A ‘torn bag mechanism’ of small extracellular vesicle release via limiting membrane rupture of en bloc released amphisomes (amphiectosomes)
- Semmelweis University, Department of Genetics, Cell- and Immunobiology, Hungary
- ELTE Eötvös Loránd University, Department of Plant Physiology and Molecular Plant Biology, Hungary
- HUN-REN-SU Translational Extracellular Vesicle Research Group, Hungary
- Laboratory of Neuroimmunology, HUN-REN Institute of Experimental Medicine, Hungary
- ELTE Eötvös Loránd University, Department of Anatomy, Cell and Developmental Biology, Hungary
- Department of Image Analysis, 3DHISTECH Ltd, Hungary
- HCEMM-SU Extracellular Vesicle Research Group, Hungary, Hungary
Figures
Figure 1 with 1 supplement
Transmission electron microscopic detection of the release and extracellular fate of large, multivesicular extracellular vesicles (MV-lEVs) secreted by different cell lines and cells in mouse organs.
Major steps of the release of MV-lEVs were detected in the case of all tested cell lines including the immortal, non-tumorous HEK293T-PalmGFP (A, H, O), HEK293 (B, I, P), the tumorous cell lines …
Figure 1—figure supplement 1
Additional transmission electron micrographs of mouse kidney and liver sections.
For further evaluation of the presence of different extracellular vesicles (EVs) in mouse kidney (A–E) and liver (F–I), low and high magnification images are shown. Presence of individual small and …
Figure 2 with 6 supplements
Detection of conventional small extracellular vesicle (sEV) markers and the LC3 protein in HEK293T-PalmGFP cell-derived EVs.
Widely used sEV markers (CD63, CD81, ALIX, and TSG101) and LC3B were tested in multivesicular large EVs (MV-lEVs) found in the microenvironment of the releasing cells by confocal microscopy after in …
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Figure 2—source data 1
XLSX file containing data points of Figure 2G, H, I, J, K, L, M, and S.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Localization of GFP signal in HEK293T-PalmGFP cells.
The GFP positivity of the HEK293TPalmGFP cells was controlled by immunofluorescence microscopy on control (A) and 30 µM Chloroquine-treated (B) cells. Co-localization of PalmGFP and the anti-GFP …
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Figure 2—figure supplement 1—source data 1
XLSX file containing data points of Figure 2-figure supplement 1C.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Confocal microscopic images of amphiectosome release by HT29 and HepG2 cells.
The intraluminal extracellular vesicles (EVs) of multivesicular large EVs (MV-lEVs) were found to be positive for LC3B and CD81 in HepG2 (A) and HT29 cells (D). The presence of ALIX/CD81 (B) and …
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Figure 2—figure supplement 2—source data 1
XLSX file containing data points of Figure 2—figure supplement 2I.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
Additional confocal microscopic images of H9c2, HEK293T-PalmGFP, and HepG2 cells-derived multivesicular large extracellular vesicles (MV-lEVs).
Released amphiectosomes of H9c2 rat cardiomyoblast cell line were captured. They contain CD63, CD81, or LC3B positive intraluminal vesicles (ILVs) (A, B). The MV-lEVs released by HEK293T-PalmGFP (C) …
Figure 2—figure supplement 4
Qualitative western blot validation of antibodies used in immunofluorescence detection.
Whole-cell lysates from three distinct human cell lines (HEK293, HepG2, and HT29) were employed in the validation process to reduce the cell line-specific variations in this qualitative study. …
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Figure 2—figure supplement 4—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp4-data1-v1.zip
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Figure 2—figure supplement 4—source data 2
Original western blots for Figure 2—figure supplement 4, indicating the relevant bands and cell lines.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp4-data2-v1.zip
Figure 2—figure supplement 5
Characterization of the in-house generated HEK293T-PalmGFP-LC3RFP cell line.
Confocal microscopy (A) and western blot analysis (B) were employed to characterize the HEK293T-PalmGFP-LC3RFP cell line. For overnight Chloroquine treatment, 30 μM Chloroquine was applied. Punctate …
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Figure 2—figure supplement 5—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 5B.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp5-data1-v1.zip
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Figure 2—figure supplement 5—source data 2
Original western blots for Figure 2—figure supplement 5B, indicating the relevant bands, cell lines, and treatments.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp5-data2-v1.zip
Figure 2—figure supplement 6
Structures involved in amphiectosome release.
