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Assembly of Tetraspanin-enriched macrodomains contains membrane damage to facilitate repair

Abstract

Various mechanisms contribute to membrane repair1,2,3,4,5,6,7,8 but the machinery that mediates the repair of large wounds on the plasma membrane is less clear. We found that shortly after membrane damage, Tetraspanin-enriched macrodomains are assembled around the damage site. Tetraspanin-enriched macrodomains are in the liquid-ordered phase and form a rigid ring around the damaged site. This restricts the spread of the damage and prevents membrane disintegration, thus facilitating membrane repair by other mechanisms. Functionally, Tetraspanin 4 helps cells mitigate damage caused by laser, detergent, pyroptosis and natural killer cells. We propose that assembly of Tetraspanin-enriched macrodomains creates a physical barrier to contain membrane damage.

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Fig. 1: High concentrations of Tspan4 proteins are recruited to the sites of cellular membrane damage.
Fig. 2: Enrichment of Tspan4 restricts the expansion of membrane damage.
Fig. 3: Tspan4-enriched microdomains increase the resistance of membranes.
Fig. 4: Tspan4 increases cell viability following membrane damage.

Data availability

All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Z. Dong laboratory (Tsinghua University) for the generous gift of YTS cells and the F. Shao laboratory (NIBS) for their help with the pyroptosis-related work. We thank all of the members of L.Y.’s group for their helpful discussions. This research was supported by the Ministry of Science and Technology of the People’s Republic of China (grant no. 2017YFA0503404 to L.Y.) and the National Natural Science Foundation of China (grant nos 32070691 to Y.H. and 92054301 to L.Y.).

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Authors and Affiliations

Authors

Contributions

L.Y. and Y.H. conceived the experiments. L.Y. wrote the paper and supervised the project. Y.H. and X.Z. carried out the experiments. All authors discussed the manuscript, commented on the project and contributed to preparing the paper.

Corresponding author

Correspondence to Li Yu.

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The authors declare no competing interests.

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Nature Cell Biology thanks Harald Stenmark, Martin Hemler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Tspan4 proteins are highly recruited to sites of cellular membrane damage.

a L929 Tspan4–mCherry and MGC803 Tspan4–GFP cells were subjected to photodamage treatment and Tspan4 distribution at the damage site was observed by confocal microscopy. Scale bar, 5 µm. b NRK cells co-expressing Tspan4–GFP and Na/K ATPase-mCherry were photodamaged, then time-lapse images were collected by confocal microscopy. Scale bar, 5 µm. c After cells were photodamaged, PI was added into the system at different time points (0, 1, 2 and 2.5 min) and the PI intensity was traced. Representative images are showed. Green, Tspan4–GFP; red, PI. Scale bar, 5 µm. d Statistical analysis of the percentage of cells with PI entry at each time point was conducted. Mean±s.e.m., n = 12 cells for the 0 min group, n = 22 for the 1 min group, n = 24 cells for the 2 min group and n = 50 cells for the 2.5 min group from 3 independent experiments. Experiments were independently performed 3 times (a-c), and representative images are shown.

Source data

Extended Data Fig. 2 ESCRT III activity is not required for recruitment of Tspan4 to damage sites.

a NRK cells co-expressing Tspan4–GFP and Chmp4b–mCherry were subjected to detergent (25 µM digitonin) treatment. Z-projection images were collected by 3D-SIM. Yellow, Tspan4; cyan, Chmp4b–mCherry. Scale bar, 5 µm. b Tspan4–GFP overexpressing cells treated with DMSO (as control) or 10 µM BAPTA-AM were subjected to photodamage. Time-lapse images were collected by confocal microscopy. Scale bar, 5 µm. c Localization of Tspan4 and Chmp3 at the damage site was analysed by confocal microscopy after photodamage in cells co-expressing Tspan4–GFP and Chmp3–mCherry or Chmp3 1-179-mCherry. Scale bar, 10 µm. d Verification of the NRK Chmp4b knockout cell line by PCR. Experiments were independently performed 3 times (a-d), and representative images are shown.

Source data

Extended Data Fig. 3 Cholesterol depletion increases the PI influx of cells after photodamage treatment.

a-c. Tspan4–GFP NRK cells under different cholesterol depletion conditions, including 10% full cholesterol medium (FBS), cholesterol depletion medium (LPDS) and cholesterol depletion medium with 30 μM Pravastatin, were observed by confocal microscopy before and after photodamage. PI influx was used to indicate cell death. Scale bar, 5 µm. Experiments were performed 3 times, and representative images are shown. d. Statistical analysis of the PI intensity of cells under different cholesterol depletion conditions before and after photodamage treatment. Mean±s.e.m., n = 17 cells for FBS, 15 cells for LPDS and 15 cells for LPDS + Prava from 3 independent experiments.

