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A phosphate-sensing organelle regulates phosphate and tissue homeostasis

Nature volume 617, pages 798–806 (2023)Cite this article

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Abstract

Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo–Cka–JNK signalling cascade controlling tissue homeostasis.

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Fig. 1: Pi deprivation or PXo deficiency induces midgut hyperproliferation.
Fig. 2: PXo localizes to PXo bodies, a type of multilamellar organelle.
Fig. 3: PXo regulates cytosolic Pi levels.
Fig. 4: PXo bodies are sensitive to Pi availability.
Fig. 5: Proteomic and lipidomic characterization of PXo bodies.
Fig. 6: PXo interacts with STRIPAK core components and suppresses Cka in enterocytes.
Fig. 7: STRIPAK/Cka–JNK signalling mediates the hyperproliferation induced by Pi starvation or PXo knockdown.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information. Source data are provided with this paper.

Code availability

‘PXo_body_area’ is available at https://github.com/charles-xu-ru/image-analysis/blob/master/PXo_body_area. ‘PXo_size_violin’ is available at https://github.com/charles-xu-ru/image-analysis/blob/master/PXo_size_violin. ‘FRET_ratios_movie’ is available at https://github.com/charles-xu-ru/image-analysis/blob/master/FRET_ratios_movie.

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Acknowledgements

We thank L. R. Garcia at Texas A&M University for sharing the pLR364-cpFlippi-6.4 plasmid; P. Montero Llopis and the Microscopy Resources on the North Quad core (MicRoN) at Harvard Medical School for imaging support; the Electron Microscopy Facility at Harvard Medical School for EM support; the Mass Spectrometry Core at Beth Israel Deaconess Medical Center for MS support; R. Binari, P. Jouandin, L. He, J. Bosch and C. Villalta for technical assistance; and A. Petsakou, L. He, B. Mathey-Prevot and S. Ellis for comments on the manuscript. We thank N. Buchon, H. Jiang, B. Edgar, S. Hou, A. Bardin, C. Wilson, F. Pignoni, D. Pan and T. Hays for sharing fly stocks; X. Yang and C. Yu for sharing the anti-Pdm1 antibody; X. He and M. Chen for suggestions on biochemical experiments with PXo; H. Huang and F. Li for advice on 18O stable isotope tracing analysis; and M. Schernthanner and A. Gola for instructions on Imaris analysis. We thank S. Shao, T. Rapoport, S. Navdar, J. Wang, C. Allard and N. Bellono for discussion. This work is supported by the National Institute of General Medical Sciences (GM067761), NIH 5P01CA120964-09, the STARR consortium and HHMI. C.X. is supported by the C. H. Li memorial fellowship, a National Cancer Center fellowship and a Charles Revson fellowship. H.-W.T. is supported by the Human Frontier Science Program. N.P. Is an investigator of the HHMI.

Author information

Author notes

  1. Chiwei Xu

    Present address: Robin Chemers Neustein Laboratory of Mammalian Development and Cell Biology, The Rockefeller University, New York, NY, USA

  2. Jun Xu

    Present address: CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

  3. Hong-Wen Tang

    Present address: Program in Cancer and Stem Cell Biology, Duke–NUS Medical School, Singapore, Singapore

  4. These authors contributed equally: Chiwei Xu, Jun Xu

Authors and Affiliations

  1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Chiwei Xu, Jun Xu, Hong-Wen Tang, Jonathan DiRusso, Yanhui Hu & Norbert Perrimon

  2. Department of Cell Biology, Electron Microscopy Facility, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Maria Ericsson

