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The formation of migrasomes is initiated by the assembly of sphingomyelin synthase 2 foci at the leading edge of migrating cells

Nature Cell Biology volume 25, pages 1173–1184 (2023)Cite this article

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Abstract

The migrasome is an organelle of migrating cells with diverse physiological functions. How migrasome formation is initiated is unknown. We found that sphingomyelin is enriched in migrasomes and identified sphingomyelin synthase 2 (SMS2) as an essential protein for migrasome biogenesis. SMS2 assembles into immobile foci that adhere on the basal membrane at the leading edge. When cells migrate away, the SMS2 foci ‘move’ out of cells and into retraction fibres, where they become migrasome formation sites and eventually grow into migrasomes. Mechanistically, SMS2 foci seed migrasomes by converting ceramide to sphingomyelin, which is essential for migrasome formation. Furthermore, CerS5, which is required for the synthesis of long-chain ceramide, and CERT, which transports ceramide from the endoplasmic reticulum to Golgi, are both required for migrasome formation. Our data reveal the essential role of ceramide and sphingomyelin in migrasome formation and suggest that SMS2 forms basal membrane-surface-connecting structures that pre-determine where migrasomes will grow.

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Fig. 1: Screening of proteins required for the formation of migrasomes.
Fig. 2: Lipidomic analysis of migrasomes and plasma membranes.
Fig. 3: Enrichment of SM on migrasomes.
Fig. 4: SM is required for migrasome formation.
Fig. 5: SMS2 is required for migrasome formation.
Fig. 6: SMS2 foci seed migrasome formation.
Fig. 7: Immobile SMS2 foci assemble at the leading edge of migrating cells.
Fig. 8: Assembly of SMS2 foci is required for migrasome formation.

Data availability

Previously published sequences used for analysis and design are available at UNIPROT (mouse SMS2: Q9D4B1). Previously published proteomics data that were re-analysed here are available under the accession code PXD026960. Lipidomic data are available in Source Data Fig. 2. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (grant nos. 32030023 and 92054301 to L.Y.), Beijing Municipal Science and Technology Commission, Administrative Commission of Zhongguancun Science Park (grant no. Z221100003422012 to L.Y.) and Tsinghua University Initiative Scientific Research Program (grant no. 20221080007 to L.Y.). We thank the State Key Laboratory of Membrane Biology for flow cytometry analysis and facility support. We thank the SLSTU-Nikon Biological Imaging Center for assistance with using the Nikon A1RSiHD25 laser scanning confocal microscope, TIRF and software. Both pET28 His6–mCherry–NT-Lys and pET28 His6EGFP–NT-Lys were provided by the RIKEN BRC through the National Bio-Resources Project of the MEXT/AMED, Japan.

Author information

Authors and Affiliations

  1. The State Key Laboratory of Membrane Biology, Tsinghua University–Peking University Joint Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China

    Haisha Liang, Yuheng Liu, Boqi Liu, Wanqing Du & Li Yu

  2. The State Key Laboratory of Membrane Biology, Tsinghua University–Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Xinyu Ma

  3. School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Yuanyuan Zhang

  4. MOE Key Laboratory of Protein Sciences, Beijing Frontier Research Center for Biological Structures, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Nan Liu

  5. School of Life Science, Tsinghua University, Beijing, China

    Weiying Zhang & Jianhui Chen

  6. Technology Center for Protein Sciences, Tsinghua University, Beijing, China

    Xiaohui Liu

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Contributions

L.Y. and H.L. conceived the experiments. L.Y. wrote the manuscript and supervised the project. H.L. carried out the cell biology and biochemistry experiments. H.L., X.M., Y.Z. and J.C. participated in the shRNA screening work. Y.L. helped analyse bioinformatic data. N.L. assisted with depicting the SMS2 structure. H.L. and W.D. completed the statistical analysis. W.Z. and B.L. prepared the zebrafish embryos in the SMS2 inhibitor-treatment experiment. X.L. carried out lipidomic analysis. All authors discussed the manuscript and contributed to the preparation of the manuscript.

Corresponding author

Correspondence to Li Yu.

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Competing interests

L.Y. is the scientific founder of Migrasome Therapeutics Ltd. All other authors declare no competing interests.

