Delphine Delacour

Apical sorting by galectin-3-dependent glycoprotein clustering

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid‐raft‐independent apical carrier vesicles harbour the beta‐galactoside‐binding lectin galectin‐3, which interacts directly with apical cargo in a glycan‐dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin‐3‐depleted cells, dedicating a central role to this lectin in raft‐independent sorting as apical receptor. Here, we demonstrate that high‐molecular‐weight clusters are exclusively formed in the presence of galectin‐3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation‐deficient Madin‐Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross‐linking by galectin‐3 is required for apical sorting of non‐raft‐associated cargo.

The role of galectins in protein trafficking.

The galectins, a family of lectins, modulate distinct cellular processes, such as cancer progression, immune response and cellular development, through their specific binding to extracellular or intracellular ligands. In the past few years, research has unravelled interactions of different galectins with lipids and glycoproteins in the outer milieu or in the secretory pathway of cells. Interestingly, these lectins do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. Instead they use a so‐called non‐classical mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. Here, they stabilize transport platforms for apical trafficking or sort apical glycoproteins into specific vesicle populations. Modes of ligand interaction as well as the modulation of binding activities and trafficking pathways are discussed in this review.

Apical protein transport

Abstract.The plasma membrane of epithelial cells and hepatocytes is divided into two separate membrane compartments, the apical and the basolateral domain. This polarity is maintained by intracellular machinery that directs newly synthesized material into the correct target membrane. Apical protein sorting and trafficking require specific signals and different intracellular routes to the cell surface. Some of them depend on the integrity of sphingolipid/cholesterol-enriched membrane microdomains named ‘lipid rafts’, others use separate transport platforms. Certain characteristics of the heterogeneous population of apical sorting signals are described in this review and cellular factors associated with sorting and transport mechanisms are discussed.

Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo.

Epithelial cells are characterised by distinct apical and basolateral membrane domains that are separated by tight junctions. Establishment and maintenance of this polarity depend on specific gene expression and protein targeting to their correct location. Our former studies, performed with renal epithelial MDCK cells, revealed a new function for galectin-3, a member of a conserved family of lectins. There, galectin-3 is required for intracellular sorting and correct targeting of non-raft-associated glycoproteins to the apical plasma membrane. In the present study, we found transport defects of the intestinal brush border hydrolases lactase-phlorizin hydrolase (LPH) and dipeptidylpeptidase IV (DPPIV) in galectin-3-null mutant mice. We could show that, in enterocytes of wild-type mice, both glycoproteins directly interact with galectin-3 and transit through non-raft-dependent apical transport platforms. Therefore, this genetic analysis provides definitive evidence for the involvement of galectin-3 in protein intracellular trafficking in vivo. Further investigations revealed that gal3-null enterocytes also exhibit striking cytoarchitecture defects, with the presence of numerous and regular protrusions located along basolateral membranes. Moreover, β-actin and villin, two characteristic markers of brush borders, become abnormally distributed along these atypical basolateral membranes in gal3–/– mice. Taken together, our results demonstrate that, in addition to a pivotal role in apical trafficking, galectin-3 also participates in epithelial morphogenesis in mouse enterocytes.

Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells.

In epithelial cells, the β-galactoside-binding lectin galectin-3 mediates the non-raft-dependent glycoprotein targeting to the apical membrane domain. In this study, we aimed to identify intracellular compartments involved in the trafficking of galectin-3. By studying fluorescent fusion proteins in living cells, we could show that galectin-3 accumulates intracellularly in acidified endosomes. Total internal reflection fluorescence microscopy studies of the apical surface of polarized MDCK cells revealed that galectin-3 is enriched in tubular and vesicular Rab11-positive recycling endosomes in the vicinity of the apical cell surface. These endosomal organelles are candidate compartments for the association between galectin-3 and exocytic apical cargo.

Galectin-3, a Novel Centrosome-associated Protein, Required for Epithelial Morphogenesis

We investigated the role of galectin-3 on polarization of epithelial renal cells, using three-dimensional cultures of MDCK cells and also galectin-3 null mutant mouse kidneys. Collectively, data show that the absence of galectin-3 influences the stabilization of centrosomes and primary cilia, with effects on epithelial cell organization.

Galectins in epithelial functions

Galectins are a family of animal lectins comprising 15 members in vertebrates. These proteins are involved in many biological processes including epithelial homeostasis and tumor progression by displaying intracellular and extracellular activities. Hence Galectins can be found either in the cytoplasm or the nucleus, associated with membranes or in the extracellular matrix. Current studies aim at understanding the roles of Galectins in cell-cell and cell-matrix adhesion, cellular polarity and motility. This review discusses recent progress in defining the specificities and mechanisms of action of Galectins as cell regulators in epithelial cells. Physiological, cellular and molecular aspects of Galectin specificities will be treated successively.

Basal foot MTOC organizes pillar MTs required for coordination of beating cilia

Coordination of ciliary beating is essential to ensure mucus clearance in the airway tract. The orientation and synchronization of ciliary motion responds in part to the organization of the underlying cytoskeletal networks. Using electron tomography on mouse trachea, we show that basal bodies are collectively hooked at the cortex by a regular microtubule array composed of 4-5 microtubules. Removal of Galectin-3, one of basal body components, provokes misrecruitment of γ-tubulin, disorganization of this microtubule framework emanating from the basal foot cap, together with loss of basal body alignment and cilium orientation, defects in cilium organization and reduced fluid flow in the tracheal lumen. We conclude that Galectin-3 plays a crucial role in the maintenance of the microtubule organizing center of the cilium and the “pillar” microtubules, and that this network is instrumental for the coordinated orientation and stabilization of motile cilia.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.

Plasticity of the brush border - the yin and yang of intestinal homeostasis.

The brush border on the apical surface of enterocytes is a highly specialized structure well-adapted for efficient digestion and nutrient transport, whilst at the same time providing a protective barrier for the intestinal mucosa. The brush border is constituted of a densely ordered array of microvilli, protrusions of the plasma membrane, which are supported by actin-based microfilaments and interacting proteins and anchored in an apical network of actomyosin and intermediate filaments, the so-called terminal web. The highly dynamic, specialized apical domain is both an essential partner for the gut microbiota and an efficient signalling platform that enables adaptation to physiological stimuli from the external and internal milieu. Nevertheless, genetic alterations or various pathological stresses, such as infection, inflammation, and mechanical or nutritional alterations, can jeopardize this equilibrium and compromise intestinal functions. Long-time neglected, the intestinal brush-border shall be enlightening again as the central actor of the complex but essential intestinal homeostasis. Here, we review the processes and components involved in brush border organization and discuss pathological mechanisms that can induce brush border defects and their physiological consequences.