Multivesicular body (MVB, A), autophagosome (B), amphisome (C), amphiectosome (D), and secreted small extracellular vesicles (sEVs) (E) were identified by transmission electron microscopy (TEM) with …
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Figure 2—figure supplement 6—source data 1
XLSX file containing data points of Figure 2—figure supplement 6Q and R.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig2-figsupp6-data1-v1.xlsx
Figure 3 with 1 supplement
Amphiectosome release and its modulation.
Based on our data, a model of amphiectosome release was generated (A). Panel A was created with BioRender.com. According to this model, the fusion of multivesicular bodies (MVBs) and autophagosomes …
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Figure 3—source data 1
XLSX file containing data points of Figure 2I and J.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig3-data1-v1.xlsx
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3J.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig3-data2-v1.zip
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Figure 3—source data 3
Original western blots for Figure 3J, indicating the relevant bands, cell lines, and treatments.
- https://cdn.elifesciences.org/articles/95828/elife-95828-fig3-data3-v1.zip
Figure 3—figure supplement 1
Supporting information for treatments and size distribution of multivesicular large extracellular vesicles (MV-lEVs).
Relative metabolic activity was assessed through a Resazurin assay (A–E) during treatment optimization. The red dashed line indicates 100% metabolic activity, representing control cells. Results are …
Figure 4
Comparison of amphiectosomes and migrasomes.
Commonly used small extracellular vesicle (sEV) markers (CD63, CD81) and TSPAN4, a suggested migrasome marker, were tested in in situ fixed intact multivesicular large EVs (MV-lEVs) of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293 human kidney(embryonic) | ECACC (Sigma) | #85120602 RRID:CVCL_0045 | Batch No: 18E026 |
| Cell line (Homo sapiens) | HT29 Caucasian colon adenocarcinoma grade II | ECACC (Sigma) | #91072201 RRID:CVCL_0320 | Batch No: 09K003 |
| Cell line (Homo sapiens) | HepG2 human hepatocyte carcinoma | ECACC (Sigma) | #85011430 RRID:CVCL_0027 | Batch No: 19B009 |
| Cell line (Mus musculus) | HL1 mouse cardiomyocyte cell line, atrial | Merck | # SCC065 RRID:CVCL_0303 | Batch No: RD1601001 |
| Cell line (Homo sapiens) | HEK293TPalmGFP human kidney(embryonic) expressing palmitoylated GFP | Kind gift of Charles Lai https://doi.org:10.103 8/ncomms8029 | Resorted before MCB preparation | |
| Cell line (Homo sapiens) | HEK293T- PalmGFPLC3RFP human kidney(embryonic) expressing palmitoylated GFP and RFP tagged LC3 | This paper | See Materials and methods | |
| Cell line (Mus musculus) | BMMC bone marrow-derived mast cells | Primary cell culture https://doi.org/10.1 002/jev2.12023 | ||
| Cell line (Rattus norvegicus) | H9C2 (2-1) rat cardiovascular , Myoblast | ECACC (Sigma) | #88092904, RRID:CVCL_0286 | Batch No: 17A028 |
| Transfected construct | LentiBrite RFP-LC3 Lentiviral Biosensor | Merck | 17-10143 | Batch No: 3530171 |
| Biological sample (Mus musculus) | own animal house | C57BL/6 RRID:MGI:2159769 | male, 12 weeks of age | |
| Antibody | rabbit polyclonal anti-CD63 (Cterminal) | Sigma/Merck | SAB2109138 | IF (1:200) WB (1:500) |