  3. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    Jui-Hsia Weng

  4. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Wenzhe Ma

  5. Department of Medicine, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    John M. Asara

  6. Department of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA

    John M. Asara

  7. Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Norbert Perrimon

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Contributions

C.X. performed most experiments including proliferation phenotyping, immunostaining, MARCM, FRET and bioinformatic analysis. J.X. performed experiments characterizing CDF feeding, enterocyte turnover/apoptosis, PXo expression, PXo body biogenesis and PXo body lipidomic/proteomic profiles. H.-W.T. characterized PXo degradation mechanisms and performed biochemical experiments (western blots, co-immunoprecipitation and affinity purification). M.E. performed EM. J.-H.W. designed and prepared the antigen for the anti-PXo antibody. J.D. helped with staining and performed phospholipid feeding experiments. Y.H. performed lipidomic heatmap and complex enrichment analysis for LC–MS/MS data. W.M. helped with OptiPrep gradient fractionation. J.M.A. analysed the proteomic and lipidomic LC–MS/MS data. C.X. and N.P. conceptualized the study, designed the experiments, analysed the data and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Chiwei Xu or Norbert Perrimon.

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Nature thanks Bruce Edgar, Osamu Nureki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Related to Fig. 1. Characterization of Pi starvation and PXo knockdown.

a,b, pH3 staining of midguts fed 7d normal or PFA food. c,d, pH3 staining of midguts fed 9d CDF containing standard (100%) or 10% Pi. e, RT-qPCR measurement of MFS10 knockdown in the midgut when RNAi (VDRC line) is expressed 5d ubiquitously. The representative results with n = 3 technical replicates are presented. P = 0.0217. f,g, Midguts with 2d induced H2B-RFP expression in ECs, followed by 9d on normal or PFA food. h,i,j,k, Apoptag staining of midguts expressing Luc RNAi or PXo RNAi in ECs for 7d, with normal food (h,k), PFA, or the last 2d on Bleo. l,m, pH3 staining of midguts expressing Luc RNAi or PXo RNAi in ECs for 7d. n,o, pH3 staining and r, mitosis quantification of midguts with PXo RNAi (VDRC line) expression in ECs for 7d. n = 8 (Ctrlw), 9 (PXo-iv). P = 0.0193. p,q, pH3 staining of midguts expressing Luc RNAi or PXo RNAi in progenitors for 8d. s, Mitosis of midguts with PXo RNAi (NIG) expression in ECs for 9d. n = 12 (Ctrlw), 15 (PXo-iN). P = 7.94E-6. t, Mitosis of midguts expressing Luc RNAi or PXo RNAi alone, or together with p35 in progenitors for 9d, with or without the last 2d on Bleo. n = 14 (Luc-i Normal, Luc-i p35, PXo-i p35), 13 (PXo-i Normal, Luc-i Bleo, PXo-i Bleo). P values from left to right: 9.97E-8, 3.00E-3, 1.92E-7. u, Mitosis of midguts with or without PXo RNAi (NIG) expression in progenitors for 8d, fed normal food (P = 5.78E-5) or 2d Bleo (P = 0.0237). From left to right: N = 14, 13, 8, 12. v, Mitosis of midguts expressing GFP or p35 alone, or together with PXo RNAi in ECs for 9d. From left to right: n = 12, 10, 12, 14; P = 3.09E-6, 5.05E-3, 2.07E-7. w, Mitosis of midguts expressing Luc RNAi or PXo RNAi in progenitors, and fed normal, PFA, or Pi food for 7d. From left to right: n = 8, 9, 8, 7, 8, 9; P = 0.0132, 1.00, 1.40E-3, 1.18E-3, 1.00. x, Mitosis of midguts with or without PXo-HA expression in ECs for 8d, fed normal food or 2d Bleo. From left to right: n = 12, 12, 13, 13; P = 3.85E-7, 0.456, 3.85E-7. Data are mean ± s.e.m. P values are from two-tailed Welch’s t test (e), two-tailed Mann-Whitney u test (r,s,t,u,v,x, between Luc-i and PXo-i Normal in w) or one-way ANOVA with Bonferroni’s multiple comparison test (w). Arrowheads highlight pH3+ examples (a–d,l–q). Scale bars, 50 µm.

