Migrasomes are recently discovered cellular organelles that form as large vesicle-like structures on retraction fibres of migrating cells. While the process of migrasome formation has been described before, the molecular mechanism underlying migrasome biogenesis remains unclear. Here, we propose that the mechanism of migrasome formation consists of the assembly of tetraspanin- and cholesterol-enriched membrane microdomains into micron-scale macrodomains, which swell into migrasomes. The major finding underlying the mechanism is that tetraspanins and cholesterol are necessary and sufficient for migrasome formation. We demonstrate the necessity of tetraspanins and cholesterol via live-cell experiments, and their sufficiency by generating migrasome-like structures in reconstituted membrane systems. We substantiate the mechanism by a theoretical model proposing that the key factor driving migrasome formation is the elevated membrane stiffness of the tetraspanin- and cholesterol-enriched macrodomains. Finally, the theoretical model was quantitatively validated by experimental demonstration of the membrane-stiffening effect of tetraspanin 4 and cholesterol.
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The main computational codes used for the simulations in this paper are available at https://github.com/benzucker-tau/Migrasome-simulation. Further codes in surface evolver and codes in Matlab used for analyses are available from the corresponding authors upon reasonable request.
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The authors are grateful to members of the Kozlov and Yu groups for helpful discussions. This research was supported by the Ministry of Science and Technology of the People’s Republic of China (2016YFA0500202 and 2017YFA0503404) to L.Y., the National Natural Science Foundation of China (31430053 and 31621063), the Natural Science Foundation of China international cooperation and exchange program (31561143002), the Independent Research of Tsinghua University (20161080135) to L.Y., and the Israel Science Foundation grant 1066/15, EU consortium InCeM to M.M.K., who also holds the Joseph Klafter Chair in Biophysics.
The authors declare no competing interests.
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Integrated supplementary information
a, mRNA levels of Tspan4 in Tspan4-GFP-expressing NRK cell lines and their parent cells were measured by real-time PCR. Data shown represent mean±SEM (n=3 independent experiments). b, Cells with stable Tspan4-GFP expression were treated with or without deglycosylation mix, then lysed and analyzed by western blot. c, mRNA levels of 9 TSPAN genes in the MGC-803 cell line were measured by real-time PCR and the values were normalized by the TSPAN4 mRNA level. Data shown represent mean±SEM (n=3 independent experiments). d, Verification of the NRK Tspan4 knockout cell line by PCR. e, The average number of migrasomes per 100 µm RF in wild type and Tspan4-KO NRK cells. Data shown represent mean±SEM, n=100 cells for each group. ***P<0.001. f, Statistical analysis of migrasome numbers in wild type L929 and Tspan4-KO L929 cell lines. Data shown represent mean±SEM; n=173 cells for WT L929; n=168 cells for Tspan4-KO L929. NS= No significance. Unprocessed blots related to b were presented in Supplementary Fig. 6. Experiments related to a-f were independently repeated 3 times. The quantifications in e and f are pooled from these independent experiments. Two-tailed unpaired t-test method was used for statistical analysis. The statistical details for a, c, e and f were available in Supplementary Table 4.
a, Tspan4-GFP-expressing NRK cells were subjected to FRAP (fluorescent recovery after photo-bleaching) analysis. Migrasomes in the growth phase and the steady phase were imaged by confocal microscopy. Scale bar, 2 µm. b, Area changes of growth-phase and steady-phase migrasomes in (a) during FRAP analysis. c, The normalized mean fluorescence intensity of Tspan4-GFP on growth-phase and steady-phase migrasomes in (a) during FRAP assays. d, Time-lapse images of a RF from a Tspan4-GFP-expressing NRK cell obtained in the course of a FRAP assay using resonant scanning mode confocal microscopy. Scale bar, 2 µm. e, Time-lapse images of a RF taken by resonant scanning mode confocal microscopy of Tspan4-mMaple3 photoconverted from green to red by 405 laser irradiation. Scale bar, 2 µm. Experiments related to a-c were independently repeated 26 times. The quantifications in b and c are one representative experiment. Experiments related to d were independently repeated 5 times. Experiments related to e were independently repeated 8 times.