| Antibody | mouse monoclonal anti-CD63 | Santa Cruz Biotechnology | MX-49.129.5 clone: sc-5275 RRID:AB_627877 | IF (1:200) TEM (1:50) WB (1:1000) |
| Antibody | rabbit polyclonal anti-CD81 | Sigma/Merck | SAB3500454 RRID:AB_10640751 | IF (1:200) WB (1:2500) |
| Antibody | mouse monoclonal anti-CD81 | Invitrogen | MA5-13548 clone: 1.3.3.22 RRID:AB_10987151 | IF (1:100) WB (1:100) |
| Antibody | rabbit polyclonal anti-TSG101 | Sigma/Merck | HPA006161 RRID:AB_1080408 | IF (1:200) WB (1:1000) |
| Antibody | rabbit polyclonal anti-ALIX (Cterminal) | Sigma/Merck | SAB420047 | IF (1:200) WB (1:1000) |
| Antibody | rabbit monoclonal anti-LC3B | Sigma/Merck | ZRB100 clone: 12K5 | IF (1:200) TEM (1:50) WB (1:1000) |
| Antibody | rabbit monoclonal anti-LC3A | Sigma/Merck | ZRB1125 clone: 3J12 | IF (1:200) WB (1:1000) |
| Antibody | rabbit polyclonal anti-TSPAN4 | Bioss | BS-9413R | IF (1:200) |
| Antibody | mouse monoclonal anti-Rab7 | Sigma/Merck | R8779 clone: Rab7117 RRID:AB_609910 | IF (1:200) WB (1:1000) |
| Antibody | mouse monoclonal anti-α-tubulin | Sigma/Merck | T9026 clone: DM1A RRID:AB_477593 | IF (1:200) |
| Antibody | mouse monoclonal anti-GFP | Sigma/Merck | G6539 clone: GFP-20 RRID:AB_259941 | IF (1:200) WB (1:1000) |
| Antibody | mouse monoclonal anti-RFP | Invitrogen | MA5-15257 clone: RF5R RRID:AB_10999796 | WB (1:1000) |
| Antibody | goat antimouse IgGATTO550 | Sigma/Merck | 43394 RRID:AB_1137651 | IF (1:1000) |
| Antibody | goat antirabbit IgG- ATTO647N | Sigma/Merck | 40839 RRID:AB_1137669 | IF (1:1000) |
| Antibody | goat antimouse Star 635P | Abberior | ST635P-1001–500UG RRID:AB_2893232 | IF (1:500) |
| Antibody | goat antirabbit Star 580 | Abberior | ST580-1002-500UG RRID:AB_2910107 | IF (1:500) |
| Antibody | goat polyclonal anti-rabbit IgG Fc (HRP) | abcam | ab97200 RRID:AB_10679899 | WB (1:10,000) |
| Antibody | goat polyclonal anti-mouse IgG Fc (HRP) | abcam | ab97265 RRID:AB_10680426 | WB (1:10,000) |
| Antibody | goat polyclonal anti-rabbit IgG (whole molecule) 10 nm gold preadsorbed | abcam | ab27234 RRID:AB_954427 | TEM (1:50) |
| Antibody | goat polyclonal anti-mouse IgG (whole molecule) 5 nm gold preadsorbed | Sigma/Merck | G7527 RRID:AB_259955 | TEM (1:50) |
| Other | CF488A conjugated Wheat Germ Agglutinin (WGA) | Biotium | 29022-1 | Lot Number: 21C0224-1149057 |
| Chemical compound, drug | Bafilomycin A1 | Sigma/Merck | B1793 | Lot Number: 0000190389 |
| Chemical compound, drug | Colchicine | Serva | 77120.02 | Lot Number: 190300 |
| Chemical compound, drug | Chloroquine diphosphate | Invitrogen | P36236 C | Lot Number: 2441325 |
| Chemical compound, drug | Rapamycin | Sigma/Merck | R0395 | Lot Number: 0000084976 |
| Chemical compound, drug | Cytochalasin B | Sigma | C2743 | Lot Number: 037M4083V |
| Chemical compound, drug | FBS | Biosera | FB-1090/500 | Lot Number: 015BS575 |
| Other | TFF Easy | HansaBioMed Life Sciences | HBM-TFF/1 | |
| Software, algorithm | LASX | Leica | Leica Application Suite X 3.5.5.19976 | |
| Software, algorithm | ZEN Blue | Zeiss | ZEN 2.3 lite | |
| Software, algorithm | iTEM | Olympus | iTEM 5.1 | |
| Software, algorithm | ImageJ | https://imagej.n et/ij/ | v1.54g | |
| Software, algorithm | Prism9 | GraphPad | GraphPad Prism 9.4.1 | |
| Software, algorithm | BioRender | https://www.biorender.com/ |
