MBI Weekly Meeting Seminar
Time: 9.30am-10.30am
Date: Friday, 31 August 2018
Venue: Level 5 Seminar Room, T-Lab
EpCAM, a key regulator of tissue biomechanics, acts as a cortical organizer of cell contractility
By Dr. Delacour Delphine, CNRS Researcher, Institut Jacques Monod, France
In monolayered epithelia, differentiated cells are polarized and display a specific apico-basal organization. To maintain cell polarization as well as cohesion of neighboring cells within the epithelial monolayer, cells developed diverse adhesion complexes. However, the epithelial monolayer is subjected to remodeling during organism development or tissue repair. In addition, cell renewal, cell rearrangements and cell extrusion occur to maintain the monolayer homeostasis. Consequently, the epithelium has to constantly maintain its compartmentalization and to collectively adapt to its microenvironment to maintain its polarized and cohesive state. In recent years, accumulating experimental evidences have led to recognition that mechanical properties of tissues have the power of directing a variety of cell functions including stem cell proliferation, cell migration and differentiation. Various reports argue in favor of a participation of physical properties of the substrate, such as substrate rigidity, in the cell response for regulation of the epithelial monolayer integrity. Very little is known about the influence of the epithelial tissue geometry per se.
Our aim is to study the influence of the tissue geometry on epithelial organization and to determine the regulatory mechanisms. These questions are addressed by developing integrated approaches at the cell biological, biochemical and biophysical levels. As a tissue model, we use the intestinal epithelium, which constitutes a very good model to approach these different questions. It has a simple and regular finger-like architecture, where proliferative and differentiate cells are distributed in distinct areas, crypts and villi, respectively. We mainly focus on the involvement of a protein candidate: EpCAM (Epithelial Cell Adhesion Molecule). Specifically expressed in monolayered epithelia in physiological conditions, EpCAM was among the first discovered cancer markers in the 1970s, and changes in its expression induce severe perturbation of epithelial tissue arrangement. Direct evidence of EpCAM involvement in epithelial morphogenesis came from a clinical study where mutations in EPCAM and subsequent loss of EpCAM expression have been correlated with the development of a rare infantile enteropathy, the CTE (Congenital Tufting Enteropathy). The CTE intestinal epithelium displays unique morphological abnormalities, materialized by formation of aberrant focal stacks of pseudo-multilayered epithelial cells, named “tufts”, making it an appealing pathological model that we use to understand the mechanisms of actions of EpCAM.
Using an intestinal cell line and 3D biomimetic substrates that recapitulate intestinal physical constraints, we showed that “tuft”-like structures appear when EpCAM-deprived cells are specifically grown on 3D synthetic villi, testifying of an enhanced mechanical stress provided by the particular topography of the monolayer substrate. EpCAM-silenced cells cultured on 3D synthetic villi can thus phenocopy CTE cellular and tissue defects. Moreover, EpCAM loss-of-function abrogates polarized monolayer arrangement and a spatial perturbation of actomyosin occurs on villus-like 3D substrates. The alteration of epithelial contractile homeostasis is directly correlated to the development of tissue lesions, since tissue-scale defects could be erased with contractility inhibitors. Data consequently tend to support a direct implication of EpCAM in the epithelial response to physical properties of the microenvironment.
In intestinal cell line and mouse and human intestinal biopsies, we scrutinize the requirement of EpCAM for correct tissue organization and homeostasis. First, we have showed that the absence of EpCAM provokes an unusual apico-basal polarity defects in mutant monolayers, which stem from inappropriate actomyosin activity at tricellular junctions. Second, we test EpCAM’s participation in early epithelial cell-substrate adhesion process and consequences on cell migration. The absence of EpCAM impairs proper single cell spreading on collagen-coated substrates with deleterious impact on front-rear polarity and migratory behaviour. In addition to a decreased protrusive activity, the loss of EpCAM changes the morphometry of focal adhesions and abrogates proper stress fibre maturation. These modifications stem from a dysfunction of actomyosin regulation and a blocking of activated Rho zone at the cell cortex. In fact, EpCAM acts upstream of the actomyosin apparatus and directly potentializes activated Rho recycling within the protrusion for efficient epithelial cell spreading. In parallel, we investigate EpCAM’s participation in the homeostasis of the intestinal tissue. Within the proliferative compartment of mouse and human intestines, EpCAM is enriched in bottom crypts, more precisely at stem cell basolateral side, suggesting its specific requirement for stem cell-substrate and cell-cell adhesions. Moreover, the absence of EpCAM in CTE patients leads to clear disorganization of stem cell arrangement. Experiments pursued by using mouse EpCAM-KO 3D organoid primary cultures or CTE 3D organoid primary cultures demonstrate that EpCAM expression is required for correct de novo growth and maintenance of the intestinal stem cell niche. In-progress combination of organoids and optimized biomimetic 3D culture systems will allow more systematic analyses and live cell imaging of the intestinal niche and the differentiated epithelium along intestinal architecture.
Altogether, our work reveals that EpCAM acts as a key regulator of epithelial morphogenesis and homeostasis along tissue architecture by regulating actomyosin activity and actin cytoskeleton arrangement at the cell cortex.
