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Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation

Abstract

The plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether the membrane tension of organelles regulates their functions, notably intracellular traffic. The endosomal sorting complexes required for transport (ESCRT)-III complex is the major membrane remodelling complex that drives intra-lumenal-vesicle (ILV) formation on endosomal membranes. Here we used a fluorescent membrane-tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when the membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro and correlates with increased ILV formation in ESCRT-III-decorated endosomes in vivo. Finally, we find that the endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and endosome trafficking, leading us to conclude that membrane tension can control organelle functions.

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Fig. 1: Hypertonic shock triggers fast and transient CHMP4B recruitment on endosomes.
Fig. 2: Specificity for ESCRT-subunit relocalization following hypertonic shock.
Fig. 3: Hypertonic shock decreases the membrane tension of endosomes.
Fig. 4: LLOMe-induced endosomal damage decreases membrane tension and triggers ESCRT recruitment for reparation.
Fig. 5: A decrease in membrane tension increases the CHMP4B polymerization rate in vitro.
Fig. 6: Tension-induced CHMP4B recruitment on endosomes triggers ILV formation.
Fig. 7: Hypertonic shock-induced ESCRT recruitment leads to EGF incorporation into ILVs.

Data availability

Source images of the figures are available at https://doi.org/10.5281/zenodo.3833867. Other data that support the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the ACCESS automated microscopy, high-content and high-throughput screening facility—and in particular D. Moreau—the Bioimaging platforms, the mass spectrometry platform, the nuclear magnetic resonance platform, the bioimaging core facility and the electron microscopy core facility of the University of Geneva for constant support. S.M. acknowledges support from the Swiss National Science Foundation (grant no. 200020 175486). J.G. acknowledges support from the Swiss National Science Foundation (grant no. 31003A_159479) and LipidX from the Swiss SystemsX.ch Initiative. A.R. acknowledges funding from the Human Frontier Science Program (Young Investigator grant no. RGY0076/2009-C), the Swiss National Fund for Research (grant nos 31003A_130520, 31003A_149975 and 31003A_173087) and the European Research Council Consolidator Grant (grant no. 311536).

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Authors

Contributions

The project was designed by V.M., J.G. and A.R. based on the first observations made by G.M. V.M. carried out all of the experiments and analyses. J.L. performed some experiments with V.M., in particular CHMP4B purification. A.G. and S.M. designed and synthesized the Lyso Flipper and FliptR molecules. V.M., J.G. and A.R. wrote the paper, with corrections from all co-authors.

Corresponding authors

Correspondence to Jean Gruenberg or Aurélien Roux.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Hypertonic shock causes relocalization of ESCRT-III to early endosomes (related to Fig. 1).

a, Western blot analysis for several ESCRT and endosomal markers of light-membrane (LM), heavy-membrane (HM) and cytosol (CYTO) fractions in control or hypertonic treated cells (830 mOsm). Full blots are available as source data. b, c, Representative confocal images of Hela cells expressing CHMP4B-GFP before and 10 min after incubation with medium containing 0.25 M NaCl (b) or hypotonic medium (c). Images representative of 2 experiments. d, Fluorescence recovery after photobleaching curves of CHMP4B-GFP fluorescence on endosomes under control and hypertonic conditions (830 mOsm), in Hela CHMP4B-GFP cells transfected or not the dominant-negative mutant of VPS4B (DN VPS4B) were incubated with an isotonic (330 mOsm) or hypertonic (830 mOsm) solution for 5 min. Shaded areas correspond to the SEM. (N values are: CHMP4B = 15; CHMP4B hyper=17; CHMP4B + DN VPS4 = 8; CHMP4B + DN VPS4 hyper=13). e, Immunofluorescence confocal images of Hela MZ cells stained for endogenous CHMP4B after incubated 10 min incubation in isotonic or hypertonic (~830 mOsm). f) Mean intensity of CHMP4B-GFP punctae over time. Shaded areas: mean ± SD (N = 722 punctae from 3 independent replicates). g, Confocal images of Hela CHMP4B-GFP loaded with EGF-Alexa 647 for 15 min (blue, EE) and for 15 min followed by a 2 h chase with EGF-Alexa 594 (red, LE) under control condition (upper panel) or after 10 min hypertonic shock (bottom panel). Images are representative of 3 experiments. h, Confocal images of Hela-CHMP4B-GFP cells after a 10min-hypertonic shock labelled with antibodies against the indicated markers (arrows indicate colocalization). Bar: 5 µm. Images are representative of 3 experiments. i, Quantification of CHMP4B-GFP fluorescence intensity within the Transferrin-A594 mask in the experiment shown in Fig. 1k. Mean±SD: shaded area (N = 100 Transferrin -positives endosomes) j, Number of CHMP4B-GFP punctae with time during a 15 min hypertonic shock (~830 mOsm), followed by a 15 min isotonic recovery (~330 mOsm). Shaded areas: mean ± SD (N = 38 cells from 3 independent replicates).