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Extended Data Fig. 2 Related to Fig. 1. Characterization of PXo expression and phenotype in ISC/EB lineage.

a, PXo genomic locus and target regions of reagents in this study, including 3 RNAi lines (from NIG, VDRC, or BDSC, as labeled by dashed squares) and 3 pairs of RT-qPCR primers (P1, P2, P3), a Gal4 knock-in allele, a 2xHA tag knock-in, and an insertional mutation (PL48). b, RT-qPCR measurement of PXo knockdown efficiency of three PXo RNAi lines expressed ubiquitously in the midgut for 5d. Luc RNAi was the control for PXo RNAi from BDSC, Ctrlv was the control for PXo RNAi (VDRC), Ctrlw was the control for PXo RNAi (NIG). The representative results with n = 3 technical replicates are presented. P values from left to right: 1.52E-3, 0.0305, 2.52E-4, 0.0236, 2.61E-3, 9.74E-3. c, RT-qPCR measurement of PXo expression in heterozygous midguts carrying one PXoPL48 mutant allele or its wild type revertant allele PXorev. The representative results with n = 3 technical replicates are presented. P values from left to right: 0.0162, 0.0405. d, PXoGal4-driven GFP co-staining with esg-lacZ. The single GFP channel is presented on the right. Red, yellow, white arrowheads mark examples of progenitor, EC, EE, respectively. e, Anti-PXo immunoblot of S2R+ cell lysates expressing GFP or GFP-PXo. Tubulin was the loading control. Gel source data in Supplementary Fig. 1e. f,g, Anti-PXo staining of midguts with or without 5d ubiquitous PXo RNAi (NIG line) expression. h, Midguts with endogenous PXo tagged by 2 copies of HA epitope (PXo2xHA) were co-stained with anti-HA and anti-PXo antibodies. A magnified view of the highlighted region is presented in (h’). The separate channels of PXo and HA stainings are shown on the right. i,j, Midguts expressing GFP in adult ISCs/EBs and their progenies (i.e. the ISC/EB lineage) were fed 10d normal food or PFA and stained for EC marker Pdm1. k,l,m,n,o,p, Midguts expressing Luc RNAi or PXo RNAi in the ISC/EB lineage (labeled by GFP expression) for 10d were stained for Pdm1, EE marker Pros, and apoptosis marker cleaved caspase 3 (CCasp3). q,r,s,t, Pdm1 or Pros staining of midguts with GFP+ MARCM clones derived from wild type (n = 135 clones) or PXoPL48 mutant progenitors (n = 150) 10d after clone induction (dpi). Cell number per clone is quantified in (u). The PXorev allele generated by PBase-mediated precise excision of the inserted piggyBac transposon from mutant PXoPL48 allele was used as an additional control (n = 134). P < 1E-14 comparing PXoPL48 mutant versus Ctrl or PXorev wild type groups. v, Mitosis quantification of midguts expressing 8d Luc RNAi versus PXo RNAi in EEs, with normal food (P = 0.686) or 2d Bleo (P = 0.0179). n = 10 (Luc-i Normal), 12 (other groups). w, Mitosis of midguts expressing Luc RNAi or PXo RNAi in ISCs for 9d, with normal food (P = 0.771) or 2d Bleo (P = 6.50E-4). n = 10 per group. Data are mean ± s.e.m. P values are calculated from two-tailed Welch’s t test (b,c) or two-tailed Mann-Whitney u test (u,v,w). The single red channels are presented in grayscale below merged images (i–t). White arrowheads highlight examples of traced cell differentiation. Scale bars, 50 µm (d,h,i–t), 10 µm (f,g,h’).

