Intracellular transport complexes in cell division dynamics and epithelial tubules organization

La division cellulaire est essentielle à l’organisation des tissus épithéliaux prolifératifs.Elle doit donc être finement régulée, à la fois dans l’espace et dans le temps, de l’échelle cellulaire à l’échelle tissulaire pour assurer l’organisation et le maintien de l’intégrité des tissus. Si de nombreux acteurs moléculaires essentiels à la division cellulaire ont été étudiés depuis des années, il reste encore beaucoup à faire pour caractériser l’ensemble des complexes protéiques qui régulent finement chaque étape de la division. De plus, l’influence de la division cellulaire sur l’organisation des tissus épithéliaux tubulaires in vivo est encore trop peu étudiée.Les tubules épithéliaux qui se composent d’une monocouche polarisée courbéeentourant une cavité centrale unique constituent les unités fonctionnelles de nombreuxorganes, notamment le rein. Comprendre comment la division cellulaire influence lecomportement de l’épithélium tubulaire en 3 dimensions (3D) et in vivo est donc de laplus haute importance.Les protéines de la machinerie de transport intra-flagellaire (IFT) ont longtemps étéétudiées pour leurs rôles dans l’assemblage et le maintien du cil primaire ainsi que pour leur contribution à l’organisation des tubules rénaux. Cependant, divers travaux, dont ceux de l’équipe, ont montré que les protéines IFT ont aussi des rôles non-ciliaires, notamment au cours de la division cellulaire pour permettre l’orientation du fuseau mitotique. Au regard des divers rôles extra-ciliaires découverts ces dernières années, lesprotéines IFT pourraient être impliquées dans d’autres mécanismes essentiels à ladivision cellulaire et à l’intégrité des tissus.Dans ce contexte, l’objectif de ma thèse a consisté à caractériser de nouveauxmécanismes moléculaires contrôlant la dynamique des cellules en division maiségalement à étudier la contribution de la division cellulaire à l’organisation des tubulesépithéliaux. Deux aspects principaux ont été abordés : (1) la caractérisation de nouveaux rôles rôles pour les protéines IFT au cours de la division cellulaire, (2) l’étude de l’impact de perturbations de la cytokinèse, la dernière étape de la division cellulaire, sur l’organisation de tubules rénaux.En utilisant des cultures 2D et 3D de cellules rénales, mon travail de thèse a permis lacaractérisation de deux nouveaux rôles extra-ciliaires pour les protéines IFT au cours de la division cellulaire. En effet, les protéines IFT jouent un rôle dans l’organisation et la dynamique du fuseau mitotique en début de mitose mais également dans l’organisation du sillon de clivage en cytokinèse. D’autre part, en utilisant l’embryon de poisson zèbre comme modèle d’étude de l’épithélium tubulaire rénal in vivo, mon travail a également permis de caractériser la contribution de la cytokinèse à l’organisation des tubules rénaux, et plus précisément au maintien de la taille de la lumière.Globalement, l’ensemble de ces travaux ont révélé deux nouvelles fonctions desprotéines IFT au cours de la division cellulaire : une fonction en début de mitose qui fournit de nouveaux éléments sur leur implication dans l’intégrité et la dynamique du fuseau mitotique et un rôle en anaphase, en interaction avec la kinésine MKLP2, dans laconstriction du sillon de clivage. Ce mécanisme est essentiel à la bonne organisationspatiale de la cytokinèse ainsi qu’au bon positionnement de la lumière dans les culturesde cellules rénales en 3D. Ces travaux ont également révélé l’importance d’une régulation précise de la contractilité cellulaire au sillon de clivage en cytokinèse pour le maintien de la taille de la lumière des tubules de rein in vivo en contrôlant la ré-intercalation des cellules filles en sortie de division.Cell division is essential for the organization of proliferative epithelial tissues. It musttherefore be tightly regulated, both in space and in time, from the cellular scale to thetissue scale to ensure tissue integrity organization and maintenance. If cell division hasbeen studied for a long time, a lot remains to be done to characterize all the molecularplayers that tightly control each stage of cell division. Moreover, the contribution of celldivision to epithelial tubule organization in vivo has yet to be fully characterize.Epithelial tubules, which consist of a curved polarized monolayer surrounding a singlecentral cavity, are the functional units of many organs, including the kidney.Understanding how cell division influences the behavior of tubular epithelium in 3dimensions (3D) and in vivo is therefore important.Proteins of the intraflagellar transport (IFT) machinery have long been studied for theirroles in primary cilium assembly and maintenance as well as for their contribution tokidney tubule organization. However, different studies, including ours, showed that IFTproteins also have non-ciliary roles, especially during cell division in mitotic spindleorientation. In view of the various extra-ciliary roles discovered in recent years, IFTproteins could be involved in other mechanisms essential for cell division and tissueintegrity.In this context, the objective of my thesis was to characterize new molecular mechanisms controlling cell division dynamics but also to study the contribution of cell division to epithelial tubule organization. Two main aspects were addressed: (1) the characterization of new roles for IFT proteins during cell division, (2) the study of the impact of cytokinesis perturbations, the last step of cell division, on kidney tubule organization.Using 2D and 3D cultures of kidney cells, I characterized two new extra-ciliary roles forIFT proteins during cell division. Indeed, IFT proteins play a role in mitotic spindleorganization and dynamics at the start of mitosis but also in cleavage furrow ingression in cytokinesis. On the other hand, using the zebrafish embryo as a model to study kidney tubular epithelium in vivo, I characterized the contribution of cytokinesis to kidney tubule organization, and more precisely to lumen size maintenance.Overall, this work has revealed two new functions of IFT proteins during cell division: afunction at the start of mitosis which provides new information on their involvement inmitotic spindle integrity and dynamics and a role in anaphase, interacting with the kinesin MKLP2, in cleavage furrow ingression. This mechanism is essential for proper spatial organization of cytokinesis as well as proper lumen positioning in 3D kidney cell cultures. This work has also revealed the importance of precise regulation of cell contractility during cleavage furrow ingression in cytokinesis for maintaining lumen size, in kidney tubules, in vivo, by controlling daughter cell re-intercalation at the exit of cell division

