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Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow

Nature Cell Biology volume 18, pages 657–667 (2016)Cite this article

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Abstract

Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo. PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1–AMPK–mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases.

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Figure 1: Fluid flow induces autophagy in MDCK cells.
Figure 2: Inhibition of ciliogenesis impairs flow-induced autophagy.
Figure 3: Inhibition of autophagy impairs regulation of cell volume.
Figure 4: Blockage of autophagic flux increases the size of tubular cells.
Figure 5: Inhibition of urinary fluid flow decreases the autophagic response and increases the size of tubular cells.
Figure 6: Impairment of autophagic activity and deregulation of cell size in PC-deficient kidney tubules.
Figure 7: Fluid-flow-induced autophagy and cell-volume regulation is dependent on the LKB1–AMPK–mTOR pathway.

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Acknowledgements

We thank F. Bienaimé, T. Blanc, C. Nguyen and C. Bauvy for assistance. We also thank T. Yoshimori (Osaka University, Osaksa, Japan) for providing GFP–LC3 and RFP–GFP–LC3 constructs. We are grateful to A. M. Cuervo (Albert Einstein College, Bronx, New York, USA) for sharing KECs and IFT88−/− KECs. We acknowledge the design of the transgenic mouse strains by L. Goldstein, San Diego, California, USA (Kif3atm1Gsn), R. Koesters, Heidelberg, Germany (Tg(Pax8–rtTA2S∗M2)1Koes), and D. Tenen, Boston, USA (Tg(TetOCre)1Dgt). We also acknowledge the Necker Institute Imaging Facility, the Fondation Imagine, the Cochin Imaging Electron Microscopy facility, and the Animal Histology and Morphology Core Facility (SFR Necker INSERM US24, CNRS UMS 3633). The work was supported by institutional funding from INSERM, CNRS, and University Paris Descartes and grants from ANR and INCa to P.C. N.D. is supported by a fellowship from Association pour la Recherche sur le Cancer (ARC), and A.V. was supported by an ERA-EDTA fellowship. E.W.K. is supported by the DFG (KU1504-5/1) and Else–Kröner–Fresenius Stiftung (2011_A87).

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Author notes

  1. Idil Orhon and Nicolas Dupont: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Necker Enfants-Malades (INEM), INSERM U1151-CNRS UMR 8253, Université Paris Descartes-Sorbonne Paris Cité, Paris F-75993, France

    Idil Orhon, Nicolas Dupont, Mohamad Zaidan, Valérie Boitez, Martine Burtin, Thierry Capiod, Gérard Friedlander, Fabiola Terzi & Patrice Codogno

  2. Institut Cochin, INSERM U1016-CNRS UMR 8104, Université Paris Descartes-Sorbonne Paris Cité, Paris F-75014, France

    Alain Schmitt

  3. Department of Nephrology, University Medical Center, Albert-Ludwig-University of Freiburg, D-79106 Freiburg, Germany

    Amandine Viau & E. Wolfgang Kuehn

  4. INSERM UMR 1185, Université Paris-Sud 11, Kremlin-Bicêtre F-94276, France

    Isabelle Beau

  5. Center for Biological Signaling Studies (bioss), Albert-Ludwig-University, D-79104 Freiburg, Germany

    E. Wolfgang Kuehn

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  1. Idil Orhon
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Contributions

I.O., N.D., M.Z., M.B. and V.B. performed most of the experiments with the exception of electron microscopy experiments, which were performed by A.S. I.B. contributed to the initial part of the project. A.V. and E.W.K. provided the mouse tissues. I.O., N.D., M.Z., T.C., G.F., F.T. and P.C. conceived and planned the experiments and interpreted data. I.O., N.D. and P.C. wrote the manuscript.

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Correspondence to Patrice Codogno.

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Integrated supplementary information

Supplementary Figure 2 Kinetics of LC3-II and actin expression determined by western blot analyses of WT and IFT88−/− KECs subjected to fluid flow.

(a) WT KEC and (b) IFT88−/− KECs were subjected to flow from 4 h to 4 days (d0, h4, d1, d2, d4) with or without chloroquine (CQ) then LC3-II and actin levels were determined by immunoblotting independently repeated three times. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 3 Recruitment of ATG16L1 to the basal body on fluid flow induction.

(a–d) WT KEC were subjected to flow or not (d0) for 1 day (d1) then fixed and labelled with γ-tubulin and ATG16L1 antibodies. (a) Cells were analysed by confocal fluorescence microscopy. Arrowheads indicate co-localization of ATG16L1 (green) and γ-tubulin (red). (b) 3D reconstruction of WT KECs subjected to flow or not (dark blue, small cells; white, large cells). (c) Two-fluorescence channel line tracings corresponding to dashed lines in the merge of panel A. (d) Recruitment of ATG16L1 to basal bodies was analysed by quantification of ATG16L1+ basal bodies using ImageJ; n = 125 cells from 5 fields of view per experiment pooled from 3 independent experiments. Data in (d) are means ± s.e.m. Statistical significance was determined using an unpaired Student’s t-test; P values are shown.

Supplementary Figure 4 Ultrastucture analyses of WT and IFT88−/− KECs subjected to fluid flow.

(a–c) WT KEC and IFT88−/− KECs were subjected to flow or not (d0) for 1 day (d1) then fixed and processed for ultrastructure electron microscopy. (a) Representative images. (b) Magnification of double membrane structures (autophagosome) shown in the white inset in panel A. (c) Quantification of autophagic vacuoles (AV) in WT KEC and IFT88−/− KECs with and without (ctrl) flow; n = 15 cells from a single experiment that was independently repeated 3 times. (d) Immunogold electron microscopy for LC3. Data in (c) are means ± s.e.m. Statistical significance was determined using an unpaired Student’s t-test; P values are shown.

