Engineered migrasomes provide a robust and thermally stable vaccination platform
- The State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, China
- The State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, Institute of Immunology, School of Basic Medical Sciences, Tsinghua University, China
- Clinical Stem Cell Research Center, Peking University Third Hospital, China
- School of Basic Medical Sciences, Tsinghua University, China
- Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structures, School of Life Science, Tsinghua University, China
- Migrasome Therapeutics, China
Figures
Figure 1 with 3 supplements
Hypotonic stimulation-induced migrasome-like structures.
(A–M) The biogenesis of migrasome-like structures in NRK cells stably expressing Tspan4-GFP. In these images, the fluorescence intensity of Tspan4-GFP is shown in a color map scale from purple (low) to yellow (high). (A) Image series of the biogenesis of migrasome-like structures. Cells were treated with hypotonic Dulbecco’s phosphate-buffered saline (DPBS) with an osmolarity of 76.3 mOsmol and imaged using a confocal microscope. Scale bar, 10 μm. (B) Representative confocal images of cells treated with DPBS with various osmolarities. 305 mOsmol represents an isotonic condition in which no migrasome-like structures were observed. DPBS diluted to 152.5, 76.3, or 50.8 mOsmol was used to achieve hypotonic stimulations of different magnitude. The size of migrasome-like structures increased as the osmolarity was reduced. Scale bars, 10 μm (upper panels) and 5 μm (lower panels). (C) For each migrasome-like structure in (B), the whole lifetime, including the growth and shrinkage, was recorded by time-lapse imaging. The largest diameter reached during the lifetime was measured. The average diameter of migrasome-like structures increased significantly as the osmolarity was reduced. For hypotonic stimulation at 152.5, 76.3, or 50.8 mOsmol, n=194, 261, 165 migrasome-like structures, respectively. Data were plotted as mean ± SD. (D) Illustration of the experimental setup for real-time imaging of the induction of migrasome-like structures. (E) Image series of cells treated with hypotonic DPBS in two different approaches. The osmolarity of DPBS was reduced to 76.3 mOsmol by either one single step (upper panel) or five steps with 1-minute intervals (lower panel). For the step-wise reduction, the osmolarity was lowered by 25% in each step. Imaging time points were counted from the final stimulation step. Migrasome-like structures induced by the stepwise protocol showed significantly enhanced stability. Scale bars, 5 μm. (F) Statistical analysis of growth curves of migrasome-like structures in (E). Data were plotted as mean + SD. For single step stimulation, n=16 cells; for step-wise stimulation, n=13 cells. (G) Representative confocal images showing the effect of latrunculin A (LatA) treatment on the biogenesis of migrasome-like structures. Cells were pre-incubated with 0, 0.25, 0.5, or 1 μM LatA for 10 minutess and then treated with a five-step hypotonic stimulation as described in (E). LatA enhanced the biogenesis of migrasome-like structures in a dose-dependent manner. The white line indicates the boundary of the cell body. Scale bars, 10 μm. (H) Statistical analysis of the number of migrasome-like structures per cell in (G). Data were plotted as mean ± SD, for LatA treatment at 0, 0.25, 0.5, or 1 μM, n=10, 12, 14, or 9 cells were analyzed, respectively. (I) Relative mRNA level of SWELL1 analyzed by qPCR. SWELL1 expression was significantly reduced in SWELL1-knockdown (KD) cells compared to control cells. Data were plotted as mean + SD, n=3. (J) Representative confocal images of control or SWELL1-KD cells. Cells were treated with a five-step hypotonic stimulation at 2-minute intervals. The osmolarity was reduced by 1/6 in each step. Migrasome-like structures are shown in the inserts. Scale bars, 10 μm. Inset scale bars, 5 μm. (K) Statistical analysis of the diameter of migrasome-like structures in (J); data were plotted as mean ± SD, n=63 for control cells and 167 for SWELL1-KD cells. (L) Representative confocal images showing the effect of extracellular cations on the biogenesis of migrasome-like structures. Before stimulation, the culture medium was replaced by modified DPBS in which either Na+, K+, or Cs+ was the only cation source. Cells were then treated with a five-step hypotonic stimulation at 2-minute intervals. The osmolarity was reduced by 1/6 in each step. Scale bars, 10 μm (upper panels) and 5 μm (lower panels). (M) Statistical analysis of the diameter of migrasome-like structures in (l); data were plotted as mean ± SD, n=18 for Na+, 119 for K+, and 203 for Cs+. (N) Multiple primary cell types and cell lines are capable of producing eMigrasomes. Cells were stained with WGA-AF488 (Thermo, W11261) after hypotonic induction of eMigrasome with our protocol. Z-stack image series were captured and sum-slices projections were applied. Scale bars, 10 μm (upper panels) and 5 μm (lower panels). For all statistical analyses in this figure, P values were calculated using a two-tailed unpaired nonparametric test (Mann–Whitney test). P value<0.05 was considered statistically significant. ***P<0.001; ****P<0.0001.
