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
Intestinal absorption of nutrients from the diet relies on both the strong polarity of the intestinal epithelial cells and the array of microvilli forming an absorptive brush border (BB) at their apical pole. Previous studies suggested that membrane trafficking plays a major role in the maintenance of the apico-basal polarity in several epithelial models. Since mutations in membrane trafficking factors have been linked to rare genetic disorders affecting the absorptive function of the intestinal epithelial cells, uncovering the molecular mechanisms by which membrane trafficking controls the maintenance of the BB and the polarity in the enterocytes is pivotal in understanding the pathophysiological mechanisms of intestinal absorption diseases and identifying new therapeutic targets.
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.
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.
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.
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.
First, the high-resolution dynamic in vivo map of the BB greatly enhances our understanding of how a BB is built, which will be instrumental in developmental biology but also to understand how genetic or environmental challenges affect the BB structure and function. Second, the combined use of C. elegans and intestinal organoids contributed to the state of the art at various research scales. Indeed, the characterization of the new pathway controlled by the V0-ATPase during the action confirms and extends the major and conserved role played by apical membrane trafficking in the maintenance of the BB and epithelial cell polarity. Most importantly, we discovered that V0-ATPase depletion induces an MVID-like phenotype in both C. elegans and in mouse, which strongly suggests that the V0-ATPase plays a major role in the appearance of the cellular and functional defects of the MVID disease. These data provide the scientific and medical communities with new paradigms to understand the pathophysiological mechanisms of MVID and new targets to cure this disorder.
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.
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.