Source data

Extended Data Fig. 2 Hypertonic shock or LLOMe treatment leads to ESCRT protein relocalization (related to Figs. 12,4).

a, Hela-CHMP4B-GFP cells were incubated for 15 min with 6 nm BSA-Gold and subjected to a hypertonic shock for 10 min. and cells were processed for immune-electron microscopy, as in Fig. 1k. The gallery shows representatives micrographs of endosomes after immuno-gold labelling of cryo-sections with anti-CHMP4B antibodies followed by 10 nm Gold-Protein A. Please note that the size of endosomes treated or not under hypertonic conditions cannot be compared in these micrographs. Indeed, we were unable to obtain nice thin cryo-sections after fixation in a paraformaldehyde-glutaraldehyde mixture containing sucrose (in order to keep osmolarity constant). Thus, we had to use normal fixative to obtain appropriate sections, but some endosome swelling occurred. Images from a single immuno-gold labelling experiment. b, c, Representatives automated confocal images of Hela MZ cells after immunostaining of the indicated ESCRT subunits under the following conditions: (b) untreated and 30 min hypertonic shock, (c) 30 min treatment with LLOMe. Quantifications in Fig. 1l and Fig. 2n were obtained from similar images. Images representative of 3 experiments. Scale bars: 10 μm.

Extended Data Fig. 3 ESCRT protein relocalization after hypertonic or LLOMe treatments (related to Figs. 2,4).

Automated confocal images of Hela cells expressing CHMP4B-GFP (green) after hypertonic shock for 30 min (a) or LLOMe treatment for 30 min (b). Cells were labelled with antibodies against the indicated ESCRT subunits (red). Images representative of 3 experiments. Scale bars: 10 μm.

Extended Data Fig. 4 Role of ALIX and TSG101 in the redistribution of CHMP4B after hypertonic shock or LLOMe treatment (related to Figs. 2,4).

Cells expressing CHMP4B-GFP were transfected with control siRNAs (siVSV) or with siRNAs against ALIX or TSG101, treated with hypertonic medium (0.5 M sucrose) or with 0.5 mM LLOMe for the indicated time, and then imaged by automated confocal microscopy. The micrographs represent CHMP4B-GFP distribution and correspond to the quantification shown in Fig. 2q and Extended Data Fig. 6f. Images are representative of 3 experiments. Bar: 20 μm.

Extended Data Fig. 5 ESCRT are recruited to late endosomes after membrane injury and are necessary for repair (related to Fig. 4).

a, Hela cells were treated for the indicated time with 0.5 mM LLOMe, stained with LysoTracker and imaged by automated confocal microscopy. Images representative of 3 experiments. Scale bars: 10 μm. b, Confocal images of CHMP4B-GFP distribution during a pulse-chase experiment with 0.5 mM LLOMe. Images representative of 3 experiments. Scale bars: 10μm. c, The graph shows the normalized mean fluorescence intensity of LysoTracker and CHMP4B-GFP that illustrates the effect of the treatment of HeLa cells with 0.5 mM LLOMe (See also Supplementary Video 3). Shaded areas correspond to SEM from (N = 26 ROIs in 10 cells from 2 independents movies). d, The graphs show the number of CHMP4B-GFP punctae per cell in cells treated with 0.5 mM LLOMe (upper graph, shaded area represents SD, N = 8 cells), as well as the mean intensity of the punctae (shaded area represents SEM, N = 43 ROIs in 8 cells). e, Hela MZ were transfected with control siRNA (siVSV) or siRNAs against ALIX, TSG101 or both ALIX and TSG101, and treated with 0.5 mM LLOMe for the indicated time. Acidic compartments were revealed using LysoTracker and imaged by automated confocal microscopy. The graph shows the mean number of LysoTracker punctae per cell. (N = 3 independent replicates represented by dots on the graph with mean ± SEM. Analysis by automated microscopy with a total of 72 fields per condition, with 10-40 cells per field.). f, Confocal images of LysoTracker staining before and 10 min after 0.5 mM LLOMe treatment. The data correspond to Fig. 2. Images representative of 4 experiments. Bars: 10 μm.

Source data

Extended Data Fig. 6 Low membrane tension facilitates CHMP4B association with GUVs (related to Fig. 5).