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Figure 1—source data 1
Original statistical data for Figure 1C, F, H, I, K and M.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig1-data1-v1.xlsx
Figure 1—video 1
Time-lapse movie showing the biogenesis of migrasome-like structures.
Related to Figure 1A. Scale bar, 10 μm.
Figure 1—video 2
The dynamics of migrasome-like structures induced by single-step approach.
Related to the upper panel of Figure 1E. Scale bar, 10 μm.
Figure 1—video 3
The dynamic of migrasome-like structures induced by a stepwise approach.
Related to the lower panel of Figure 1E. Scale bar, 10 μm.
Figure 2 with 1 supplement
Mechanistic and morphologic similarity between migrasomes and eMigrasomes.
(A) Representative confocal images showing the effect of Tspan4-GFP in the biogenesis of migrasome-like structures. NRK cells were transiently transfected with mCherry vector or Tspan4-mCherry. The two populations of transfected cells were mixed in a 1:1.5 ratio in a test tube and then seeded in a confocal chamber. Cells were pre-incubated with 2 μM LatA for 10 minutess and then treated with a three-step hypotonic stimulation with 2-minute intervals. In each step, the osmolarity was reduced by 1/6 (16.7%). WGA-AF647 (Thermo, W32466) was then added to stain migrasome-like structures. Z-stack images were captured for further analysis. Scale bars, 10 μm. Inset scale bars, 5 μm. (B) Statistical analysis of the number of migrasome-like structures per cell in NRK cells transiently transfected with mCherry vector or Tspan4-mCherry in (A). Data were plotted as mean ± SD, n=53, 53 cells, respectively. (C) Statistical analysis of the number of migrasome-like structures per cell in NRK cells transiently transfected with mCherry vector (n=26 cells) or CD82-mCherry (n=44 cells) in Figure 2—figure supplement 1A. Data were plotted as mean ± SD. (D) Statistical analysis of the number of migrasome-like structures per cell in NRK cells transiently transfected with mCherry vector (n=31 cells) or Tspan1-mCherry (n=44 cells) in Figure 2—figure supplement 1B. Data were plotted as mean ± SD. (E) Representative confocal images showing the effect of cholesterol extraction on migrasome-like structures. NRK cells stably expressing Tspan4-GFP were stimulated to generate migrasome-like structures as described in (A). Cells were then incubated with 10 mM MβCD or buffer supplied with an equal volume of control solvent (H2O) for 30 minutes before imaging. Z-stack images were captured for further analysis. Scale bars, 10 μm. Inset scale bars, 5 μm. (F) Statistical analysis of the number of migrasome-like structures per cell in control cells (n=56) or cells treated with 10 mM MβCD (n=58) in (E). Data were plotted as mean ± SD. (G) Representative confocal images showing the effect of sphingomyelin depletion on the biogenesis of eMigrasomes. NRK cells stably expressing Tspan4-GFP were incubated with DMSO or 25 μM SMS2-IN-1 for 16 hours. Cells were then treated and imaged as described in (A). Scale bars, 10 μm. Inset scale bars, 5 μm. (H) Statistical analysis of the number of migrasome-like structures per cell in control cells (n=51) or cells treated with 25 μM SMS2-IN-1 (n=46) in (G). Data were plotted as mean ± SD. (I) Representative confocal images showing a cell generating natural migrasomes (left) and eMigrasomes (right). Migrasomes and eMigrasomes are morphologically similar. The fluorescence signal of Tspan4-GFP is highly enriched in both migrasomes and eMigrasomes. Scale bars, 10 μm. Inset scale bars, 5 μm. For all statistical analyses in this figure, P values were calculated using a two-tailed unpaired nonparametric test (Mann–Whitney test). P value<0.05 was considered statistically significant. ***P<0.001; ****P<0.0001.
