Transmission electron microscopic detection of the release and extracellular fate of large, multivesicular EVs secreted by different cell lines and cells in mouse organs

Major steps of the release of large, multivesicular EVs 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 HepG2 (C,J,Q) and HT29 (D,K,R), the beating cardiomyocyte cell line HL1 (E,L,S) and the primary suspension of bone marrow derived mast cells (BMMCs) (F,M,T). The different phases of EV secretion were also captured in the circulation of mouse kidney (V) and liver (W,X). According to the electron micrographs, we found evidence for the budding (A-G,X) and secretion (H-N,V,W) of the multivesicular large EVs (MV-lEVs). We also detected the extracellular rupture of the limiting membrane of the released MV-lEVs with the escape of the intraluminal vesicles (ILVs) by a “torn bag mechanism” (O-U,V). Although it is not always clear whether the secreted MV-lEVs have a single or double limiting membrane, several micrographs suggest the presence of the double membrane (Y-AF) in the secreted MV-lEVs. In the case of BMMCs (Y), the release phase of a multivesicular structure is captured. The bottom portion of this structure embedded in the cytoplasm is surrounded by a single membrane (white arrow head) while the upper (budding) portion is covered by double membrane (asterisk).

Detection of conventional 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 MV-lEVs found in the microenvironment of the releasing cells by confocal microscopy after in situ fixation (A-F). Normalized fluorescence intensities were calculated in order to determine the relative localization of the limiting membrane (PalmGFP), the conventional sEV markers and the LC3B signal (G-L). Fluorescence intensity peaks of sEV markers were largely overlapping with each other, while the LC3B signal and the sEV markers showed separation. Co-localization rates were also calculated (M). The sEV markers co-localized with one another as no significant difference was found among them. In contrast, low co-localization rates were detected between the “classical” sEV markers and LC3B (one-way ANOVA, p<0.0001, n=8-26 confocal images). Real time release of LC3 positive sEVs by the “torn bag mechanism” was studied in the case of HEK293T-PalmGFP-LC3RFP cells by Elyra7 SIM2 super-resolution live cell imaging (N,O). Images were recorded continuously, and selected serial time points are shown. LC3 positive, red fluorescent small particles were released within a 5 min timeframe (O) and are indicated by white arrows. Presence of CD63 and LC3B were detected in the case of a separated sEV fraction using immunogold TEM. HEK293T-PalmGFP derived sEV fraction is shown by negative-positive contrast without immune labelling (P). In double-labelled immunogold TEM images (Q,R), distinct LC3B positive (Q) and CD63 positive (R) sEVs were found. However, CD63-LC3B double positive EVs were not detected. Black arrowheads indicate 10 nm gold particles identifying LC3B, while white arrowheads show 5 nm gold particles corresponding to the presence of CD63. Quantitative analysis of TEM images was performed (S), and the diameters of different EV populations were determined. The LC3B negative population was significantly smaller than the LC3B positive one (p<0.0001, t-test; n=79-100). No difference was detected when the immunogold labelled sEV fraction (either LC3B positive or negative, LC3B+/-) and the unlabeled sEV fraction (sEV) were compared (p<0.05, t-test, n=112-179).

Confocal microscopic analysis of HEK293T-PalmGFP and HepG2 cells derived lEVs; HEK293T-PalmGFP cells

The MV-lEVs released by HEK293T-PalmGFP (A) and HepG2 cells (B) were tested for CD63 sEV and Rab7 late endosomal markers. Both CD63 and Rab7 were present in the ILVs. The GFP positivity of the HEK293T-PalmGFP cells was controlled by immunofluorescence microscopy on control (C) and Chloroquine-treated (D) cells. Co-localization of Palm-GFP and the anti-GFP signal was calculated in the plasma membrane and in the cytoplasm (E) and Pearson’s correlations were visualized. The green fluorescence in the plasma membrane was clearly GFP-dependent in the case of control (Ctrl Membrane) and Chloroquine-treated (Chloro Membrane) samples, while in the cytoplasm, the correlation was significantly weaker (Ctrl Cytoplasm and Chloro Cytoplasm). Within the cytoplasm, the correlation was stronger in Chloroquine-treated cells, suggesting that the phagophore membrane may accumulate Palm-GFP (p < 0.0001, t-test; n = 45).