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Nature Cell Biology thanks Graça Raposo, Kwang Pyo Kim, 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 Relative abundance of the glycoceramides CerG1, CerG2 and CerG3.

a–c, Glycoceramides CerG1 (a), CerG2 (b) and CerG3 (c) with different fatty-acid-chain lengths and saturation status in migrasomes (blue), plasma membranes (red) and cell bodies (grey). Lipid extracts from migrasomes, plasma membranes and cell bodies were analysed by LC–MS/MS. The relative abundance of each lipid species was calculated using its chromatographic area divided by the total areas of all identified lipids under the positive-ion mode. Data are the mean ± s.d. (technical replicates, n = 3).

Source data

Extended Data Fig. 2 Enrichment of SM in migrasomes from multiple cell types.

Confocal images of MC38 cells, TSPAN4–GFP-expressing NRK cells, BJ cells and primary MEF cells. mCherry–NT-Lys was used to label the distribution of SM on these cells. The MC38, BJ and primary MEF cells were also stained with WGA488 to label migrasomes. Scale bars, 10 μm.

Extended Data Fig. 3 Cert are required for migrasome formation.

a, L929 cells expressing TSPAN4–GFP were infected with control (NS) or two Cert-shRNA lentiviruses and stained with mCherry–NT-Lys. Green, TSPAN4–GFP; red, mCherry–NT-Lys. Scale bar, 10 µm. b,c, qPCR analysis of Cert expression in cells from Fig. 4e and Extended Data Fig. 3a. Data are the mean ± s.d., n = 3 independent experiments. ****P < 0.0001; two-tailed unpaired Student’s t-test. d, Statistical analysis of migrasome numbers from Extended Data Fig. 3a. n = 150 cells per group from three independent experiments. Data are presented as the median with the interquartile range. ****P < 0.0001 (NS versus Cert-KD-2; NS versus Cert-KD-3); two-tailed unpaired non-parametric test (Mann–Whitney test). e, L929 cells stably expressing TSPAN4–GFP were infected with lentivirus carrying non-specific-shRNA (NS) or Cert-shRNA (Cert-KD-1) constructs. Cells were stained with anti-ceramide (α-Ceramide) and observed by TIRF. Green, TSPAN4–GFP; red, anti-ceramide. Scale bars, 10 µm. f, The number of ceramide foci in RFs per cell from Extended Data Fig. 3e was statistically analysed. NS, n = 180 cells, Cert-KD-1, n = 157 cells. Data (from three independent experiments) are presented as the median with the interquartile range. ****P < 0.0001; two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant.

Source data

Extended Data Fig. 4 CerS5 are required for migrasome formation.

a, L929 cells expressing TSPAN4–GFP were infected with control (NS) or three different Cers2-shRNA (Cers2-KD-1, Cers2-KD-2 and Cers2-KD-3) lentivirus. Cells were stained with mCherry–NT-Lys and observed. Green, TSPAN4–GFP; red, mCherry–NT-Lys. Scale bar, 10 µm. b, L929 cells expressing TSPAN4–GFP were infected with control (NS-2) or two different Cers5-shRNA lentiviruses (Cers5-KD-2 and Cers5-KD-3). Compared with Cers5-KD-1 (Fig. 4j), there is one more enhancer in the Cers5-KD-2 and Cers5-KD-3 plasmids; thus, a second NS (NS-2) was applied as the control for Cers5-KD-2 and Cers5-KD-3. Cells were stained with mCherry–NT-Lys and observed. Green, TSPAN4–GFP; red, mCherry–NT-Lys. Scale bar, 10 µm. c, qPCR analysis of Cers2 expression in cells from Extended Data Fig. 4a. Data are the mean ± s.d.; n = 3 independent experiments. ****P < 0.0001; two-tailed unpaired Student’s t-test. d, Statistical analysis of migrasome numbers from Extended Data Fig. 4a. Data (from three independent experiments) are presented as the median with the interquartile range. P values, two-tailed unpaired non-parametric test (Mann–Whitney test): NS (n = 201 cells) versus Cers2-KD-1 (n = 151 cells), P = 0.3021; NS versus Cers2-KD-2 (n = 151 cells), P = 0.4921; NS versus Cers2-KD-3 (n = 150 cells), P = 0.1974 (ns, non-significant). The data in the NS group were same as that in Fig. 4k. e,f, qPCR analysis of the Cers5 mRNA levels in the cells from Fig. 4j and Extended Data Fig. 4b. Data are the mean ± s.d.; n = 3 independent experiments. ****P < 0.0001; two-tailed unpaired Student’s t-test. g, Statistical analysis of migrasome numbers from Extended Data Fig. 4b. Data (from three independent experiments) are presented as the median with the interquartile range. ****P < 0.0001 (NS-2 (n = 201 cells) versus Cers5-KD-2 (n = 151 cells); NS-2 versus Cers5-KD-3 (n = 150 cells)); two-tailed unpaired non-parametric test (Mann–Whitney test).

