Periodic Reporting for period 1 - GUTPOLAR (Membrane trafficking as a link between cell polarity and intestinal absorptive function: from C. elegans to mammalian miniguts)
Reporting period: 2019-05-06 to 2021-05-05
The GUTPOLAR action aimed to better understand how this cellular transport mechanism coordinates the apical localization of polarity and BB components in intestinal cells, notably by focussing on the trans-species function of the V0-ATPase complex, which seems to play a major role in this process.
The overall objectives were i) to study the role of the V0-ATPase complex and its genetic partners in polarity and BB maintenance in vivo using the C. elegans intestine as a model and ii) to establish and use mouse intestinal organoids to study the role of the V0-ATPase in polarity maintenance in an advanced mammalian intestine model.
Using various genetic and imaging tools in vivo (C. elegans) and ex vivo (intestinal organoids), we demonstrated that the V0-ATPase complex plays a major and conserved role in intestinal BB and polarity maintenance. Notably, we showed that loss of this complex recapitulates the rare genetic absorption disorder Microvillus inclusion disease (MVID) in both models, which paves the way to study its implication in the aetiology of this disease.
These results have been disseminated through scientific publications, presentations at scientific conferences as well as through informal meetings with collaborators and website/social medias. They have also been exploited to better assess the role of the V0-ATPase in MVID, notably through a new study funded by a patients’ foundation and through further analyses in patients, in collaboration with the team’s medical collaborators.
- Description of the work performed and main results:
1) Generation of a high-resolution dynamic map of the BB in vivo. To reach this objective, we combined CRIPSR-CAS9 endogenous tagging and state-of-the-art super-resolution imaging systems to study the (co)localization, the apical expression as well as the dynamics of BB components during C. elegans intestinal development. This led to a detailed and quantitative description of the dynamic recruitment of BB components during microvilli assembly in vivo and uncovered that mature BBs are very stable, a characteristic that has never been shown before (Bidaud-Meynard et al., in preparation-a)
2) V0-ATPase controls an apical recycling pathway needed for BB maintenance. Using various photonic (super-resolution, live imaging) and transmission electron microscopy (TEM) techniques as well as epistasis experiments, we performed a detailed analysis of the effect of V0-ATPase silencing on trafficking, polarity and BB maintenance. This led to the characterization of a new trafficking pathway, involving the endocytic-recycling and vesicular fusion components RAB-11 and SNAP-29, respectively, that is required for BB and polarity maintenance. Furthermore, these results demonstrated that V0-ATPase silencing induces an MVID-like phenotype in worms, characterized by a microvillus atrophy and cytoplasmic microvillus inclusions (Figure 1) (Bidaud-Meynard et al., Development, 2019).
3) Set up mouse intestinal organoids culture to study V0-ATPase-dependent polarity and BB maintenance mechanisms in mammals. To reach this objective, the ER performed a secondment in the lab of Henner Farin (Georg-Speyer-Haus, Frankfurt, Germany) to learn organoid culture technology. Then, the ER established organoids culture at the host institution (IGDR, Rennes, France) and knocked out V0-ATPase and V1-ATPase (as a negative control) complex subunits as well as Myo5b (as a positive control), the most-mutated MVID-causing gene, by an inducible CRISPR-CAS9 technology.
4) As in C. elegans, V0-ATPase knockout (KO) in organoids also induces an MVID-like phenotype. An in-depth phenotypical analysis (super-resolution imaging/immunohistochemistry analysis of BB, trafficking and apical transporters markers as well as ultrastructural analysis by TEM) allowed to show that V0-ATPase KO, as in C. elegans, induces an MVID-like phenotype in murine intestinal organoids (e.g. cytoplasmic microvillus inclusions, Figure 2) (Bidaud-Meynard et al., in preparation-b). These results demonstrate the conserved function of the V0-ATPase complex in BB and polarity maintenance and suggest that this complex is involved in MVID pathophysiology, which is now investigated in collaboration with our medical collaborators.
Hence, the results of the GUTPOLAR action will be directly exploited by both basic science and medical communities. Furthermore, this project is a perfect example of how a basic science project can be translated into the clinic. This will help i) the global population and patients suffering from intestinal diseases to better appreciate the work of scientists in laboratories and the tremendous help of UE funded MSCA-IF fellowships in this process and ii) French and European governments to design rare disease policies and research funding.