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A phosphoinositide conversion mechanism for exit from endosomes

Nature volume 529, pages 408–412 (2016)Cite this article

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Abstract

Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity1,2. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles3, and on lysosomes4, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P)5, are a hallmark of the endosomal system1,2. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans6. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

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Figure 1: Impaired exocytosis from endosomes in cells from patients with XLCNM.
Figure 2: Tf exocytosis from endosomes requires MTM1-mediated PI(3)P hydrolysis.
Figure 3: Endosomal accumulation of PI(3)P and PI(3)P effector proteins inhibits exocytosis from endosomes.
Figure 4: PI(3)P to PI(4)P conversion is required for exocyst-dependent endosomal exocytosis.

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Acknowledgements

We thank L. Pluska for initial studies on PI4K2α-exocyst, I. Ganley for fluorescently labelled p40-Phox, M. Ringling, M. Mühlbauer, L. von Oertzen, and S. Zillmann for technical assistance, and A. Marat for comments. This work was supported by grants from the German Research Foundation (SFB740/C8 to V.H. and SFB958/A11 to M.K.) and Agence Nationale de la Recherche (ANR-14-CE12-0009/13-BSV2-0004 to J.L.).

Author information

Authors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Berlin, 13125, Germany

    Katharina Ketel, Michael Krauss, Dmytro Puchkov & Volker Haucke

  2. Department of Translational Medicine and Neurogenetics, Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, Strasbourg University, Illkirch, 67404, France

    Anne-Sophie Nicot & Jocelyn Laporte

  3. Freie Universität Berlin, Faculty of Biology, Chemistry and Pharmacy, Berlin, 14195, Germany

    Marnix Wieffer & Volker Haucke

  4. European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, 69117, Heidelberg, Germany

    Rainer Müller, Devaraj Subramanian & Carsten Schultz

  5. NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, 10117, Germany

    Volker Haucke

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Contributions

K.K., D.P., M.K. and A.-S.N. performed experiments; R.M., D.S., and J.L. contributed reagents; K.K., M.W., M.K., C.S., and V.H. designed research. K.K. and V.H. wrote the manuscript.

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Correspondence to Volker Haucke.

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Extended data figures and tables

Extended Data Figure 1 Characterization of XLCNM patient phenotype.

a, HeLa cells treated with scrambled (scr) siRNA or depleted of MTM1 (MTM1-KD) expressing eGFP–Rab (Rab5, Rab8) or labelled for the indicated protein were co-labelled using β1-integrin-specific antibodies. Co-localization of β1-integrin with late endosomal Rab7 and recycling endosomal TfR in MTM1-KD cells is indicated by arrowheads. Scale bar, 10 μm; magnified insets, 1 μm. Representative images from one of two independent experiments (for TfR) or from one experiment with 10–20 images per condition are shown. b, Quantitative analysis of TfR levels per cell area of XLCNM H31 cells and healthy controls (n = 3). c, Impaired Tf exocytosis from endosomes in XLCNM G92-628 cells (patient 2) monitored by TIRF microscopy. Quantified are Tf exocytic events normalized to cell area. n, number of independent experiments with 15–30 images (b) or 5 videos (c) analysed per condition per experiment. Mean ± s.e.m.; **P < 0.01, unpaired two-tailed t-test; NS, non-significant.

Extended Data Figure 2 Transferrin endosomes in MTM1-depeleted cells.