Supplementary Figure 5 Effect of fluid flow on ciliogenesis.

(a–d) WT KECs were not subjected to flow (d0) or were subjected to flow for 4 h (h4) or 4 days (d4) then fixed, labelled with acetylated-tubulin (red) and LC3 antibody (green), then analysed by fluorescence microscopy. (a) Representative images of ciliated cells. (b) Number of ciliated cells was quantified using Imaris software. Not significant indicated by ‘ns’. n = 45 cells pooled from 3 independent experiments. (c) Representative images. (d) Cilium length was quantified using Imaris software; n = 45 cells pooled from 3 independent experiments. Data in (b) and (d) are means ± s.e.m. Statistical significance was determined using an unpaired Student’s t-test; P values are shown.

Supplementary Figure 6 Effect of inhibition of ciliogenesis on flow-induced autophagy.

(a) WT KEC and IFT88−/− KECs were subjected to flow or not from 4 h to 4 days (d0, h4, d1, d2, d4). Mean cell volumes were determined by microscopy; ∗, P < 0.05, n = 125 cells from 5 fields of view per experiment pooled from 3 independent experiments. Not significant indicated by ‘ns’. (b–d) KECs engineered to inducibly express an shRNA designed to inhibit expression of kif3a were treated or not with IPTG and subjected to flow for 4 days (d4) or not (d0). (b) Reductions in levels of Kif3a on induction of shRNA expression were confirmed by immunoblotting independently repeated three times. (c) KECs treated or not with IPTG were fixed, labelled with LC3 antibody, and analysed by fluorescence microscopy. Number of endogenous LC3 puncta per cell were quantified using ImageJ software; n = 125 cells from 5 fields of view per experiment pooled from 3 independent experiments. (d,e) KECs engineered to inducibly express shRNA targeting kif3a were treated or not with IPTG and subjected to flow for 4 days (d4) or not (d0). (d) 3D reconstructions of cells (dark blue indicates small cells and white indicates large cells). (e) Mean cell volumes; n = 125 cells from 5 fields of view per experiment pooled from 3 independent experiments. Data in (a), (c), and (e) are means ± s.e.m. Statistical significance was determined using an unpaired Student’s t-test; P values are shown. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 Western blot analysis of KECs expressing shRNAs targeting Atg5 or Atg16L1.

Expression of shRNA targeting (a) Atg5 or (b) Atg16L1 in KECs treated or not with IPTG and subjected to flow for 4 days reduced levels of ATG5 and ATG16L1, respectively, as shown by immunoblotting independently repeated three times. (c) KECs engineered to inducibly express shRNA targeting Atg5 were treated or not with IPTG and subjected to flow from 4 h to 4 days (d0, h4, d1, d2, d4), then LC3-II and actin levels were determined by immunoblotting. (d) KECs engineered to inducibly express shRNA targeting Atg16L1 were treated or not with IPTG and subjected to flow from 4 h to 4 days (d0, h4, d1, d2, d4), then LC3-II and actin levels were determined by immunoblotting independently repeated three times. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 8 Role of Hedgehog signalling and PC2 calcium channel in fluid flow-induced autophagy.

(a) Time course of expression of Gli1 and Ptch1in WT KECs subjected to flow from 4 h to 4 days (4 h, d1, d2, d4) or not (d0). As a positive control, cells were treated with purmophamine for 24 h. Gli1 and Ptch1 mRNA levels were quantified by real-time RT-PCR, normalized with respect to the β-actin gene, and presented as fold increases; from n = 3 independent experiments as shown in i. Not significant indicated by ‘ns’. (b) Cells were subjected to flow for 4 h (h4) or not (d0) or treated with purmophamine for 24 h, then fixed and stained for Smo (green) and acetylated-tubulin (red) to evaluate localization of Smo to the axoneme of cilia. Cells were then analysed by fluorescence microscopy. (c) KECs engineered to inducibly express an shRNA targeting Pkd2 were treated or not with IPTG, and Pkd2 mRNA levels were quantified by real-time RT-PCR, normalized with respect to the β-actin gene, and presented as fold increases; from n = 3 independent experiments as shown in i. (d,e) KECs engineered to inducibly express an shRNA targeting Pkd2 were treated with IPTG (right panel) or not (left panel), labelled with Fura-2, and analysed by fluorescence microscopy. Black arrowheads indicate when cells were subjected to flow or to ATP (to enable evaluation of cell viability). (f,g) KECs engineered to inducibly express an shRNA targeting Pkd2 were treated or not with IPTG and subjected to flow for 4 h or not (d0) with or without CQ. LC3-II/actin ratios visualized by immunoblotting were quantified by densitometry; ∗, P < 0.05, from n = 3 independent experiments as shown in i. (h,i) KECs engineered to inducibly express shRNA targeting Pkd2 were treated or not with IPTG and subjected to flow for 4 days (d4) or not (d0). (h) 3D reconstructions of cells (dark blue indicates small cells and white indicates large cells). (i) Mean cell volumes; n = 125 cells from 5 fields of view per experiment pooled from 3 independent experiments. Data in (a), (c), (g), and (i) are means ± s.e.m. Statistical significance was determined using an unpaired Student’s t-test; P values are shown. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Table 1 Lentivirus vectors and shRNAs.
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Orhon, I., Dupont, N., Zaidan, M. et al. Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow. Nat Cell Biol 18, 657–667 (2016). https://doi.org/10.1038/ncb3360

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