Source data

Extended Data Fig. 3 Related to Fig. 2. Further analysis of PXo localization.

a,b,c,d,e,f,g, GFP staining of different organs from adult flies with ubiquitous expression of GFP-PXo for 5d. h,i,j,k,l,m,n,o, Co-staining of ubiquitously expressed GFP-PXo with various markers in the midgut, including Cnx99a (ER), RFP-KDEL (ER lumen), Hrs (early endosome), Rab7 (late endosome), ATP5A (mitochondria), GMAP (cis-Golgi), GalT-TagRFP (trans-Golgi), and WGA (glycans). 3D view is presented. p,q, P-Cho staining of midguts with ubiquitously expression of GFP-PXo for 5d and collected 2h post P-Cho injection. A non-injection group was included as the negative control. 3D view of confocal image is presented. r, Co-staining of ubiquitously expressed GFP-PXo with Avidin (ex vivo incubation). 3D view of confocal image is presented. s, Co-localization analysis with the quantification of Mander’s coefficient between GFP-PXo and Nile Red, ManII-TagRFP, or GalT-TagRFP. n = 4 midguts per group. P values from bottom to top: 1.16E-10, 1.14E-10. t, Shortest distance between each labeled PXo body and P-Cho (2h post injection), dextran (feeding), or avidin (feeding). n = 185 (P-Cho group), 149 (dextran), 122 (avidin) PXo bodies from 4 midguts were analyzed. P < 1E-14. Data are mean ± s.e.m. P values are from one-way ANOVA with Bonferroni’s multiple comparison test (s, t). Separate green and red channels are presented below merged images (h–r). Scale bars, 50 µm (a–g), 10 µm (h–r).

Source data

Extended Data Fig. 4 Related to Fig. 2. Analysis of the dependency of PXo bodies on other canonical organelles.

a, EM of midgut frozen section with immunogold-labeled GFP-PXo (examples highlighted with arrowheads) expressed in progenitors. b, Midgut EM shows unlabeled PXo bodies mainly in ECs (examples highlighted with arrowheads). A progenitor cell, as circled out by dashed line, can be distinguished from ECs, based on its basal localization, high electron density, lack of microvilli, and lack of mitochondria (“mt”) cristae. A magnified view of the squared region, as presented in (b’), shows a PXo body in the progenitor. c, Midgut EM distinguishes the membranous PXo bodies (highlighted with arrowhead) from lipid droplets (“LD”). d,e, EM shows PXo body fragmentation in midguts with ubiquitous γCOP knockdown for 7d. f,g,h,i,j, HA staining of midguts expressing PXo-HA together with Luc RNAi, Sar1 RNAi, Arf1 RNAi, γCOP RNAi, or Pmm2 RNAi in ECs for 7d. Lethal giant larvae (Lgl) marks cell border. k,l,m,n,o,p, EM showing PXo bodies in ECs expressing Luc RNAi, Pink RNAi, Parkin RNAi, Lamp1 RNAi, or Abca RNAi for 7d, with PXo body size quantification in (q). n = 110 (Abca-i1) or 120 (other groups). P values from bottom to top: 1.00, 0.747, 0.0162, 1.00, 1.00. r,s,t,u, EM showing PXo bodies in ECs expressing Luc RNAi (n = 70), Rab7 RNAi (n = 60), or Rab11 RNAi (line #1: n = 70; line #2: n = 80) for 3d, with quantification in (v). P values from bottom to top: 1.00, 0.490, 1.00. Data are mean ± s.e.m.. P values are calculated from one-way ANOVA with Bonferroni’s multiple comparison test. Scale bars, 1 µm (a,b’, c–e, k–p, r–u), 10 µm (b), 50 µm (f–j).