a, Efficient siRNA-mediated depletion of MTM1 from HeLa cells as demonstrated by immunoblot analysis. Lysates of HeLa cells expressing HA–MTM1 were taken as a control to assure MTM1 antibody specificity. For blot source data, see Supplementary Fig. 1. b–d, Time course of Tf internalization was measured radioactively and the ratio between Tf uptake and surface Tf levels over time was quantified in HeLa cells depleted of MTM1 (MTM1-KD) compared with scrambled (scr) siRNA-treated controls. While Tf endocytic rates remained unchanged in MTM1-KD cells (c, d), Tf uptake and surface level were significantly decreased in MTM1-depleted cells (b), indicative of defective Tf recycling upon loss of MTM1. b–d, Mean ± s.e.m., n = 4 independent experiments, each experiment done in technical triplicates; NS, non-significant, *P < 0.05, unpaired, two tailed t-test. e, f, Quantitative analysis of TfR levels by confocal imaging (TfR levels area; e) or flow cytometry (TfR levels per cell; f) of (scr) control or MTM1-KD cells (mean ± s.e.m., n = 3 independent experiments; NS, non-significant, unpaired two-tailed t-test). g, h, Impaired Tf exocytosis monitored by TIRF microscopy in MTM1-depleted HeLa cells is restored by re-expression of eGFP–MTM1 WT, but not inactive eGFP–MTM1 (C375S) or eGFP. g, Snapshot of a single exocytic event in control cells with characteristic time course of appearance, brightening, and spreading of the fluorescent signal. Scale bar, 400 nm. h, Kymographs of Tf fluorescence signal over 28 μm. Arrowheads indicate time of plasma membrane fusion. i, Surface TfR labelled with Tf-Alexa647 (at 4 °C) does not co-localize with intracellular TfR in control or MTM1-depleted HeLa cells. Scale bar, 2 μm. j, Maximum intensity projections of z-stacks acquired by confocal imaging confirm the perinuclear accumulation of Tf-ligand and TfR in controls and accumulation at the cell periphery in MTM1-depleted cells as shown in Fig. 2c. Scale bar, 5 μm. k, Electron micrographs of MTM1-depleted HeLa cells treated with Tf-HRP. Scale bar, 200 nm. l, Accumulation of Tf-HRP positive endosomes in MTM1-depleted cells (mean ± s.e.m., n = 10 cells, *P < 0.05, one-sample t-test), m, while size of TfR-HRP positive endosomes remained unchanged (mean ± s.e.m., n = 52 (scr) and 90 (MTM1-KD) endosomes from 10 cells; NS, non-significant, one-sample t-test). g–k, Representative images from one of three (g, h) or two (i) independent experiments or from one experiment with ten images (j) or ten cells (k) per condition are shown.

Extended Data Figure 3 Endosomal trafficking in MTM1-depleted cells and effects of depletion of other MTM family members.

a, EGF degradation monitored over 120 min in HeLa cells using radioactive 125I-labelled EGF was unaltered upon loss of MTM1 (MTM1-KD) compared with scrambled (scr) treated controls (mean ± s.e.m., n = 3 independent experiments, each experiment done in technical triplicates). b, Quantitative analysis of EGFR levels normalized to cell area of (scr) control or MTM1-KD cells (EGFR per unit area; n = 3). c, Secretion of GFP–reporter construct from the Golgi complex is not affected in MTM1-depleted C1 cells. Fifteen minutes after initiating secretion, GFP is enriched in the Golgi complex. GFP levels were normalized to cell number and Golgi content (n = 3). d, LC3B level remain unchanged in HeLa cells depleted of MTM1 compared with (scr) control cells (LC3B per unit area; n = 3). e, Autophagy can be induced in HeLa cells depleted of MTM1 by starvation and BafA1 treatment, monitored by LC3B-specific antibody labelling (scale bar, 10 μm). f, g, Internalized cholera toxin (Ctx) is retrogradely transported to the Golgi complex in HeLa cells treated with scrambled (scr) siRNA or depleted of MTM1 (MTM1-KD). f, Quantified are Pearson’s coefficients between internalized Ctx-CF568 and the Golgi complex, assessed by GM130-specific antibody labelling (n = 3). g, Scale bar, 10 μm; magnified insets, 1 μm. h, Cholera toxin uptake quantified as the percentage of cells with internalized Ctx is unchanged upon loss of MTM1 (n = 3). i, Efficient siRNA-mediated depletion of eGFP-tagged catalytically active myotubularin-related (MTMR) proteins (MTMR1, MTMR2, MTMR4, MTMR7) from HeLa cells as demonstrated by immunoblot analysis. For blot source data, see Supplementary Fig. 1. j, Depletion of MTMR1, but not MTMR2, MTMR4, or MTMR7, phenocopied TfR mislocalization observed in MTM1-depleted HeLa cells (n = 3). k, Inhibition of Tf exocytosis in cells depleted of MTMR1, but not MTMR2, MTMR4, or MTMR7, compared with (scr) controls (Tf exocytic events per unit area; n = 3). n, number of independent experiments with 15–30 images (b–d, f, h, j) or five videos (k) analysed per condition per experiment. Mean ± s.e.m.; NS, non-significant, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. e, g, Representative images from one of two (e) or three (g) independent experiments are shown.