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Figure 2—source data 1
Original statistical data for Figure 2B, C, D, F and H.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The effect of tetraspanin expression on the formation of migrasome-like structures.
(A) NRK cells were transiently transfected with mCherry vector or CD82-mCherry. The cells were then treated and imaged as described in Figure 2A. Scale bar, 10 μm. Inset scale bars, 5 μm. (B) NRK cells were transiently transfected with mCherry vector or Tspan1-mCherry. The cells were then treated and imaged as described in Figure 2A. Scale bar, 10 μm. Inset scale bars, 5 μm.
Figure 3 with 1 supplement
Isolation and characterization of eMigrasome.
NRK cells stably expressing Tspan4-GFP were used in all experiments in this figure if not otherwise specified. (A) Schematic illustration showing the process of eMigrasome induction, isolation, and purification. (B) Confocal image (left), threshold edge (middle), and centroid map (right) of purified eMigrasomes. Image processing and analysis were performed using ImageJ. The Hough circle transform plugin was applied to recognize and transform thresholded edges into binned objects representing individual eMigrasomes. Scale bar, 10 μm. Inset scale bar, 5 μm. (C) Statistical analysis of the radius of purified eMigrasomes. Measurement was performed using the map generated by Hough circle transformation analysis. 4725 particles were analyzed and the data were binned to plot the distribution of eMigrasomes radius. (D) TEM micrograph of negatively stained purified eMigrasomes. Scale bar, 1 μm. (E) Cryo-EM micrograph of purified eMigrasomes. Scale bar, 200 nm. (F) Western blot showing the protein level of several markers in cell bodies (Cell) and eMigrasomes (eMig). An equal amount of protein was loaded in each lane. (G) Representative time-lapse confocal images showing the high permeability of eMigrasomes to Cy5 at 1.5 hours post purification (left) and the gradual increase in the permeability of eMigrasomes to 40 kDa dextran-TMR (right). Scale bars, 5 μm. (H) Statistical analysis of the percentage of eMigrasomes (eMigs) that were permeable to 40 kDa dextran-TMR at the indicated time points. For each time point, eMigrasomes from three different views were analyzed (data were plotted as mean ± SD, n=3). From left to right, 336, 493, 812, 830, and 631 eMigrasomes were analyzed. (I) Representative confocal images of eMigrasomes after sitting at room temperature for 0, 3, 7, or 14 days. Aliquots of eMigrasomes were stored in EP tubes as pellets at room temperature for the indicated time, then resuspended and dropped into a confocal chamber before imaging. Z-stack image series were captured and sum-slices projections were applied. Scale bar, 10 μm. Inset scale bar, 5 μm. (J) Number of eMigrasomes after storage at room temperature for 0, 3, 7, or 14 days. Aliquots of eMigrasomes (7 × 106 eMigrasomes per tube in black, 3 × 106 eMigrasomes per tube in green) were stored in EP tubes as pellets at room temperature for the indicated time, then resuspended and stained with WGA561 before counting by FACS. (K) Time-lapse image series showing purified eMigrasomes treated with 10 mM MβCD or control buffer. A 10 μl drop of concentrated eMigrasomes was settled in a confocal chamber, then sealed and maintained at 37°C during imaging. Buffer containing 10 mM MβCD or control solvent was added to the drop using the equipment illustrated in Figure 1D. Scale bars, 10 μm (left panels) and 5 μm (right panels).
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Figure 3—source data 1
Original western blots for Figure 3F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig3-data2-v1.zip
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Figure 3—source data 3
Original statistical data for Figure 3C, H and J.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig3-data3-v1.xlsx
Figure 3—video 1
4D time-lapse movie showing the biogenesis of eMigrasomes.
Related to Figure 3. Scale, each grid represents 20 μm in x, y, or z axis.
Figure 4
eMigrasomes as an antigen carrier for vaccination.
