GUTPOLAR action led to four major scientific achievements, which enhance our understanding of the mechanisms underlying intestinal BB and apico-basal polarity establishment and maintenance as well as intestinal pathophysiology: 1) the generation of the first high-resolution dynamic map of the intestinal BB in vivo, 2) the characterization of a new V0-ATPase-dependent trafficking pathway that is necessary for the maintenance of polarity and BB in the C. elegans intestine, 3) the establishment of mouse intestinal organoids, used to establish the conservation of the V0-ATPase function and 4) the characterization of an MVID-like phenotype upon V0-ATPase depletion in both models, which suggests that this complex may be involved in the aetiology of this disorder.
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.