Source data

Extended Data Fig. 5 shRNA-resistant constructs restore the migrasome formation in relevant knockdown cells.

a, L929 cells stably expressing TSPAN4–GFP were infected with lentiviruses carrying non-specific-shRNA (NS) or another two Sgms2-shRNAs (Sgms2-KD-2 and Sgms2 -KD-3). Cells were stained with mCherry–NT-Lys and observed by confocal microscopy. Green, TSPAN4–GFP; red, mCherry–NT-Lys. Scale bar, 10 µm. b,c, qPCR analysis of the cells from Fig. 5a and Extended Data Fig. 5a. Sgms2 expression was reduced significantly by shRNA expressed from lentiviral constructs. Data are the mean ± s.d.; n = 3 independent experiments. ****P < 0.0001 (NS versus Sgms2-KD-1; NS versus Sgms2-KD-2; NS versus Sgms2-KD-3); two-tailed unpaired Student’s t-test. P < 0.05 was considered statistically significant. d, Statistical analysis of migrasome numbers from Extended Data Fig. 5a. n = 150 cells per group from three independent experiments. Data are presented as the median with the interquartile range. ****P < 0.0001 (NS versus Sgms2-KD-2; NS versus Sgms2-KD-3); two-tailed unpaired non-parametric test (Mann–Whitney test); P < 0.05 was considered statistically significant. e, qPCR analysis of the cells from Extended Data Fig. 5f. Sgms2 and Cert expression were reduced significantly by shRNAs expressed from lentiviral constructs. Data are the mean ± s.d. of three independent experiments. ****P < 0.0001 (NS versus Sgms2-KD-1; NS versus Cert-KD-1); two-tailed unpaired Student’s t-test; P < 0.05 was considered statistically significant. f, L929 cells stably expressing TSPAN4–GFP were infected with non-specific-shRNA (NS), Sgms2-shRNA (Sgms2-KD-1) or Cert-shRNA (Cert-KD-1) lentivirus. For the rescue experiment, Sgms2-KD-1 cells were further infected by lentivirus carrying a SMS2–3×Flag construct. The Cert-KD-1 cells were further infected by lentivirus carrying a CERT-M6–3×Flag construct, which is resistant to Cert-KD-1. Cells were stained with mCherry–NT-Lys and observed by confocal microscopy. Green, TSPAN4–GFP; red, mCherry–NT-Lys. Scale bar, 10 µm. g, Western blot showing the protein expression level of SMS2–3×Flag in Sgms2-KD-1 cells and CERT-M6–3×Flag in Cert-KD-1 cells. h, Statistical analysis of the migrasome numbers from Extended Data Fig. 5f. NS, n = 200 cells; other groups, n = 150 cells per group. Data (from three independent experiments) are presented as the median with the interquartile range. ****P < 0.0001 (NS versus Sgms2-KD-1; NS versus Cert-KD-1; Sgms2-KD-1 versus Sgms2-KD-1 + SMS2-3xFlag; Cert-KD-1 versus Cert-KD-1 + CERT-M6–3×Flag); two-tailed unpaired non-parametric test (Mann–Whitney test); P < 0.05 was considered statistically significant.