(A) Schematic illustration of strategies for loading proteins of interest (POI) onto eMigrasomes. The protocol includes the construction of antigen-encoding plasmids, the construction of engineered cell lines stably expressing the antigens, and the production of antigen-loaded eMigrasomes. Membrane proteins, which already carry a transmembrane (TM) domain, are overexpressed in cells (top). Cytosolic proteins are membrane tethered by tagging with the TM sequence and polybasic tail of STX2 (bottom). Cells are then treated with hypotonic buffer to enrich the POI on the surface of eMigrasomes. (B) Representative confocal image of a cell expressing PD1-mCherry and Tspan4-GFP. The transmembrane protein PD1-mCherry was loaded onto eMigrasomes as shown in the top part of (A). Scale bars, 10 μm. Inset scale bars, 5 μm. (C) Representative confocal image of a cell expressing membrane-tethered OVA-mCherry (mOVA-mc) and Tspan4-GFP. To create an extracellular membrane-tethered form of OVA (mOVA), the sequence of OVA was fused to the C-terminus of a truncated form of mouse STX2, in which only the transmembrane region and a polybasic tail remained. The mCherry tag was fused to the C-terminus of OVA to trace the localization of this fusion protein. The soluble protein OVA was loaded onto the membrane of eMigrasomes, as shown in the bottom part of (A). Scale bars, 10 μm. Inset scale bars, 5 μm. (D) Representative confocal image of eMigrasomes isolated from MCA-205 cells stably expressing Tspan4-GFP and mOVA-mCherry (eM-OVA). Scale bar, 10 μm. Inset scale bar, 5 μm. (E) Western blot showing the amount of full-length mOVA-mCherry protein in host cell and eM-OVA. Cell lysate (C) containing 1 μg total protein and purified eM-OVA samples containing 0.1, 0.3, or 1 μg total protein were loaded. The protein-immobilized PVDF membrane was firstly incubated with anti-OVA antibody and then stripped and re-blotted with anti-mCherry antibody. The antigen mOVA-mCherry was highly enriched in eMigrasomes compared to host cells. MCA-205 cells stably expressing Tspan4-GFP and mOVA-mCherry were used for all experiments in the rest of this figure if not otherwise specified. (F) Amount of OVA-specific IgG in mouse serum on day 14 after intravenous (i.v), nasal, or subcutaneous (s.c) immunization with eM-OVA (20 µg/mouse). OVA-specific IgG was quantified by ELISA. Data were plotted as mean ± SD, n=4 mice were analyzed for each group. (G) ELISA quantification of OVA-specific IgG in sera from wild-type (WT) mice on day 14 after tail intravenous immunization with eM-OVA at the indicated dose. Data were plotted as mean ± SD. For 1, 3, 10, 20 µg/mouse eM-OVA immunization, n=5 mice; for 40 µg/mouse eM-OVA immunization, n=6. (H) ELISA quantification of OVA-specific IgG in sera from WT mice on day 14 after tail intravenous immunization with eM-OVA (20 µg/mouse, n=6 mice) or intraperitoneal immunization with Alum/OVA (n=5 mice). Data were plotted as mean ± SD. (I) Titer analysis of OVA-specific IgG1, IgG2b, IgG2c, and IgG3 in the sera from mice immunized with eM-OVA (20 µg/mouse, n=6 mice) or Alum/OVA (n=5 mice). Serum samples were collected on day 14. Each dot represents an individual serum sample. Data were plotted as mean ± SD. (J) Illustration of the experimental setup for assaying the stability of eM-OVA. (K) Immunoblotting analysis of the amount of OVA protein in samples of eM-OVA that were left at room temperature for 0, 3, 7, or 14 days. 2 µg protein was loaded in each lane. (L) ELISA quantification of OVA-specific IgG in sera from WT mice on day 14 after tail intravenous immunization with eM-OVA stored at room temperature for 0 days (D0, n=5 mice), 3 days (D3, n=5 mice), 7 days (D7, n=6 mice), or 14 days (D14, n=5 mice). Data were plotted as mean ± SD. 20 µg eM-OVA was injected per mouse. For all statistical analyses in this figure, P values were calculated using a two-tailed unpaired nonparametric test (Mann–Whitney test). P value<0.05 was considered statistically significant. n.s. P>0.05; *P<0.05; **P<0.01.
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Figure 4—source data 1
Original western blots for Figure 4E and K, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4E and K.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig4-data2-v1.zip
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Figure 4—source data 3
Original statistical data for Figure 4F, G, H, I and L.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig4-data3-v1.xlsx
Figure 5
An eMigrasome-based vaccine induces a strong humoral protective response against SARS-CoV-2.