a, Images of a GUV held in the aspiration pipette while purified CHMP4B-A488 is released from the ejection pipette. Binding is clearly visible 2 min after CHMP4B ejection. Bars: 10μm. Images representative of 5 experiments. b, c, This example shows that the force exerted on the bead (measured with optical tweezers) increases with time (b), as does CHMP4B binding on the GUV (c), and thus that the force increase is correlated with CHMP4B binding. d, Images of CHMP4B-A488 binding on the GUV surface when the buffer containing CHMP4B is hypertonic or hypotonic. Bars: 10μm. Images representative of 2 experiments. e, f, Tension was released by a decrease in the aspiration of the tongue with the aspiration pipette. In (e), the upper image shows the CHMP4B fluorescence on the GUV 7 min after the beginning of protein ejection, and the bottom image 7 min after the release of membrane tension (note the disappearance of the tongue). Bars: 10 μm. Images representative of 3 experiments. (f) Quantification of the experiment shown in (e); lines are linear fits. g, Same experiments than in Fig. 5c but with Snf7 instead of CHMP4B (at the same concentration). Mean±SEM (N = 13 GUVs). h, GUVs were incubated for 30 min with 1 µm Snf7 in isotonic buffer, and then the solution was replaced with exactly the same solution (+SNF7). Then, at 56 min, the solution was replaced again but with hypertonic buffer also containing 1 µm Snf7 (+SNF7 Hyper, blue part of the graph). Mean±SEM (N = 20 GUVs). i, The upper kymograph shows GUVs containing low (125 mM) sucrose, which were then incubated with CHMP4B under hypertonic conditions in the presence of 250 mOsm sucrose; N = 44 GUVs. The lower kymograph shows GUVs containing high (500 mM) sucrose, which were incubated with CHMP4B under isotonic conditions in the same buffer (500 mM sucrose); N = 54 GUVs. The mean intensity of CHMP4B-Alexa 488 on the bilayer over time is shown in the right panel; binding rate is 1/τ = 0.07837 for 125 mM sucrose, and 1/τ = 0.03522 for high sucrose, as indicated. Mean±SEM. j, Volumes of single GUV before (black) and after a 10 min hypertonic shock (red) (N = 20 GUVs, bars represent the mean).

Source data

Extended Data Fig. 7 Low membrane tension promotes CHMP4B polymerization on LUV membranes (related to Fig. 5).

a, b, Cryo-electron micrographs of LUVs incubated for 2 h with 1 μM CHMP4B-GFP in hypotonic (a) or hypertonic (b) solution. Bars: 100 nm. Images are representative of 2 experiments. c, d, Electron micrographs after negative staining of LUVs incubated for 2 h with 1 μM CHMP4B-GFP in hypotonic (c) or hypertonic (d) solution. Bars: 100 nm. Images are representative of 2 experiments.

Extended Data Fig. 8 EGF triggers endosomal membrane tension decrease in vivo (related to Fig. 6).

a, b, As in Fig. 4, the panel (a) shows FIB-SEM micrographs of cells loaded with BSA-gold. The electron density of early endosomes (EE) or late endosomes (LE) labelled with BSA-gold was measured before (blue) and after (red) a 10 min hypertonic shock (b). Mean±SD (N are respectively EE control:121; EE Hyper: 124; LE control:120; LE Hyper:98 from 2 independent replicates, Kruskal–Wallis test: P < 1.10-15). c, Lyso Flipper lifetime measurements on RAB5Q79L endosomes before and 10 min after 200 ng/ml EGF treatment. Thin lines: independent images from 4 experiments, thick red line: mean ± SEM, two-tailed paired t-test P = 0.0000025902. d, Lyso Flipper lifetime measurements in MDA cells before and 20 min after 200 ng/ml EGF treatment. Thin lines: independent images from 5 experiments, thick red line: mean ± SEM, two-tailed paired t-test P = 0.0000496474.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Hypertonic shock triggers fast relocalization of CHMP4B to endosomes. Confocal time-lapse movie recorded at speed of 1 frame per 10 s of CHMP4B–GFP during hypertonic treatment. Images are representative of five experiments (related to Fig. 1).

Supplementary Video 2

Hypertonic shock triggers transient relocalization of CHMP4B to endosomes. Confocal time-lapse movie recorded at a speed of 1 frame per 5 min of CHMP4B–GFP during hypertonic treatment. This movie corresponds to the time series shown in Fig. 1a. Images are representative of three experiments (related to Fig. 1).

Supplementary Video 3

LLOMe triggers massive relocalization of CHMP4B. Confocal time-lapse movie recorded at a speed of 1 frame per 30 s of CHMP4B–GFP during 0.5 mM LLOMe treatment. Images are representative of five experiments (related to Fig. 4).

Supplementary Video 4

LLOMe triggers loss of endosomal acidity and quick relocalization of CHMP4B to endosomes. Confocal time-lapse movie recorded at speed of 1 frame per 2 min of CHMP4B–GFP cells stained with LysoTracker during 0.5 mM LLOMe treatment. Images are representative of two experiments (related to Fig. 4).

Supplementary Video 5

A decrease in membrane tension trigger by hypertonic buffer increases CHMP4B polymerization rate in vitro. Confocal time-lapse movie recorded at speed of 1 frame min−1 of CHMP4B–A488 on GUVs during isotonic, followed by hypertonic incubation. Images are representative of six experiments (related to Fig. 5).

Supplementary Video 6

A decrease in membrane tension trigger by hypertonic buffer increases CHMP4B polymerization on LBPA-containing GUVs. Confocal time-lapse movie recorded at speed of 1 frame min−1 of CHMP4B–A488 on DOPC:DOPE:LiverPI:LBPA GUVs during isotonic followed by hypertonic incubation. Images are representative of four experiments (related to Fig. 5).

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Mercier, V., Larios, J., Molinard, G. et al. Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation. Nat Cell Biol 22, 947–959 (2020). https://doi.org/10.1038/s41556-020-0546-4

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