Source data

Extended Data Fig. 6 SMS2 inhibitors blocked migrasome formation in multiple cell lines and in zebrafish embryos.

a–e, SMS2 inhibitors (SMS2-IN-1, SMS2-IN-2 and Ly93) block migrasome formation. L929 cells, MC38 cells, TSPAN4–GFP-expressing NRK cells, BJ cells and primary MEF cells were treated with SMS2 inhibitors. Information about the inhibitor concentrations is in Methods. Except for TSPAN4–GFP-expressing NRK cells and TSPAN4–GFP-expressing L929 cells, the cells were stained with WGA488 to label migrasomes. Cells were observed by confocal microscopy. Scale bars, 10 µm. f, Representative images of migrasomes in zebrafish embryos treated with DMSO and 100 µM SMS2-IN-1. Z-stack images were collected from zebrafish gastrulas to quantify the number of migrasomes. Migrasomes labelled with PH–mCherry were visualized in gastrulas. The Z-stack images of the gastrulas were acquired by Andor Dragonfly spinning disk confocal microscopy. Twenty-five slices were acquired at 2-μm intervals from the animal pole. Nine images were combined for each slice. The arrows indicate migrasomes. Scale bar, 10 μm. g, Statistical analysis of the migrasome numbers in zebrafish embryos treated with DMSO and SMS2-IN-1. Data from four independent experiments were pooled. DMSO, n = 51 embryos; SMS2-IN-1, n = 54 embryos. Data are presented as the median with the interquartile range. ****P < 0.0001; two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant. h–v, Statistical analysis of the migrasome numbers in the DMSO (control) or inhibitor-treated groups from Extended Data Fig. 6a–e. Migrasome number per cell (Mig number/cell); L929 cells (L929); BJ cells (BJ); MC38 cells (MC38); primary MEF cells (pMEF); NRK cells expressing TSPAN4–GFP (NRK); DMSO (D); SMS2-IN-1 (IN-1); SMS2-IN-2 (IN-2). Data are presented as the median with the interquartile range. n = 150 cells per group from three independent experiments. ****P < 0.0001 (DMSO versus Ly93; DMSO versus SMS2-IN-1; DMSO versus SMS2-IN-2); two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant.

Source data

Extended Data Fig. 7 The recruitment of cholesterol in Sgms2-knockdown cells was restored following the addition of SM.

a, L929 cells stably expressing TSPAN4–GFP were infected for more than 24 h with lentivirus carrying non-specific-(NS) or Sgms2-shRNA (Sgms2-KD-1) constructs and then subjected to puromycin selection for 48 h. Cells were supplemented with vehicle or 5 µM SM. Cells were fixed with 4% PFA, stained with filipin III (which binds specifically to cholesterol) and observed by confocal microscopy. Scale bars, 10 µm. b, qPCR analysis of Sgms2 expression in the cells from Extended Data Fig. 7a. Sgms2-shRNA significantly reduced Sgms2 expression. Data are the mean ± s.d., n = 3 independent experiments. ****P < 0.0001; two-tailed unpaired Student’s t-test. P < 0.05 was considered statistically significant. c, Statistical analysis of summed filipin III intensity per RF length from Extended Data Fig. 7a. NS + Vehicle, n = 157 cells; NS + SM, n = 164 cells; Sgms2-KD-1 + Vehicle, n = 153 cells; Sgms2-KD-1 + SM, n = 162 cells. Data are from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. ****P < 0.0001 (NS + Vehicle versus Sgms2-KD-1 + Vehicle; Sgms2-KD-1 + Vehicle versus Sgms2-KD-1 + SM); two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant.

Source data

Extended Data Fig. 8 SMS2 foci were observed in multiple cell lines.

Immunostaining of endogenous SMS2 in MC38 cells, TSPAN4–GFP-expressing NRK cells, BJ cells and primary MEF cells. MC38 cells, BJ cells and primary MEF cells were stained with WGA488 to label migrasomes. Scale bars, 5 μm.

Extended Data Fig. 9 SMS2 can be found both on lysosomes and on foci located on the basal membrane.

a, Confocal image of an L929 cell stably expressing SMS2–GFP and LAMP1–mCherry. Scale bars, 10 μm. b, Three-dimensional imaging of the cell used in Extended Data Fig. 9a. Top view in maximum intensity projection mode (left). Side view (middle); enlarged region of interest from the middle panel (right). In the right panel, the cell body is outlined by the thick white dashed line. Green, SMS2–GFP; red, LAMP1–mCherry. Scale bars, 10 µm. c, TIRF image of an L929 cell stably expressing SMS2–GFP and LAMP1–mCherry. Scale bar, 10 μm. d, Live-cell imaging of an L929 cell stably expressing SMS2–mCherry and Paxilin–GFP. Green, Paxilin–GFP; red, SMS2–mCherry. Scale bars, 10 µm. SMS2 foci are clearly not part of focal adhesions labelled by Paxilin–GFP.