(A) Representative confocal images showing the presence of Spike-mCherry (S-mc) in engineered cells and isolated eMigrasomes. Scale bars, 10 μm. Inset scale bars, 5 μm. (B) Western blot showing the amount of full-length Spike-mCherry protein in eM-S. A titration (1, 2, 5, 10, 20, or 50 ng) of recombinant spike protein was loaded as standards. Purified eM-NC or eM-S samples containing 1 μg total protein were loaded. (C) Representative confocal images showing the presence of integral spike protein in isolated eMigrasomes. Scale bars, 10 μm. Inset scale bars, 5 μm. (D) Schematic diagram of the experimental procedure for immunization with Spike-loaded eMigrasomes (eM-S) and collection of serum. (E) Illustration of the rVSV-venus-SARS-CoV-2 system. VSV-Venus-SARS-CoV-2 was mixed with vaccinated mice sera. Vero-TMPRSS2 cells were infected with the reporter virus/serum mixture with an MOI of 0.01. 40 hours post-infection, the Venus-positive infected cells were quantified to estimate the NT50 value for each serum. (F) Spike-specific IgG was quantified in WT mice immunized with eM-S (20 µg/mouse, i.v.) at different time points. Data were plotted as mean ± SD. Each symbol represents one individual animal, n=6 mice were analyzed. (G) Neutralization curves are presented for sera from primary-vaccination and boost-vaccination. Data were plotted as mean + SEM, n=6 mice were analyzed. Nonlinear regression was performed using the equation for the normalized response versus the inhibitor, incorporating a variable slope. (H) NT50 of individual mouse in vaccinated groups was compared by P-value (paired t-test) and is indicated. Dotted lines represent assay limits of detection. Each line represents an individual mouse, n=6 mice were analyzed.
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Figure 5—source data 1
Original western blots for Figure 5B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5B.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig5-data2-v1.zip
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Figure 5—source data 3
Original statistical data for Figure 5F–H.
- https://cdn.elifesciences.org/articles/97621/elife-97621-fig5-data3-v1.xlsx
Author response image 1
eMigrasome-based vaccination showed similar efficacy compared with adjuvanted recombinant spike protein The amount of S1-specific IgG in mouse serum was quantified by ELISA on day 14 after immunization.
Mice were either intraperitoneally (i.p.) immunized with recombinant Alum/S1 or intravenously (i.v.) immunized with eM-NC, eM-S or recombinant S1. The administered doses were 20 µg/mouse for eMigrasomes, 10 µg/mouse (i.v.) or 50 µg/mouse (i.p.) for recombinant S1 and 50 µl/mouse for Aluminium adjuvant.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Rattus norvegicus) | NRK | ATCC | CRL-6509 | |
| Cell line (Homo sapiens) | HEK 293T | ATCC | CRL-3216 | |
| Cell line (Mus musculus) | MCA-205 | STR profiling, authenticated by Procell Life Science & Technology | ||
| Cell line (Cercopithecus aethiops) | Vero | ATCC | CCL-81 | |
| Cell line (H. sapiens) | ADSC | Procell | CP-H202 | Human Adipose-derived Mesenchymal Stem Cells |
| Cell line (H. sapiens) | BMSC | Procell | CP-H166 | Human Bone Marrow Mesenchymal Stem Cells |
| Antibody | Anti-ovalbumin (mouse monoclonal) | Santa Cruz | sc-80589 | ELISA (1:4000– 1:512,000), WB (1:1000) |
| Antibody | Anti-Spike (mouse monoclonal) | Sino Biological | 40591-MM42 | ELISA (1:5000– 1:640,000), WB (1:1000) |
| Antibody | Anti-Spike (chimeric MAb) | Sino Biological | 40150-D001 | IF (1:100) |
| Recombinant DNA reagent | pB-mOVA-mCherry (plasmid) | This paper | pB-mCherry derived plasmid | |
| Recombinant DNA reagent | pB-S-mCherry (plasmid) | This paper | pB-mCherry derived plasmid | |
| Recombinant DNA reagent | pB-Tspan4-GFP (plasmid) | Jiao et al., 2021 | pB-GFP derived plasmid | |
| Peptide, recombinant protein | Ovalbumin | Sigma | A5503 | |
| Chemical compound, drug | Latrunculin A | Cayman | 10010630 | 0.25–2 µM |
| Other | Imject Alum | Thermo Fisher | 77161 | |
| Other | Fibronectin | Sigma | F0895 | 10 µg/ml for imaging; 4 µg/ml for eMigrasome preparation |
| Other | 40 kDa Dextran-TMR | Thermo Fisher | D1842 | (25 μg/ml) |
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Engineered migrasomes provide a robust and thermally stable vaccination platform
eLife 13:RP97621.
https://doi.org/10.7554/eLife.97621.3