Source data

Extended Data Fig. 5 Related to Fig. 3. PXo structure and live imaging for cytosolic Pi.

a, AlphaFold structure prediction of rice PHO1-2, with color-scaled visualization of the prediction confidence score. The pairwise structure alignment between PXo and PHO1-2 was shown on the right, with aligned regions superposed on one another and highlighted in color. The summary of amino acid sequence alignment was shown at the bottom, with aligned regions highlighted in color. b, A single Z-stack of confocal image of midguts expressing PXo-HA and FLIPPi together in the ECs for 7d. Separate channels of HA and YFP (FLIPPi) stainings are presented on the right of merged images. c, Co-localization analysis with the quantification of the PXo-FLIPPi and FLIPPi-PXo Mander’s coefficients. n = 9 per group. d,e, Kinetics of the average FRET ratio (cpVenus/CFP) in the midguts expressing FLIPPi ubiquitously (tubts) or specifically in ECs (Myo1Ats), progenitors (esgts), ISCs (Dlts) or EBs (Su(H)ts) for 3–7 days. The imaging videos were captured for ~10 min. The time points adding an extra final concentration of 30 mM Pi (~300s, using sodium phosphate or potassium phosphate) to the AHL imaging buffer is indicated by black arrows. Note that a new steady cytosolic Pi level is reached ~5 min after the addition of extra Pi. f, Color-scaled visualization of FRET ratios in the posterior midgut with ubiquitous FLIPPi expression for 7d. The same area was imaged again 10 min after the addition of 30 mM Pi (g). h, FRET ratio quantification of midguts with ubiquitous FLIPPi expression for 7d on normal food before or after 30 mM Pi addition to the imaging buffer (n = 9), or with the last 4d on PFA food (n = 7). P values from bottom to top: 3.91E-3, 3.50E-4. i,j,k, FRET ratio visualization and quantification of ECs expressing FLIPPi alone or FLIPPi together with MFS10 RNAi for 7d, before or 10 min after 30 mM Pi addition. n = 11 per group. P values from bottom to top: 9.77E-4, 9.77E-4, 0.0173, 0.0192. l,m,n, FRET ratio visualization and quantification of midguts expressing FLIPPi alone (by crossing to Ctrlw, n = 10) or FLIPPi together with PXo RNAi (NIG line, n = 11) in ECs for 6d. P values from bottom to top: 1.95E-3, 4.03E-4. o,p, FRET ratios visualization of progenitors expressing FLIPPi alone or together with PXo-HA for 7d. q, FRET ratio quantification of midguts expressing FLIPPi alone (n = 9), FLIPPi together with PXo-HA (n = 9), or FLIPPi together with PXo RNAi (NIG line, n = 11) for 7d, before or after Pi addition. P values from bottom to top: 0.0210, 0.936, 0.0114, 0.778. Data are mean ± s.e.m. P values are from Wilcoxon signed rank test for same sample comparison before and after Pi addition in (h,k,n), two-tailed Mann-Whitney u test between different samples (h,k,n) or one-way ANOVA with Bonferroni’s multiple comparison test (q). Scale bars, 10 µm (b), 200 µm (f,g,i,j,l,m,o,p).

Source data

Extended Data Fig. 6 Related to Fig. 4. EM analysis for the dependency of PXo bodies on Pi availability and PXo expression.

a,b, EM of wild type (genotype: OreR) midguts with or without PFA feeding for 4d, along with magnified views of highlighted regions in the bottom and PXo body size quantification in (c). n = 17 PXo bodies were analyzed per group. P = 1.28E-5. d,e, EM of midguts with or without ubiquitous MFS10 RNAi expression for 5d, along with a magnified view of highlighted regions in the bottom and PXo body size quantification in (f). n = 91 (Ctrlv), 89 (MFS10-iv). P = 1.38E-8. g,h, EM of midguts with or without ubiquitous PXo RNAi (NIG line) expression for 5d, along with magnified views of highlighted regions in the bottom and PXo body size quantification in (i). n = 40 PXo bodies per group. P = 1.30E-9. Data are mean ± s.e.m. P values are from two-tailed Mann-Whitney u test (c,f,i). Scale bars, 4 µm (a,b,d,e,g,h, top images), 1 µm (a,b,d,e,g,h, bottom images).

