Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast

Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.


Loïc Verlingue

Rémi Trimbour

Nicolas Sompairac

Marianyela Petrizzelli

Vincent Noel

Cristobal Monraz

Jane Merlevede

Aziz Fouché

Inna Kuperstein

Maria Rodrigo Riestra

Autophagy-mediated metabolic effects of aspirin.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b or Bcln1) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.

Reciprocal regulation of Aurora kinase A and ATIP3 in the control of metaphase spindle length.

Maintaining the integrity of the mitotic spindle in metaphase is essential to ensure normal cell division. We show here that depletion of microtubule-associated protein ATIP3 reduces metaphase spindle length. Mass spectrometry analyses identified the microtubule minus-end depolymerizing kinesin Kif2A as an ATIP3 binding protein. We show that ATIP3 controls metaphase spindle length by interacting with Kif2A and its partner Dda3 in an Aurora kinase A-dependent manner. In the absence of ATIP3, Kif2A and Dda3 accumulate at spindle poles, which is consistent with reduced poleward microtubule flux and shortening of the spindle. ATIP3 silencing also limits Aurora A localization to the poles. Transfection of GFP-Aurora A, but not kinase-dead mutant, rescues the phenotype, indicating that ATIP3 maintains Aurora A activity on the poles to control Kif2A targeting and spindle size. Collectively, these data emphasize the pivotal role of Aurora kinase A and its mutual regulation with ATIP3 in controlling spindle length.

Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia.

Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo.

Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes.

Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B

The functional determinants of H3K4me3, their potential dependency on histone H2B monoubiquitination, and their contribution to defining transcriptional regimes are poorly defined in plant systems. Unlike in Saccharomyces cerevisiae, where a single SET1 protein catalyzes H3K4me3 as part of COMPlex of proteins ASsociated with Set1 (COMPASS), in Arabidopsis thaliana, this activity involves multiple histone methyltransferases. Among these, the plant-specific SET DOMAIN GROUP 2 (SDG2) has a prominent role.

Histone modifications during the life cycle of the brown alga Ectocarpus.

Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins.