Dominique Massey-Harroche

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.

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.

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

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.

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.

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.

Crumbs proteins control ciliogenesis and centrosome organization: what about the mechanism?

Results Crb2 and Crb3 share many protein interactions and mechanisms of compensation could exist. However our data revealed the contrary since we showed that primary cilia formation requires a threshold level of both Crb2 and Crb3. To decipher the mechanism that underlies this requirement we have focused on Crb2, which is predominantly expressed in ARPE-19 cells. Using both optical imaging and electron microscopy we showed that Crb2 is involved in cilia initiation but not cilia maintenance. Furthermore we uncovered that Crb2 acts at a very early stage of ciliogenesis, by affecting the localization of centriolar and peri-centriolar markers such as PCM-1. Conclusion Crb2 allows the efficient organization of the centrosome and associated proteins and the primary vesicle formation to promote ciliogenesis. Taken together, our data show that Crb2 is essential for the primary ciliogenesis by a still unknown mechanism.

The Crumbs3 Complex

Epithelial layers have allowed the evolution of metazoans by promoting complex multicellularity and protection from the environment. This essential role for epithelia relies on their ability to build an apical surface facing the outside world and a basolateral surface connecting cells together to coordinate their movements and resistance to stress. These epithelial features are dependent on several protein complexes for their establishment and maintenance. Among these protein complexes, one complex contains an apical transmembrane protein, Crumbs, that plays an essential role for the proper organization of tight junctions, apical morphology, or cell and tissue growth. In this chapter we will review how Crumbs3, one of the Crumbs proteins, both recruits adapter proteins essential for the building of tight or adherens junctions in vertebrates and interacts with the subapical actin cytoskeleton. These functions are broadly conserved in animals from flies to human, and it remains a challenge to understand all the molecular mechanisms by which Crumbs3 and its partners participate in the building of a functional epithelial sheet to keep body homeostasis and to allow for morphogenetic events as essential as gastrulation.

The basolateral domain of polarized cells is the privileged site for localization of annexins

The Iuminal binding protein BiP, a resident of the endoplasmic reticulum (ER), is a member of a wide class of protein termed molecular chaperone. It is structurally and functionally related to cytosolii HSP70 but differing in the presence of a signal peptide and an ER retention signal (K/HDEL). In tobacco, BiP is encoded by a multigene family and at least out member is able to complement a yeast mutant indicating to a same functional role (Denecke J., Grjldman M. H., Demolder .I., Seurick J. and Botterman J. (1991), Plonf Ceil 3, 1025-1035). Although BiP is present in detectable amount under normal conditions, it can be induced by a variety of stress (e.g. tunicamycin) rest&g in mtiokled secretory proteins (Denecke J. and Vi&e A. (1995). Methods in C&l Biol. 24, 335-348). Biochemical evidence shows its implication in the process of protein folding due to its ability to bind to polypeptides in the ER lumen (Pedrazzini E. and Vitale A. (1996), Plant Physid. Biochem. 34, 207-216) but the role of BiP in tbc quality contra1 by the ER retention of malfolded proreins is not clearly established. We were interested to alter expression of this molecular chaperone to provide more information about its function in secretion and tmhsport of proteins in plants. We set up a.functional assay for BiP based on the comparison of transient protein synthesis in the cytosol (GUS) and on the rough ER (barley n-amylase) after electroporation of tobiT protoplasrs in presence of corresponding genes. Co-nansfection experiments were carried out using both 1) a plasmid containing GUS and a-amylase genei and 2) a plasmid containing BiP construct. A positive effect of transient overexpression of BiP on the synthesis of a-amylase under ER stress has been observed. Then, we have generated transgenic tobacco plants exhibiting different levels of BiP or expressing BiP mutants which will be used as &ls to better understand Sip regul&on, protein synthesis and proteins transport in plants.