Extended Data Figure 4 Endosomal compartments in MTM1-depleted cells.

a, TfR-containing endosomal tubules accumulate in HeLa cells depleted of MTM1. Scale bar, 10 μm; magnified insets, 2 μm. b, c, HeLa cells depleted of MTM1 were labelled for the indicated protein by specific antibodies. EEA1 localizes to sub-plasma-membrane TfR accumulations, distinct from sites containing the endocytic adaptor AP-2 or the retromer component Vps26. APPL1 and LAMP1 did not localize to TfR accumulations in MTM1-depleted cells. No changes in the GM130-containing cis-Golgi compartment were observed. c, Quantification of co-localization between the indicated protein and TfR in the cell periphery calculated using Pearson’s correlation coefficient (EEA1 and VPS26: n = 4; AP2 and LAMP1: n = 3). d, Distribution of Rab proteins in HeLa cells treated with scrambled (scr) siRNA or depleted of MTM1 (MTM1-KD). Confocal images of HeLa cells expressing eGFP–Rabs (Rab4, Rab5, Rab11, Rab14, Rab35). Note the strong accumulation of early and recycling endosomal Rabs on sub-plasma-membrane TfR-containing endosomes in MTM1-KD cells. e, Quantification of co-localization between the indicated Rab protein and TfR in the cell periphery calculated using Pearson’s correlation coefficient (Rab5 and Rab11: n = 4; Rab4 and Rab14: n = 3). f, g, Distinct early and recycling endosomal compartments in MTM1-depleted cells. f, HeLa cells treated with scrambled siRNA or depleted of MTM1 and co-expressing eGFP–Rabs (Rab4, Rab11, Rab14). Rab5 was endogenously labelled using a Rab5-specific antibody. g, Pearson’s correlation coefficients between eGFP–Rabs and endogenous Rab5 in the cell periphery (n = 3). n, number of independent experiments with 15–30 images analysed per condition per experiment. Mean ± s.e.m., unpaired two-tailed t-test. a, b, d, f, Representative images from 1 of 3 (a, b: for AP2, LAMP1, APPL1, GM130; d: for Rab4, Rab14, f) or 4 (b: for EEA1, VPS26; d: for Rab5, Rab11) independent experiments or from 1 experiment with 15 images per condition (d: Rab35) are shown. b, d, f, Scale bar, 10 μm; magnified insets, 1 μm.

Extended Data Figure 5 Endosomal accumulation of PI(3)P-binding sorting nexin (SNX) proteins upon loss of MTM1 and localization of MTM1 to PI(3)P-containing endosomes.

a, Distribution of SNX proteins in HeLa cells treated with scrambled (scr) siRNA or depleted of MTM1 (MTM1-KD). Accumulation of PI(3)P-binding SNXs (eGFP-tagged SNX1, SNX3, SNX4, SNX8, SNX17, SNX27) but not eGFP-tagged SNX15 on sub-plasma-membrane TfR endosomes in MTM1-KD cells. Scale bar, 10 μm; magnified insets, 1 μm. b, Quantification of co-localization between the indicated SNX protein and TfR in the cell periphery calculated using Pearson’s correlation coefficient. Mean ± s.e.m., n = 3 independent experiments, unpaired two-tailed t-test. Numbers of images analysed: experiment 1 (scr: SNX4 n = 13, SNX15 n = 11, SNX17 n = 25; MTM1-KD: SNX4 n = 14, SNX15 n = 10, SNX17 n = 22); experiment 2 (scr: SNX4 n = 15, SNX15 n = 14, SNX17 n = 15; MTM1-KD: SNX4 n = 15, SNX15 n = 15, SNX17 n = 14); experiment 3 (scr: SNX4 n = 15, SNX15 n = 15, SNX17 n = 15; MTM1-KD: SNX4 n = 9, SNX15 n = 20, SNX17 n = 20). c, HeLa cells co-expressing eGFP–Rabs and mCherry–MTM1. Co-localization of MTM1 with early and recycling endosomal Rabs (Rab4, Rab5, Rab11, Rab14) is indicated by arrowheads. Scale bar, 2 μm. d, HeLa cells expressing eGFP–MTM1 were labelled for the indicated endosomal markers by specific antibodies. Arrowheads mark co-localization of MTM1 with the indicated proteins, whereas arrows mark MTM1-positive endosomes devoid of the indicated protein. Scale bar, 2 μm. e, Binding of HA-tagged MTM1 expressed in Hek293 cells to liposomes containing 5 mol% of the indicated phosphoinositide in flotation assays. MTM1 predominantly binds to PI(3)P-containing liposomes. Input, 30% (top) for bound and 15% (bottom) for unbound fractions. For blot source data, see Supplementary Fig. 1. a, c, d, Representative images from 1 of 3 (a, for SNX4, SNX15, SNX17) independent experiments or from 1 experiment with 6–20 images per condition (a, for SNX3, SNX8, SNX27, c, d) are shown.