Source data

Extended Data Fig. 4 Tspan4 proteins restrict damage expansion in vitro.

a Time-lapse images of Tspan4–GFP GPMVs or GFP-CAAX (as control) GPMVs in the in vitro collapse assay. Green, GFP; red, Rho-PE; yellow, merge. Scale bar, 5 µm. b Diagram of the method used to measure fluorescence enrichment. c Images were collected by confocal microscopy after Tspan4-OE GPMVs, NRK GPMVs and NRK Tspan4-KO GPMVs were used in the in vitro collapse assay. Scale bar, 10 µm; zoom in, 2 µm. d Time-lapse imaging of rupture events in Tspan4–GFP GPMVs after detergent treatment. Yellow arrow, rupture site. Scale bar, 5 µm. Experiments were independently performed 3 times (a and c-d), and representative images are shown.

Extended Data Fig. 5 Tspan4 and Tspan5 enhance cell survival following membrane damage.

a Cell death was analysed by PI permeability in NRK Tspan4-OE, NRK WT and NRK Tspan4-KO cells after detergent treatment. Cells were imaged by confocal microscopy. Scale bar, 10 µm. b Statistical analysis of absolute PI intensity in NRK Tspan4-OE, NRK WT and NRK Tspan4-KO cells after detergent treatment. Mean±s.e.m., n = 29 cells for Tspan4-OE, 42 cells for WT and 33 cells for Tspan4-KO from 3 independent experiments. c Verification of the Tspan4-KO Tspan5 knockout cell line by PCR. d Tspan4-OE (Tspan4-overexpression), WT, and Tspan4/Tspan5-KO cells pre-stained by FM 1-43 (5 μM) were subjected to photodamage treatment and the FM 1-43 signal was monitored. Time-lapse images were collected by confocal microscopy and displayed as heatmap images. Scale bar, 10 µm. e Statistical analysis of FM 1-43 fluorescence intensity of the total cell substrate during and after photodamage treatment. Mean±s.e.m. n = 19 cells for Tspan4-OE, 30 cells for WT, 32 cells for Tspan4/Tspan5-KO, 17 cells for Tspan4-OE no-damage, 21 cells for WT no-damage and 9 cells for Tspan4/Tspan5-KO no-damage from 3 independent experiments. Statistical analysis of cell viability in each group (NRK Tspan4-OE, NRK WT, NRK Tspan4-KO, NRK Tspan4/Tspan5 double-knockout cells) after PBS or LPS electroporation treatment. Mean±s.e.m., n = 4 independent experiments. Unpaired t-test was used.

Source data

Extended Data Fig. 6 Tspan4 protects cancer cells from killing by NK cells.

Killing of MGC803 Tspan4–BFP cells (a), MGC803 WT cells (b), or MGC803 Tspan4-KO cells (c) by YTS NK cells was observed by confocal microscopy. Cell death was indicated by PI influx. Yellow, BFP; cyan, CD56–APC; red, PI. Scale bar, 10 µm. Experiments were performed 5 times (a-c), and representative images are shown.

Supplementary information

Supplementary Information

Supplementary figure with legends.

Reporting Summary

Supplementary Video 1

A Tspan4–GFP-expressing cell during and after photodamage treatment. Video of a Tspan4–GFP-expressing NRK cell during and after photodamage was conducted by confocal microscopy at a speed of 1 frame per 2 s. Green, Tspan4–GFP. The yellow circle in the second frame indicates the exact time and area of laser damage. Yellow arrow, damage site. Video display rate, 20 f.p.s. Scale bar, 5 µm.

Supplementary Video 2

A Tspan4–GFP-expressing cell during and after photodamage treatment in the presence of PI. Video of a Tspan4–GFP-expressing NRK cell in the presence of PI to indicate membrane permeability during and after photodamage. Imaging was conducted by confocal microscopy at a speed of 1 frame per 4 s. Green, Tspan4–GFP.

Supplementary Video 3

A Tspan4–GFP-expressing cell during and after photodamage treatment in the presence of PI. Merge channel. Red, PI; green, Tspan4–GFP. The yellow circle in the second frame indicates the exact time and area of laser damage. Yellow arrow, damage site. Video display rate, 20 f.p.s. Scale bar, 5 µm.

Supplementary Video 4

Partially opened structures in Tspan4 proteoliposomes. Three-dimensional-tilt movie of Tspan4 proteoliposomes conducted by transmission electron microscopy (FEI Titan Krios 300 kV). Yellow arrow, partially opened site. Video display rate, 5 f.p.s. Scale bar, 10 nm.

Supplementary Video 5

Partially opened structures in Tspan4 proteoliposomes. Reconstructed tomographic Z-stack images (from bottom to top) of Tspan4 proteoliposomes. Yellow arrow, partially opened site. Video display rate, 5 f.p.s. Scale bar, 10 nm.

Source data

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 5

Unprocessed gels.

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Huang, Y., Zhang, X., Wang, HW. et al. Assembly of Tetraspanin-enriched macrodomains contains membrane damage to facilitate repair. Nat Cell Biol 24, 825–832 (2022). https://doi.org/10.1038/s41556-022-00920-0

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