding provides clues for the development of bioremediation strategies in marine ecosystems and/or bioinspired processes to recycle this critical metal.

Methylmercury exposure of the sponge O. lobularis induces strong tissue and cell defects.

Mediterranean marine biota suffers from various anthropogenic threats. Among them, pollutants such as mercury (Hg) represent important environmental issues that are exacerbated by bioaccumulation and bioamplification along food webs via its organic form, monomethylmercury (MMHg). To date, very little is known regarding the impact of mercury on Porifera and the few available studies have been exclusively focused on Demospongiae. This work studies the effect of MMHgCl at different biological levels of Oscarella lobularis (Porifera, Homoscleromorpha). Bioaccumulation assays show that MMHgCl significantly accumulated in sponge tissues after a 96-h exposure to 0.1 µg L-1. Toxicity assays (LC5096h) show a sensibility that depends on life-stage (adult vs bud). Additionally, we show that the exposure to 1 µg L-1 MMHgCl negatively impacts the epithelial integrity and the regeneration process in buds, as shown by the loss of cell-cell contacts and the alteration of osculum morphogenesis. For the first time in a sponge, a whole set of genes classically involved in metal detoxification and in antioxidant response were identified. Significant changes in catalase, superoxide dismutase and nuclear factor (erythroid-derived 2)-like 2 expressions in exposed juveniles were measured. Such an integrative approach from the physiological to the molecular scales on a non-model organism expands our knowledge concerning sensitivity and toxicity mechanisms induced by MMHg in Porifera, raising new questions regarding the possible defences used by marine sponges.

Evolution and cell physiology. 4. Why invent yet another protein complex to build junctions in epithelial cells?

The formation of the first epithelium was an essential step for animal evolution, since it has allowed coordination of the behavior of a cell layer and creation of a selective barrier between the internal medium and the outside world. The possibility of coupling the cells in a single layer has allowed morphogenetic events, such as tube formation, or gastrulation, to form more complex animal morphologies. The invention of sealed junctions between cells has allowed, on the other hand, creation of an asymmetry of nutrients or salts between the apical and the basal side of the epithelial layer. Creation of an internal medium has led to homeostasis, allowing the evolution of more complex physiological functions and the emergence of sophisticated animal shapes. During evolution, the origins of the first animals coincided with the invention of several protein complexes, including true cadherins and the polarity protein complexes. How these complexes regulate formation of the apicolateral border and the adherens junctions is still not fully understood. This review focuses on the role of these apical polarity complexes and, in particular, the Crumbs complex, which is essential for proper organization of epithelial layers from Drosophila to humans.

PALS1 is essential for retinal pigment epithelium structure and neural retina stratification.

The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.

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.

Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6.

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end-stage renal failure. The disease is caused by mutations in NPHP1-9 genes, which encode the nephrocystins, proteins localized to cell-cell junctions and centrosome/primary cilia. Here, we show that nephrocystin mRNA expression is dramatically increased during cell polarization, and shRNA-mediated knockdown of either NPHP1 or NPHP4 in MDCK cells resulted in delayed tight junction (TJ) formation, abnormal cilia formation and disorganized multi-lumen structures when grown in a three-dimensional collagen matrix. Some of these phenotypes are similar to those reported for cells depleted of the TJ proteins PALS1 or Par3, and interestingly, we demonstrate a physical interaction between these nephrocystins and PALS1 as well as their partners PATJ and Par6 and show their partial co-localization in human renal tubules. Taken together, these results demonstrate that the nephrocystins play an essential role in epithelial cell organization, suggesting a plausible mechanism by which the in vivo histopathologic features of NPH might develop.

Epithelial trafficking: new routes to familiar places.

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.