Extended Data Figure 6 Sub-plasma-membrane endosomal TfR accumulations are selectively enriched in PI(3)P.

a, HeLa cells were labelled for PI(3)P using different eGFP–2xFYVE domain concentrations, starting with 2.25 μg ml−1 (1) and subsequent 1:3 dilutions of (1) (0.75 μg ml−1 (1:3), 0.25 μg ml−1 (1:9), 0.083 (1:27)). PI(3)P levels per cell area were quantified. The titration curve is shown and the linear range indicated by the blue box. b, HeLa cells treated with DMSO or 2 μm wortmannin were stained for PI(3)P using purified eGFP–2xFYVE. Note the absence of eGFP–2xFYVE staining in cells treated with the broad-spectrum phosphoinositide 3-kinase inhibitor wortmannin. c, PIPx stainings in MTM1-depleted HeLa cells: eGFP–2xFYVE was used to detect PI(3)P. PI(4)P, PI(3,4)P2 and PI(4,5)P2 were detected with specific antibodies against the corresponding PIPx species. Sub-plasma-membrane TfR accumulations are selectively enriched in PI(3)P. d, Decreased PI(3)P levels in HeLa cells overexpressing WT but not mutant inactive (C375S) mCherry–MTM1. Depletion of PI(3)P is consistent with PI(3)P 3-phosphatase activity of MTM1. Cells were labelled for PI(3)P using eGFP–2xFYVE domain or Phox-AF488 as indicated and the relative levels of PI(3)P normalized to cell area were quantified (n = 3). e, Increase of PI(3)P in MTM1-depleted HeLa cells. Cells were labelled for PI(3)P using Phox-AF488 and the relative levels of PI(3)P normalized to cell area were quantified (n = 3). f, HeLa cells were labelled for PI(3)P using eGFP–2xFYVE domain and Phox-AF488 as indicated. g, Efficient silencing of SNX4 and SNX17 in HeLa cells as demonstrated by immunoblotting. h, Co-depletion of MTM1 and SNX4 or SNX17 reveals additional pools of PI(3)P inaccessible in cells only depleted of MTM1. Relative levels of PI(3)P in cells treated with the indicated siRNAs (n = 3). i, Efficient co-depletion of Vps34 and MTM1 in HeLa cells as demonstrated by immunoblotting. j, MTM1-KD cells or (scr) controls were treated with VPS34-IN1 for the indicated times and labelled for β1-integrin. Pharmacological inhibition of Vps34 rescues β1-integrin accumulation in MTM1-depleted HeLa cells within 60 min, assessed by β1-integrin-specific antibody labelling. n, number of independent experiments with 15–30 images analysed per condition per experiment. Mean ± s.e.m., *P < 0.05, **P < 0.01, unpaired two-tailed t-test. b, c, f, j, Representative images from 1 of 3 (c) independent experiments or from 1 experiment with 10–15 images per condition (b, f, j) are shown. b, c, f, j, Scale bar, 10 μm; magnified insets, 1 μm. For blot source data, see Supplementary Fig. 1.