Source data

Extended Data Fig. 7 Related to Fig. 4. Mechanistic analysis of Pi-sensitivity for PXo and PXo bodies.

a,b,c, Midguts with GFP-PXo expression in ECs (driven by Myo1AGal4), with the last 4d on normal, PFA, or Pi food before dissection. d, e,f, Midguts with ubiquitous GFP-PXo expression for 7d, with the last 4d on CDF containing standard (100%), 20%, or 10% Pi. The staining for junctional protein Discs large (Dlg) marks cell borders. g,h,i,j, Anti-PXo staining of midguts with ubiquitous expression of GFP-Lamp1 or GFP-Atg8 (autophagosome marker) under normal feeding (g,h) or 4d PFA feeding conditions (i,j). A single Z-stack of confocal image is presented. Separate green and red channels are presented below merged images. Arrowheads highlight examples of co-localization. k,l, m,n, S2R+ cells expressing GFP-PXo with or without 24 hrs treatment of PFA, PFA together with BafA1, or PFA together with MG132. o,p, Midguts ubiquitously expressing GFP-PXo△SPX for 7d, with the last 4d on normal or PFA food, were stained for GFP and Dlg. q, Immunoblot of midgut lysates with ubiquitous expression of GFP-PXo△SPX for 7d, with or without the last 4d on PFA. Tubulin was the loading control. Quantification of relative normalized anti-GFP IB intensity is presented. Gel source data in Supplementary Fig. 1f. Scale bars, 50 µm (a–f, o,p), 10 µm (g–n).

Extended Data Fig. 8 Related to Fig. 5. Further examination of PXo’s relevance to intestinal barrier integrity or lipid metabolism.

a,b, Smurf assay to evaluate the barrier function of midguts expressing Luc RNAi, PXo RNAi ubiquitously for 10 days. Prolonged Tsp2A RNAi expression was used as a positive control. c,d, Representation of different lipid categories in the previously characterized lipidomes of Drosophila larval midguts or adult whole bodies. e,f,g,h, GFP staining and PXo density quantification of midguts with ubiquitous GFP-PXo expression for 7d, with the last 3d on normal food (n = 17), or food containing additional 1% (n = 13, P = 0.0393) or 5% PC (n = 13, P = 3.40E-8). Data are mean ± s.e.m. P values are from one-way ANOVA with Bonferroni’s multiple comparison test. Scale bars, 50 µm.

Source data

Extended Data Fig. 9 Related to Fig. 5. PXo body extraction and purification methods.

a, A centrifugation tube with midgut lysate OptiPrep gradient after ultracentrifugation, alongside with the cartoon created from BioRender.com depicting 14 gradient layers (fractions). b,c,d,e, Merged bright field and GFP fluorescent images of microscopic slides prepared from 15 µl aliquots of separated Layers 8–11. The refraction index (RI) for each Layer is also measured. f, RI measurement of 10%–30% serial concentrations of OptiPrep solutions, along with their calculated density. g, EM of a dissociated PXo body alongside a mitochondria (mt) from combined Layers 9-10. h, IB of the same volume of lysates from 14 separate Layers for GFP-PXo and ATP5A. Gel source data in Supplementary Fig. 1g. i,j,k,l,m,n, ATP5A and GMAP staining of midgut homogenate before (Input) and after immunoprecipitation (IP) of nanobody-conjugated Chromotek-GFP-Trap agarose beads, along with the flowthrough (Supernatant). o,p, Whole midguts with ubiquitous GFP-PXo expression for 5d, with the last 2d on normal food or PFA food. Scale bars, 50 µm (b,c,d,e), 1 µm (g), 10 µm (i,j,k,l,m,n), 1 mm (o,p).