Confocal microscopic images of amphiectosome release by HT29 and HepG2 cells

The intraluminal EVs of amphiectosomes were found to be positive for LC3B and CD81 in HT29 cultures (A) and HepG2 cells (H). In the case of HT29 cells, phases of the “torn bag mechanism” were captured, including a secreted intact amphiectosome (B), MV-lEV with ruptured limiting membrane releasing internal vesicles (C, white arrow), an inside-out secreted amphiectosome (D, white arrow), and an amphiectosome with a fully disintegrated limiting membrane and released sEVs (E). The presence of TSG101/CD63 (F), ALIX/CD81 (G), and LC3B/CD81 (H) was also examined in the released amphiectosomes of the HepG2 cell line.

Western blot validation of antibodies used in immune-fluorescence detection

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 the study. Protein bands lacking posttranslational modifications are noted as “w/o PTM.” Numerous posttranslational modifications of CD63 and CD81 are widely recognized.

Characterization of the 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 treatments, 30 μM Chloroquine was applied. Punctate LC3 fluorescence observed in (A) corresponds to autophagosomes. In panel (B), whole-cell lysates from HEK293T-PalmGFP (lane i), HEK293T-PalmGFP-LC3RFP (lane ii) and Chloroquine-treated HEK293T-PalmGFP-LC3RFP (lane iii) samples were examined. Gray arrows indicate LC3I, while black arrows highlight LC3II. Full Western Blot images can be find in Fig.3_S2 supplementary figure.

Structures involved in amphiectosome release

Multivesicular body (MVB, A), autophagosome (B), amphisome (C), amphiectosome (D) and secreted sEVs (E) were identified by TEM with and without immunogold labelling of HEK293T-PalmGFP cell cultures. White arrowheads (5 nm gold particles) indicate CD63 and black arrowheads (10 nm gold particles) show LC3B. While MVBs (F) were LC3B negative (K), we detected CD63 positivity on the surface of the ILVs (K). In an autophagosome (G), the limiting membrane layers were double positive for CD63 and LC3B (L). In contrast, the internal membranes of autophagosome were CD63 single positive (L). In the case of an amphisome (H), heterogeneous membrane structures were visible with variable size and morphology. The ILVs were either CD63 or LC3B positive (M). The amphiectosomes were located in the extracellular space and contained ILVs of different size and shape (I). The ILVs of amphiectosome (as in case of amphisome), were either CD63 or LC3B positive (N). Secreted sEVs (J) with immunogold labelling (O), were also found to be either CD63 or LC3B positive. Release of an amphiectosome is shown (P) with CD63 and LC3B immunogold signals. Higher magnification of the insert is indicated by the black rectangle. It shows either CD63 or LC3B positive ILVs. Size distributions of ILVs of MVBs, amphisomes and amphiectosomes were determined on Epon-embedded ultrathin sections (Q). Although the ILV sizes differed significantly (one-way ANOVA, ****: p<0.0001, n=73, 138 and 595, respectively), the majority of ILVs had a diameter between 40-100 nm. The diameter of LC3B positive and negative ILVs of amphiectosomes was assessed on TEM images of immunogold labelled ultrathin sections (R). LC3B negative ILVs were significantly smaller than the LC3B positive ones, while the ILVs in the Epon-embedded sections did not differ from the LC3B positive ones (one-way ANOVA, p <0.001, n= 595, 101 and 70, respectively).

