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

corrigéInternational audienceBACKGROUND 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 in electron microscopy. We next performed Myo5B depletion in 3D-grown human Caco2 cells forming cysts and we found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore we observed that a majority of cyst 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 tricho-hepato-enteric 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

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

Nectins rather than E-cadherin anchor the actin belts at cell-cell junctions of epithelia

Cell-cell junctions support the mechanical integrity of epithelia by enabling adhesion and tension transmission between neighboring cells. The prevailing mechanistic dogma is that E-cadherin supports and transmits mechanical tension between cells through actin belts in a region named the zonula adherens . Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that the zonula adherens consists of E-cadherin and nectin belts that are separated by about 150 nm along the apico-basal direction, the nectin belt being in the immediate vicinity of the actin belt. The segregation of nectins and E-cadherin increases as the tissue matures. Our data redefine the structure of the zonula adherens and show that nectins, rather than E-cadherin, are the major connectors of actin belts in epithelia

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

International audienceEpithelial 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

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 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 STED microscopy on Caco-2 cells, human and murine intestinal samples. Proteins organize in submicrometric clusters, with PAR3 overlapping with the tight junction (TJ) while PALS1-PATJ and aPKC-PAR6β 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 weakly overlaps with other polarity proteins. This organization at the nanoscale level significantly simplifies our view on how polarity proteins could cooperate to drive and maintain cell polarity

CRB3 and ARP2/3 regulate cell biomechanical properties to set epithelial monolayers for collective movement

Summary Several cellular processes during morphogenesis, tissue healing or cancer progression involve epithelial to mesenchymal plasticity that leads to collective motion (plasticity?). Even though a rich variety of EMP programs exist, a major hallmark unifying them is the initial breaking of symmetry that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodelled correlating with the build-up of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behaviour, but how forces are regulated during the breaking of symmetry at the onset of EMP remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during the epithelial-mesenchymal plasticity remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of symmetry of the EMP. CRB3 interacts with ARP2/3 and controls the remodelling of actin throughout the monolayer via the modulation of the Rho-/Rac-GTPase balance. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during EMP

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

International audience: 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

International audienceEpithelial 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 STED 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-PAR6β 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-PAR6β 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

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

International audienceAlthough 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-βII-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 βII 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

L’éponge marine Oscarella lobularis un modèle pour comprendre l’origine et l’évolution des épithéliums

In order to investigate the origin and evolution of epithelia, we are developing a model of sponge belonging to the homoscleromorph class. Sponges are one of the earliest emerged animal lineages and are therefore a key group for understanding the early steps of animal evolution. We aim to characterize the molecular mechanisms involved in the establishment of epithelia in our model in order to decipher, in a comparative approach, the mechanisms that are conserved across animals and thus probably acquired in their last common ancestor from those specific to our model. To address these central issues, we rely on a set of complementary approaches: comparative genomics and transcriptomics, classical histology, electron microscopy, immunofluorescence, biochemistry and proteomics.Afin d’émettre des hypothèses sur l’origine et l’évolution des épithéliums, nous développons un modèle d’éponge appartenant à la classe des homoscléromorphes. Les éponges sont l’une des lignées ayant émergé le plus précocement au cours de l’évolution des animaux, elles constituent donc un groupe clé pour comprendre les premières étapes de l’évolution animale. Nous cherchons à caractériser les mécanismes moléculaires qui concourent à la mise en place des épithéliums dans notre modèle afin d’en déduire, dans une approche comparative, les mécanismes qui sont conservés à l’échelle des animaux et donc acquis très probablement chez leur dernier ancêtre commun et ceux spécifiques à notre modèle. Afin de répondre à ces questions fondamentales, nous nous appuyons sur un ensemble d'approches complémentaires : génomique et transcriptomique comparées, histologie classique, microscopie électronique, immunofluorescence, biochimie et protéomique