Extended Data Fig. 10 Related to Fig. 5. Validation of identified PXo body proteins.

a, α-Spec staining of midguts with ubiquitous expression of GFP-PXo. A single Z-stack of confocal image is presented, with separate green and red channels presented below merged images and white arrowheads highlighting examples of co-localization. b,c,d,e, Co-staining of GFP-PXo with hts-mCherry, SERCA-Tomato, Ogdh-Flag, or Idh-Flag ubiquitously expressed in the midgut. A single Z-stack of confocal image is presented, with separate green and red channels presented below merged images and white arrowheads highlighting examples of co-localization. f,g,h, Co-staining of EC-expressed PXo-HA with Idh3a-GFP expressed in a fosmid (f), or endogenously tagged GFP-Treh (g) or Ance-GFP (h). A single Z-stack of confocal image is presented, with separate green and red channels presented below merged images and white arrowheads highlighting examples of co-localization. i,j, Co-staining of PXo-HA and GFP-Vha55 or Mipp1-GFP expressed in ECs. A single Z-stack of confocal image is presented, with separate green and red channels presented below merged images and white arrowheads highlighting examples of co-localization. Scale bars, 10 µm.

Extended Data Fig. 11 Related to Fig. 6. Comparison of how PXo affects Cka expression in ECs and in progenitors.

a,b, Flag-Cka staining of midguts expressing Luc RNAi or PXo RNAi in progenitors for 9d. A magnified view of regions highlighted with white dashed squares is presented in (a’,b’). The red channel images of anti-Flag staining are in grayscale below the merged images. White arrowheads highlight examples of progenitors that are differentiating towards ECs, as judged by their extended morphology, and enlarged nuclei size (b,b’). c,d,e,f, Midguts expressing BFP-Cka together with Luc RNAi or PXo RNAi in ECs for 4d or 8d. g,h, Midguts expressing BFP-Cka together with Luc RNAi or PXo RNAi in progenitors for 5d. The single-channel images of BFP-Cka fluorescence are presented in grayscale below merged images. Scale bars, 50 µm (a,b,c,d,e,f,g,h), 10 µm (a’,b’).

Extended Data Fig. 12 Related to Fig. 7. STRIPAK and JNK signaling mediate the hyperproliferation phenotype caused by PXo knockdown in ECs and in progenitors.

a,b, Puc-lacZ staining of midguts expressing Luc RNAi or PXo RNAi in progenitors for 7d. c,d,e, Midguts expressing Luc RNAi or PXo RNAi in ECs for 7d were stained for phosphorylated p38 (p-p38) and the cell border marker Dlg. Midguts fed with Bleo food for 2d were included as a positive control. f, Mitosis of midguts expressing PXo RNAi alone (n = 14) or PXo RNAi together with Cka RNAi (VDRC line, n = 15), Mob4 RNAi (VDRC, n = 14), or Strip RNAi (VDRC, n = 14) in ECs for 8d. P values from bottom to top: 1.50E-6, 3.60E-6, 8.08E-6. g, Mitosis of midguts expressing PXo RNAi together with Luc RNAi (n = 14), Strip RNAi (BDSC line, n = 14), mts RNAi (BDSC, n = 12), or Pp2A-29B RNAi (BDSC, n = 14) in ECs for 8d. P values from bottom to top: 1.86E-9, 5.40E-10, 3.69E-11. h, Mitosis of midguts expressing PXo RNAi together with Luc RNAi, Cka RNAi (BDSC), Mob4 RNAi (2 different BDSC lines), or bsk RNAi in progenitors for 9d. From left to right: n = 9, 10, 9, 11, 10; P = 1.07E-12, 3,33E-10, 4.93E-13, 2,72E-12. i, Mitosis of midguts expressing PXo RNAi alone (n = 12), or PXo RNAi together with Cka RNAi (VDRC line, n = 12), Mob4 RNAi (VDRC, n = 10), or Strip RNAi (VDRC, n = 12) in progenitors for 9d. P values from bottom to top: 4.57E-9, 3.72E-6, 4.57E-9. j, Mitosis of midguts expressing PXo RNAi together with Luc RNAi (n = 15), Strip RNAi (BDSC, n = 15), mts RNAi (BDSC, n = 14), or Pp2A-29B RNAi (BDSC, n = 12) in progenitors for 9d. P values from bottom to top: 6.29E-6, 2.07E-8, 1.72E-7. Data are mean ± s.e.m. P values are from one-way ANOVA with Bonferroni’s multiple comparison test. Scale bars, 50 µm.