Extended Data Fig. 10 SM treatment cannot rescue the formation of SMS2 foci and migrasome formation in cells expressing SMS2-S217A mutant.

a, The SMS2 model predicted by AlphaFold is coloured by the confidence score. b, Western blot analysis to estimate the protein level of GFP-tagged SMS2 in L929 cells and L929 cells expressing WT SMS2–GFP or the mutants SMS2-S217A–GFP and SMS2-H229A–GFP. c, L929 cells expressing WT SMS2–GFP or the mutant SMS2-H229A–GFP were stained with mCherry–NT-Lys and observed by TIRF microscopy. Scale bars, 10 µm (main images) and 2 µm (magnifications). The boxed areas in the ‘Merge’ panels have been enlarged (right). d, L929 cells expressing WT SMS2–GFP or the mutant SMS2-S217A–GFP were supplemented with vehicle or 5 µM SM. After a 12-h incubation, the cells were stained with mCherry–NT-Lys and observed by TIRF microscopy. Scale bar, 10 µm. e, Statistical analysis of SMS2 foci per cell area in cells from Extended Data Fig. 10c. Data are from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. P values were calculated using a two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant. SMS2 + Vehicle (n = 172 cells) versus SMS2 + SM (n = 166 cells), *P = 0.0316; S217 + Vehicle (n = 157 cells) versus S217 + SM (n = 166 cells), ***P = 0.0007. f, L929 cells expressing WT SMS2–GFP or the mutants SMS2-S217A–GFP were supplemented with vehicle or 5 µM SM. After a 12-h incubation, the cells were stained with mCherry–NT-Lys and observed by confocal microscopy. Scale bar, 10 μm. g, Statistical analysis of the migrasome numbers in the cells from Extended Data Fig. 10e; n = 100 cells per group from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. P values were calculated using a two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant. SMS2 + Vehicle versus SMS2 + SM, P = 0.2875; S217A + Vehicle versus S217A + SM, P = 0.2182; ns, non-significant.

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Supplementary Video 1

Growth of migrasomes. TSPAN4–GFP-expressing L929 cells were stained with mCherry-tagged non-toxic lysenin (mCherry–NT-Lys) and recorded with confocal imaging at 1.5-min intervals. Scale bar, 10 µm.

Supplementary Video 2

mCherry–NT-Lys labelling L929 cells with SMase treatment. L929 cells stably expressing TSPAN4–GFP were treated with SMase (0.8 U ml−1). The cells were stained with mCherry–NT-Lys and recorded with confocal imaging at 1.5-min intervals. Scale bar, 10 µm.

Supplementary Video 3

Migrasome formation procedure of NS and Sgms2-KD-1 cells. TSPAN4–GFP-expressing NS and Sgms2-KD-1 cells were stained with mCherry-tagged non-toxic lysenin (mCherry–NT-Lys) and recorded with confocal imaging at 3-min intervals. The migrasomes indicated by arrowheads shrank back. Scale bar, 10 µm.

Supplementary Video 4

SGMS2(SMS2) foci seed migrasome formation (confocal). L929 cells stably expressing SGMS2(SMS2)–GFP and TSPAN4–mCherry were collected by confocal microscopy at 3-min intervals. Scale bar, 10 µm.

Supplementary Video 5

Assembled at the leading edge of migrating cells, SGMS2(SMS2) foci are immobile relative to the supporting surface (TIRF). L929 cells stably expressing SGMS2(SMS2)–GFP and TSPAN4–mCherry were collected by TIRF at 1-min intervals. Scale bar, 10 µm.

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Liang, H., Ma, X., Zhang, Y. et al. The formation of migrasomes is initiated by the assembly of sphingomyelin synthase 2 foci at the leading edge of migrating cells. Nat Cell Biol 25, 1173–1184 (2023). https://doi.org/10.1038/s41556-023-01188-8

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