Extended Data Figure 7 PI(3)P manipulations.

a, Co-depletion of MTM1 and Kif16b does not restore defective Tf exocytosis in MTM1-depleted cells (Tf exocytic events per unit area; n = 3). b, Co-depletion of MTM1 and Kif16b restores perinuclear TfR localization. Normalized fraction of HeLa cells with perinuclear TfR in cells treated with the indicated siRNAs (n = 3). c, Efficient co-depletion of PIKfyve and MTM1 in HeLa cells as demonstrated by immunoblotting. For blot source data, see Supplementary Fig. 1. d, Co-depletion of MTM1 and PIKfyve does not restore defective Tf exocytosis in MTM1-depleted cells (Tf exocytic events per unit area; n = 3). e, Co-depletion of PIKfyve and MTM1 is unable to restore perinuclear TfR localization. Normalized fraction of cells with perinuclear TfR in cells treated with the indicated siRNAs (n = 3). f, Co-depletion of PIKfyve and MTM1 leads to increased EEA1 density (n = 3). g, Swelling of EEA1-positive endosomes in PIKfyve-depleted and MTM1/PIKfyve co-depleted cells. Scale bar, 10 μm; magnified insets, 1 μm. Representative images from one of three independent experiments are shown. n, number of independent experiments with 15–30 images (b, e, f) or 5 videos (a, d) analysed per condition per experiment. Mean ± s.e.m.; NS, non-significant, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test.

Extended Data Figure 8 Rab11a in endosomal exocytosis of Tf.

a, Tf exocytosis was monitored by dual-colour TIRF microscopy in HeLa cells expressing GFP–Rab5a or GFP–Rab11a. Snapshots of representative Tf-positive, Rab-containing endosomes are shown. Scale bar, 400 nm. b, Efficient siRNA-mediated depletion of Rab11a from HeLa cells as demonstrated by immunoblot analysis. c, TfR mislocalizes upon loss of Rab11a in HeLa cells. Quantified was normalized fraction of cells with perinuclear TfR (n = 3). d, Impaired Tf exocytosis monitored by TIRF microscopy in Rab11a-depleted HeLa cells (Tf exocytic events per unit area; n = 3). e, Reduced endosomal exocyst association in Rab11a-depleted HeLa cells revealed by confocal imaging using Exo70-specific antibody labelling (n = 3). f, Scale bar, 10 μm; magnified insets, 1 μm. Representative images from one of three independent experiments are shown. g, Tf-Alexa647 (red) exocytosis from endosomes in HeLa cells overexpressing the PI(4)P reporter eGFP–2xPH-FAPP1 (green) analysed by dual-colour TIRF microscopy. Order of acquisition was reversed compared with Fig. 4a to exclude imaging artefacts. The 647 nm channel was imaged before acquisition of the 488 nm channel. Scale bar, 400 nm. n, number of independent experiments with 15–30 images (c, e) or 5 videos (d) analysed per condition per experiment. Mean ± s.e.m., **P < 0.01, unpaired two-tailed t-test. a, g, Representative images of one experiment with ten videos per condition (a) or two independent experiments with ten videos per condition per experiment (g) are shown.

Extended Data Figure 9 PI4K2α and exocyst associate and are required for endosomal exocytosis of Tf.

a, PI4K2α interacts directly with Sec6 and MTM1. Pull down assays were performed using GST-MTM1 or GST-Sec6 as bait to pull down HA-tagged PI4K2α from Cos-1 cells and detected using PI4K2α-specific antibody. The PI4K2α-specific band is indicated by the arrow. b, Co-immunoprecipitation of PI4K2α and exocyst components from stable Hek293. c, Reciprocal interaction between MTM1 and Sec6. Pull down assays were performed using GST-MTM1 as bait to pull down B10-tagged Sec6 or using GST-Sec6 to pull down B10-tagged MTM1 from Cos-1 cells and detected using B10-specific antibodies as indicated by arrows. d, e, Efficient depletion of PI4K2α (d) and exocyst subunits Exo70, Sec3, and Sec6 (e) as demonstrated by immunoblotting of HeLa cell lysates. f, Depletion of exocyst or PI4K2α from HeLa cells impairs Tf exocytosis monitored by TIRF microscopy. Kymographs of Tf fluorescence signal over 28 μm; arrowheads indicate time of fusion. Representative images from one of three independent experiments are shown. g, Depletion of PI4K2α or exocyst from HeLa cells causes TfR mislocalization. Normalized fraction of cells with perinuclear TfR was quantified (n = 4). h, Co-depletion of MTM1 and PI4K2α does not restore perinuclear TfR localization in MTM1-depleted HeLa cells. Normalized fraction of cells with perinuclear TfR in cells treated with the indicated siRNAs (n = 3). i, Co-depletion of MTM1 and PI4K2α does not restore defective Tf exocytosis in MTM1-depleted HeLa cells (Tf exocytic events per unit area; n = 3). n, number of independent experiments with 15–30 images (g, h) or 5 videos (i) analysed per condition per experiment. Mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. For blot source data, see Supplementary Fig. 1.

