Structure and functions of intercellular epithelial junctions: an evolutionary perspective – EvolAj

Work packages 1 and 4 rely on the strong expertise of partner 1 and on routine techniques fully

mastered in Caco2 cells by this team, therefore there are no major risks of failure for this objective.

Partner 2 in collaboration with partner 1 has developed routine use of immunofluorescence in the sponge Oscarella lobularis, as well as competition and dissociation assays 20,22,23.

Partner 1 has already developed several techniques and tools for work package 3 and the expertise of partner 2 in marine organisms will be useful for this task. Altogether, the risks concerning aim 2 and 3 seem reasonable. In addition, for each task we provide alternative approaches to maximize our chances to reach our goals.

WP1: Roles of Cadherin/Catenin and Nectin/Afadin complexes in the organization and anchoring of the actin belt in human epithelial cells.

Task 1.1: Understanding the role of the Nectin/Afadin complex in anchoring the F-actin belt.

We generated an afadin knockout in Caco-2 cells and observed detachment of the actin belt from the plasma membrane, as well as altered positioning along the apicolateral axis, proving that afadin is essential for anchoring the actin belt at the adherens junction. In contrast, the actin belt remains intact and positions itself randomly in the apicolateral region.

Task 1.2: Measure the role of afadin in transmitting epithelial forces.

The impact of this loss of actin belt anchoring on apical tension was assessed by inferring forces via the curvature of cell contacts between cells expressing afadin and those no longer expressing afadin. Cells no longer expressing afadin exhibit lower apical tension than wild-type cells. These findings were confirmed by apical laser ablation, which demonstrated a greater release of apical tension in wild-type cells. These results were published in 2024.: Mangeol P, Massey-Harroche D, Sebbagh M, Richard F, Le Bivic A, Lenne PF. 2024. The zonula adherens matura redefines the apical junction of intestinal epithelia. Proc Natl Acad Sci U S A. PMID: 38377188.

WP2: Caractérisation des complexes Cadherin/Catenin et Nectin/Afadin et leur lien avec l’anneau de F-actine chez l’éponge Oscarella lobularis.

Task 2.1: O. lobularis homologues of known bilaterian AJ components : subcellular localization and relations with the AJ-like structures.

During this project, we tested various specific antibodies targeting key proteins of polarity complexes and developed a reproducible protocol to trigger asexual reproduction of Oscarella lobularis in the laboratory, thereby producing buds.

Immunolocalization results (schematic view in Figure 1B), currently being analyzed, show that Ol-Cadherin localizes to the membranes of epithelial cells in contact with the basement membrane (identified by collagen IV), but not to adherens-like junctions (ALJs), suggesting a role in cell-matrix adhesion rather than cell-cell adhesion.

Conversely, Ol-Afadin localizes specifically to ALJs, consistent with recent results obtained in WP1, where the actin belt is associated with the Nectin/Afadin complex (NAC) rather than the Cadherin/Catenin complex (CCC). Interestingly, β-catenin, and potentially α-catenin (labels to be confirmed), also appear to colocalize with Afadine at the junctions.

In this project, we have discovered a totally unexpected feature of intestinal epithelial cells:

By generating an afadin knockout in Caco-2 cells and we have observed the detachment of the actin belt from the plasma membrane, as well as altered positioning along the apicolateral axis, proving that afadin is essential for anchoring the actin belt at the adherens junction. In contrast, the actin belt remains intact and positions itself randomly in the apicolateral region.

The impact of this loss of actin belt anchoring on apical tension was assessed by inferring forces via the curvature of cell contacts between cells expressing afadin and those no longer expressing afadin. Cells no longer expressing afadin exhibit lower apical tension than wild-type cells. These findings were confirmed by apical laser ablation, which demonstrated a greater release of apical tension in wild-type cells.

These results were published in 2024. It has been advertized on the web site of CNRS Biology: www.insb.cnrs.fr/fr/cnrsinfo/une-nouvelle-categorie-de-jonctions-cellulaires-dans-lintestin

ALB has been invited to give a talk at the annual American Digestive Disease week in San Diego in June 2025.

Mangeol P, Massey-Harroche D, Sebbagh M, Richard F, Le Bivic A, Lenne PF. 2024. The zonula adherens matura redefines the apical junction of intestinal epithelia. Proc Natl Acad Sci U S A. PMID: 38377188. This paper has been commented in Too old for hide-and-seek; cell maturation reveals hidden apical junctional organization. Jensen CC, Peifer M. Proc Natl Acad Sci U S A. 2024 Mar 19;121(12):e2401735121. doi: 10.1073/pnas.2401735121., and in Afadin-nectin forces its way to the front. Sebbagh M, Schwartz MA. J Cell Biol. 2024 May 6;223(5):e202403177. doi: 10.1083/jcb.202403177.

An ANR application has been submitted in 2025 by Carole Borchiellini to follow up the work on sponges.

In metazoans, the cohesion of epithelial tissues is ensured by specific structures, the intercellular junctions. These junctions have been mainly studied in bilaterian model organisms and their existence, molecular composition and structural organization, as well as the details of their interaction with the actin cytoskeleton, remain largely unknown in non-bilaterian animals. The aim of this project is to characterize the molecular and functional organization of intercellular junctions in two organisms belonging to metazoan lineages that diverged very early in evolution, Oscarella lobularis (Porifera, sponges) and Trichoplax adhaerens (Placozoa). These models have epithelia with very different properties such as a high stability of junctions, or on the contrary, extreme morphological plasticity. In parallel, we use a bilaterian model, the Caco2 cell line, derived from a human digestive epithelium, as a source of information on the protein complexes that compose the intercellular junctions and on their role in the recruitment of the apical actin belt common to all metazoan epithelia. We will explore the respective roles of the Cadherin/Catenin and Nectin/Afadin complexes in the anchoring of this apical actin belt and the transmission of forces between Caco2 cells. In O. lobularis and T. adhaerens, we will study the localization of proteins homologous to those that constitute junctional complexes in bilaterians and which are mostly highly conserved. Our hypothesis is that some of these homologs may also be involved in the formation of cell-cell junctions in non-bilaterians. This functional conservation will be tested by expressing the different genes of Trichoplax or Oscarella in Caco2 cells whose homologous genes have been invalidated, in order to verify whether this is sufficient to restore a wild-type phenotype. The aim of this project is to determine the conservation of epithelial adherens junctions during evolution and to deduce the type of junctions present in the common ancestor of metazoans.