Amphiectosome release and its modulation

Based on our data, a model of amphiectosome release was generated (A). According to this model, the fusion of MVBs and autophagosomes forms amphisomes. The LC3B positive membrane layer (indicated in orange) undergoes disintegration and forms LC3B positive ILVs inside the amphisome. Later, the amphisome is released into the extracellular space by ectocytosis and can be identified extracellularly as an amphiectosome. Finally, the limiting membrane(s) of the amphiectosome is ruptured and the ILVs are released as sEVs into the extracellular space by a “torn bag mechanism”. In order to further support our model on amphiectosome release and “torn bag” EV secretion different in vitro treatments were applied. Cytochalasin B, Colchicine, Chloroquine, Bafilomycin A1 and Rapamycin were used to modulate amphisome release. Points of actions are summarized (B). While Cytochalasin B inhibits actin-dependent membrane budding and cell migration, Colchicine blocks the microtubule-dependent intracellular trafficking. Chloroquine and Bafilomycin have similar, Rapamycin have opposite effects on lysosome-autophagosome or lysosome-amphisome fusion. Chloroquine and Bafilomycin inhibits lysosomal degradation while Rapamycin accelerates it. Based on confocal microscopy, Cytochalasin B (CytoB) did not alter the dynamics of amphiectosome release (C). In contrast, both Colchicine (Colch) and Rapamycin (Rapa) significantly inhibited the release of amphiectosomes, while Chloroquine (Chloro) and Bafilomycin (Bafilo) increased the release frequency. Between Chloroquine and Bafilomycin effect significant difference was not detected (C). Results are shown as mean ± SD of 3-4 independent biological replicates, analyzed by one-way ANOVA, *: p<0.05, **: p<0.01, ns: non-significant. Presence of membrane-bound (lipidated) LC3II was tested by Western blotting. The total protein content of serum-, cell- and large EV-depleted conditioned medium of HEK293T-PalmGFP (PalmGFP) and HEK293T-PalmGFP-LC3RFP (PalmGFP-LC3RFP) cells was purified by precipitation and 20 µg of the protein samples were loaded on the gel (D). The lipidated LC3II band was detected in all cases. Relative expression of control (Ctrl) and Chloro treated samples were determined by densitometry. Chloroquine treatment increased the LC3II level by approximately by two fold. Results are shown as mean ± SD of n=6 biological replicates.

Dose determination for treatments and size distribution of 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 presented as mean ± SD values of n=3-4 independent biological replicates. Student’s unpaired t-test was performed to compare control and treated cells (*: p<0.05, **: p<0.01, ****: p<0.0001). For Colchicine treatment, alterations in the microtubular network were observed through immunocytochemistry (F) and documented using an epifluorescent microscope. Changes in the diameter of released amphiectosomes under various treatments were determined on confocal microscopy images. A significant reduction in size was identified only in the case of Rapamycin treatment (G, one-way ANOVA test *: p<0.05, n= 95-101).

Unedited Western blots of Fig.2_S3 and Fig.3

Subfigure A displays the unedited Western blots utilized for the quantification in Fig.3D. Black arrows indicate the location of LC3I, while red squares represent the quantified LC3II. In Subfigure B, the unedited blots from Fig.2_S3B are presented. Black arrows denote the LC3I-RFP fusion protein, while red arrows indicate the LC3II-RFP fusion protein. The blue arrow points to the native LC3I, while the green arrow represents the native LC3II protein.

Comparison of amphiectosomes and migrasomes

Commonly used sEV markers (CD63, CD81) and TSPAN4, a suggested migrasome marker, were tested in in situ fixed intact MV-lEVs of HEK293T-PalmGFP (A,B) and HT29 (C-F) cells by confocal microscopy. Normalized fluorescence intensities were calculated in order to determine the relative localization of the limiting membrane (with PalmGFP or lactadherin labelling) and the CD63/TSPAN4 and CD81/TSPAN4 markers (G-L). In the case of HEK293T-PalmGFP-derived EVs, there were no “classical migrasomes” with TSPAN4 in their limiting membrane were found. The TSPAN4 signal was only detected intraluminally in the MV-lEVs. The limiting membranes of HT29-derived MV-lEVs were either TSPAN4 positive and negative. The co-localization rate between the limiting membrane and TSPAN4 was low in case of HEK293T-PalmGFP-derived EVs. In the case of HT29 cells, two MV-lEV populations were identified: one with low and one with high co-localization rates (M). Live cell imaging of HEK293T-PalmGFP-LC3RFP cells showed migrasomes-like (retraction fiber-associated) MV-lEVs with or without intraluminal LC3 positivity (N,O). Using TEM, we could identify structures with retraction fiber-associated migrasome morphology in the case of HL1 (P), HEK293T-PalmGFP (Q) and BMMC (R) cells. For comparison, budding of amphiectosomes of the same HL1 (S), HEK293T-PalmGFP (T) and BMMC cells (U) are shown (without being associated with long retractions fibers).