Extended Data Figure 10 Exocyst and MTM1 recruitment to endosomes depends on PI4K2α and initiates MTM1-dependent PI(3)P-dephosphorylation.

a, Depletion of PI4K2α but not of MTM1 impairs membrane recruitment of Exo70, Sec8, Sec3, and MTM1. Representative immunoblot is shown. Asterisk indicates non-specific band recognized by PI4K2α antibodies. b, Efficient membrane–cytosol fractionation verified by quantifying the membrane/total protein ratio of the palmitoylated membrane protein gadkin and cytosolic actin (mean ± s.e.m., n = 5 independent experiments). c, Depletion of PI4K2α but not of MTM1 in HeLa cells leads to decreased Exo70 level. Scale bar, 10 μm; magnified insets, 1 μm. d, Reduced endosomal exocyst association in PI4K2α-depleted cells is rescued by membrane-permeant PI(4)P but not PI(3)P revealed by confocal imaging using Exo70-specific antibody labelling (n = 4). e, Expression of mutant inactive (D308A) but not WT PI4K2α in HeLa cells reduces endosomal exocyst association revealed by confocal imaging using Exo70-specific antibody labelling (n = 3). f, PI4K2α-dependent recruitment of MTM1 is independent of its kinase activity. Expression of WT or inactive mutant (D308A) eGFP–PI4K2α recruits mCherry–MTM1 to Rab5 Q79L endosomes. Quantified are Pearson’s coefficients between mCherry–MTM1 and HA–Rab5 Q79L (n = 4). g, HeLa cells co-expressing WT or inactive, mutant (C375S) MTM1, PI4K2α, and Rab5 Q79L were labelled for PI(3)P using eGFP–2xFYVE. Inactive MTM1 localizes to PI(3)P-positive early endosomes, while this PI(3)P pool is lost upon recruitment of active MTM1. Scale bar, 1 μm. h, PI(3)P and PI(4)P localize to distinct subdomains on Rab5 Q79L endosomes labelled using eGFP–2xFYVE and a PI(4)P-specific antibody respectively. PIP localizations are indicated by arrowheads (blue: PI(3)P; red: PI(4)P; yellow: co-localization of PI(3)P and PI(4)P). Scale bar, 1 μm. i, Increased PI(4)P levels in HeLa cells overexpressing eGFP–PI4K2α WT compared with mock transfected cells. Increase of PI(4)P is consistent with phosphoinositide 4-kinase activity of PI4K2α. Shown are the relative levels of PI(4)P normalized to cell area (n = 3). j, Increase of PI(3)P in PI4K2α-depleted HeLa cells. Cells were labelled for PI(3)P using Phox-AF488 and the relative levels of PI(3)P normalized to cell area quantified (n = 3). k, Recruitment of eGFP–SNX3 and eGFP–SNX27 to dispersed endosomes in PI4K2α-depleted HeLa cells. Scale bar, 2 μm. l, eGFP–PI4K2α expressed in MTM1-depleted HeLa cells localizes to TfR-positive endosomes, assessed by TfR-specific antibody labelling and indicated by arrows. Scale bar, 10 μm; magnified insets, 1 μm. n, number of independent experiments with 15–30 images (d, e, f, i, j) analysed per condition per experiment. Mean ± s.e.m.; NS, non-significant, *P < 0.05, ***P < 0.001, unpaired two-tailed t-test. c, g, h, k, l, Representative images from 1 of 4 (c, l) independent experiments or from 1 experiment with 10–20 images per condition (g, h, k) are shown.

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Ketel, K., Krauss, M., Nicot, AS. et al. A phosphoinositide conversion mechanism for exit from endosomes. Nature 529, 408–412 (2016). https://doi.org/10.1038/nature16516

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