a, Tspan1-mCherry-, Tspan7-mCherry-, Tspan9-mCherry- and CD82-mCherry-expressing NRK cells were observed by confocal microscopy. Enrichment of Tspan proteins on migrasomes is revealed by heat map images. Scale bars, 10 µm. b, NRK cells stably expressing Tspan4-GFP were treated by MβCD (Methyl-β-cyclodextrin, 10 mM) for 0 min and 30 min. Confocal images were collected at each time point. Scale bar, 10 µm. c, Statistical analysis of migrasome numbers at each time point in images like those in (b). Data shown represent mean±SEM; n=84 cells for MβCD 0 min; n=109 cells for MβCD 30 min. ***P<0.001. d, Relative total cellular cholesterol level of cells grown under different cholesterol depletion conditions, including 10% full cholesterol medium (FBS), cholesterol depletion medium (LPDS) and cholesterol depletion medium with 30 μM Pravastatin. Data shown represent mean±SEM (n=3 independent experiments), ***P<0.001. Experiments related to a-d were independently repeated 3 times. The quantifications in c are pooled from three independent experiments. Two-tailed unpaired t-test method was used for statistical analysis. The statistical details for c and d were available in Supplementary Table 4.
Supplementary Figure 4 Cholesterol is colocalized with Tspan4 on migrasome-like structures in in vitro systems.
Z-stack images of an in vitro-reconstituted Tspan4-containing GUV labeled with Topfluor-cholesterol and stained by Cy5-SE to label Tspan4. Images were acquired by confocal microscopy, then mean intensity Z-projections were generated by ImageJ. Purple, proteins; green, Topfluor-cholesterol; red, Rhodamine-PE. Images are representative of 34 tethers from 4 independent experiments. Scale bar, 2.5 µm.
Supplementary Figure 5 Tspan4 is highly recruited to migrasome-like structures during micropipette-induced reconstitution.
a, GUVs embedded with CD63-GFP were subjected to the in vitro reconstitution assay as described in Fig. 5a. Green, proteins, red, Rhodamine-PE. Images are representative of 10 tethers from 3 independent experiments. Scale bar, 5 µm. b, Time-lapse images showing pulling-induced assembly of tetraspanin-enriched microdomains and formation of a migrasome-like structure by micropipette-induced reconstitution. 6 independent experiments were repeated. Scale bar, 10 µm; zoomed-in panels, 5 µm. c, Working model to explain the analysis method for time-lapse images of micropipette reconstitution using the Imaris Spots function. Tspan4-GFP fluorescent signals on migrasome and retraction fiber structures during micropipette reconstitution were recognized and labeled by Imaris Spots. The initial attachment point of the migrasome or retraction fiber structure was set as the origin. The distance between each spot to the origin was measured and recorded. d, Time-lapse images from (b) were analyzed using the Imaris Spots function. Tspan4-GFP signals labeled by Imaris Spots were traced in each frame and the distance from each spot to the normalized origin was calculated and plotted. 3 independent experiments were repeated. The snapshot merged image of frame 36 is shown in the lower panel; scale bar, 20 µm.
a, Unprocessed blots related to Fig. 1d. Verification of Tspan4-GFP protein expression levels in different cell lines by western blot. The sections presented in Fig. 1d were labeled by red squares. b, Unprocessed gel related to Fig. 4a. Recombinant Tspan4-GFP was analyzed on an SDS-PAGE gel and stained by Coomassie Brilliant Blue. The section presented in Fig. 4a was labeled by red square. c, Unprocessed blots related to Supplementary Fig. 1b. Cells with stable Tspan4-GFP expression were treated with or without deglycosylation mix, then lysed and analyzed by western blot. The sections presented in Supplementary Fig. 1b were labeled by red squares.
Supplementary Figures 1–6, Supplementary Note 1 and legends for Supplementary Tables and Videos.
Transcription levels of the top 10 expressed TSPAN genes in NRK cells.
Measurement of liposome radius.
Measurement of the K1 value of liposomes.
Statistics source data.
Tspan4-GFP forms fast-moving puncta on migrasome and retraction fibre. Related to Fig. 2i.
In vitro reconstitution of migrasome formation by pulling, control GUV. Related to Fig. 5b.
In vitro reconstitution of migrasome formation by pulling, Tspan4-GFP embedded GUV. Related to Fig. 5b.
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Huang, Y., Zucker, B., Zhang, S. et al. Migrasome formation is mediated by assembly of micron-scale tetraspanin macrodomains. Nat Cell Biol 21, 991–1002 (2019) doi:10.1038/s41556-019-0367-5
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