Deciphering the Antifibrotic Property of Metformin

Fibrosis is a chronic progressive and incurable disease leading to organ dysfunction. It is characterized by the accumulation of extracellular matrix proteins produced by mesenchymal stem cells (MSCs) differentiating into myofibroblasts. Given the complexity of its pathophysiology, the search for effective treatments for fibrosis is of paramount importance. Metformin, a structural dimethyl analog of the galegine guanide extracted from the “French Lilac” (Fabaceae Galega officinalis), is the most widely used antidiabetic drug, recently recognized for its antifibrotic effects through ill-characterized mechanisms. The in vitro model of TGF-β1-induced fibrosis in human primary pulmonary mesenchymal stem cells (HPMSCs), identified as CD248+ and CD90+ cells, was used to study the effects of metformin extracts. These effects were tested on the expression of canonical MSC differentiation markers, immune/inflammatory factors and antioxidative stress molecules using qRT-PCR (mRNA, miRNA), immunofluorescence and ELISA experiments. Interestingly, metformin is able to reduce/modulate the expression of different actors involved in fibrosis. Indeed, TGF-β1 effects were markedly attenuated by metformin, as evidenced by reduced expression of three collagen types and Acta2 mRNAs. Furthermore, metformin attenuated the effects of TGF-β1 on the expression of PDGF, VEGF, erythropoietin, calcitonin and profibrotic miRs, possibly by controlling the expression of several key TGF/Smad factors. The expression of four major fibrogenic MMPs was also reduced by metformin treatment. In addition, metformin controlled MSC differentiation into lipofibroblasts and osteoblasts and had the ability to restore redox balance via the Nox4/Nrf2, AMP and Pi3K pathways. Overall, these results show that metformin is a candidate molecule for antifibrotic effect and/or aiming to combat the development of chronic inflammatory diseases worldwide

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

International audienc

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles.

International audienc

IFT proteins spatially control the geometry of cleavage furrow ingression and lumen positioning

Cytokinesis relies on central spindle organization and provides a spatial landmark for lumen formation. Here, the authors show that intraflagellar transport proteins are required for the localization of the cytokinetic regulator Aurora B and subsequent cleavage furrow ingression and lumen positioning

The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces

View the article online https://www.science.org/doi/10.1126/sciadv.abn5406International audienceThree-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order