Source data

Supplementary information

Supplementary Fig. 1

Uncropped immunoblots from main and Extended Data figures. a, Fig. 4m. b,c, Fig. 6b,c. d, Fig. 6e. e, Extended Data Fig. 2e. f, Extended Data Fig. 7q. h, Extended Data Fig. 9.

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Supplementary Table 1

Proteomic analysis of purified PXo bodies. a, A summary of the proteins identified by LC–MS/MS, with two replicates of blank loading control, four replicates of negative control (GFP immunoprecipitation) and four replicates of PXo body samples (PXo immunoprecipitation). The list is ranked by enrichment score, calculated as (PXo sample total peptide number + 1)/(negative control total peptide number + 1). As the threshold for identifying candidate PXo body proteins, the enrichment score should be higher than 2 and the peptides should be identified in at least three of the PXo body samples. For one pair of PXo body and negative-control samples, we included a group with additional boiling for 10 min before loading on a gel, whose LC–MS/MS detection appears consistent with that for groups that were not boiled. b,c, A summary of proteins identified by MS of lamellar bodies extracted from rat lungs in previous studies (Ridsdale, R. et al. PLoS ONE 6, e16482 (2011); Wang, P. et al. BMC Cell. Biol. 9, 34 (2008)). d, A summary of proteins identified by LC–MS/MS of a fraction enriched in human epidermal lamellar bodies in a previous study (Raymond, A.-A. et al. Mol. Cell. Proteomics 7, 2151–2175 (2008)). The cytosoluble fractions (S100) were used as the negative control. As the threshold, the enrichment score of (LB queries + 1)/(S100 queries + 1) should be higher than 2. e, A summary of DAVID GO analysis for PXo body proteome. As the threshold, the count of proteins identified in GO terms should be at least three and the P value should be less than 0.0001.

Supplementary Table 2

Lipidomic analysis of purified PXo bodies. a, A full list of the lipids identified in purified PXo bodies under normal (Ctrl) or PFA feeding conditions, along with their classification, peak area, observed mass (ObsMz) and retention time (Rt). b, Consolidated composition of lipid categories identified in PXo bodies, compared with the previous characterization of the lipidome of the larval midgut or adult whole body of flies fed yeast food (Carvalho, M. et al. Mol. Syst. Biol. 8, 600 (2012)). c, IsoSearch analysis of gut PXo bodies purified from flies fed CDF or CDF with 18O labelling of Pi. 18O incorporation causes a shift in molecular weight, resulting in a second peak with a larger mz1 value but the same Rt. Note that IsoSearch only captures a small fraction of isotopic lipids owing to the complexity of the lipidome and the presence of peaks from similar lipid species.

Supplementary Table 3

Affinity purification LC–MS/MS analysis of PXo-interacting proteins. a, A list of GFP-interacting (control) and GFP–PXo-interacting proteins identified by affinity purification LC–MS/MS and the number of detected peptides. b, The input file we used for COMPLEAT complex enrichment analysis. c, A list of the top complexes enriched for proteins preferentially interacting with GFP–PXo, according to COMPLEAT complex enrichment analysis of our affinity purification LC–MS/MS data.

Supplementary Table 4

Fly genotypes used in each figure.

Supplementary Video 1

Midgut FRET ratios are steady in the AHL imaging buffer and decrease immediately after addition of additional Pi. FRET ratio visualization of the midgut expressing FLIPPi in enterocytes.

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Xu, C., Xu, J., Tang, HW. et al. A phosphate-sensing organelle regulates phosphate and tissue homeostasis. Nature 617, 798–806 (2023). https://doi.org/10.1038/s41586-023-06039-y

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