Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.
(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.
To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.
Transactivation response DNA-binding protein 43 (TDP-43) is a widely expressed heterogeneous nuclear ribonucleoprotein involved in regulating exon splicing, gene transcription, and mRNA stability, all vital for cell survival1,2. In neurodegenerative conditions like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a nuclear protein TDP-43 abnormally accumulates in the cytoplasm. This shift results in a loss of TDP-43 function in the nucleus and a toxic gain-of-function in the cytoplasm. Pathological accumulation of TDP-43 begins in specific regions of the brain and spinal cord, spreading through these areas in a prion-like fashion, a process closely associated with disease progression3. However, the exact mechanism by which TDP-43 pathology spreads through the brain and spinal cord remains unknown.
TDP-43 is secreted through extracellular vesicles (EVs), and elevated levels of TDP-43 are detected in the plasma and cerebrospinal fluid (CSF) of ALS and FTLD-TDP patients4,5,6. CSF from patients diagnosed with ALS and FTLD induces intracellular mislocalization and aggregation of endogenous TDP-43 in human glioma cells7. Thus, TDP-43 released extracellularly via EVs may mediate the cell-to-cell spreading of TDP-43 pathology.
Autophagy is a well-preserved cellular degradation mechanism that involves the enclosure of unwanted substances within double-membrane vesicles, known as autophagosomes, which are marked by LC3. These autophagosomes fuse with lysosomal-associated membrane protein 1 (LAMP1)-positive lysosomes to form autolysosomes (LC3+/LAMP1+), leading to the degradation of their contents8. Histological analyses indicate that autophagosomes engulfing inclusions accumulate in the neurons of sporadic ALS patients9. Some causal genes of familial ALS and FTLD-TDP are linked to the regulation of autophagy10,11,12. These findings suggest that autophagy is suppressed in ALS and FTLD-TDP patients.
The previous study indicated that TDP-43 is secreted via EVs positive for the autophagosome marker LC3-II when autophagosome-lysosome fusion is inhibited13. Dysregulation of the autophagy-lysosome pathway might cause not only the intracellular accumulation of TDP-43 but also the extracellular release of TDP-43 via EVs14. However, it remains unknown how LC3-II positive EVs are released and how significant this is in the spreading of TDP-43 pathology.
EVs are classified into large EVs (100 to 1000 nm in diameter), which are produced from cell-surface budding, and small EVs (50 to 150 nm in diameter), which are produced from the budding of endosomal membranes toward the interior of the endosome (known as exosomes) and the Golgi apparatus. To separately collect large and small EVs, we perform sequential centrifugation and collect pellets by centrifugation at 20,000 × g and 110,000 × g, respectively. The P1 EV fraction (20,000 × g pellets) is prepared to collect large EVs, and the P2 EV fraction (110,000 × g pellets) is prepared to collect small EVs15. The methodology to assess LC3-II levels in large and small EVs derived from sequential centrifugation by immunoblotting is stable and reproducible16. In addition to analyzing LC3-II levels in EVs, analyzing autolysosome and omegasome formation enhances understanding of how dysregulation of autophagy leads to the release of LC3-II positive EVs. Here, the present study shows the suppressive role of syntaxin 6 (STX6), a SNARE protein, which promotes the movement of transport vesicles to target membranes17, in the release of LC3-II positive EVs when autophagosome-lysosome fusion is inhibited.
1. Preparation of the cell, P1, and P2 EV fraction from the cultured medium of HeLa cells
2. Immunoblotting analysis
3. Analysis of the number of autophagosomes and autolysosomes
4. Analysis for omegasome formation
As shown in previous studies, Baf treatment increased the levels of TSG101 (P1: P < 0.01, P2: P = 0.012, as determined by two-way ANOVA in EZR19) and LC3-II (P1: P < 0.01, P2: P < 0.01, as determined by two-way ANOVA in EZR19) in the EV-rich fraction. Notably, Baf treatment also increased the levels of STX6 (P1: P < 0.01, P2: P < 0.01, as determined by two-way ANOVA in EZR19) in the EV fraction (Figure 1A), suggesting that STX6 may be a component of EVs. Proliferating cell nuclear antigen (PCNA) was not observed in the EV fraction. Immunoblotting of STX6 in the cell fraction revealed that STX6 levels decreased by half with its siRNA knockdown (Figure 1B). STX6 knockdown did not affect TSG101 levels in the cell and EV fractions under both vehicle- and Baf-treated conditions (Figure 1C). However, STX6 knockdown increased LC3-II levels in the cell fraction under the vehicle-treated condition, but not under the Baf-treated condition. STX6 knockdown also increased LC3-II levels in the EV fraction under the Baf-treated condition (Figure 1D). These findings suggest that the methods used to detect LC3-II in EVs by immunoblotting are sufficiently sensitive, even if the knockdown efficiency is not substantial. Additionally, these results indicate that STX6 knockdown increases intracellular autophagosome numbers and enhances the extracellular release of autophagosome-associated vesicles under Baf treatment.
To further investigate whether STX6 knockdown influences intracellular autophagosome and autolysosome numbers, autophagosomes and lysosomes were visualized using the autophagosome marker mCherry-LC3 and the lysosome marker LAMP1-GFP. Confocal live-cell imaging revealed GFP- and mCherry-positive vesicles in HeLa cells expressing LAMP1-GFP and mCherry-LC3 (Figure 2A). Autolysosomes were identified as GFP- and mCherry-positive vesicles, while autophagosomes were identified as GFP-negative and mCherry-positive vesicles. STX6 knockdown increased the number of autophagosomes and autolysosomes under the vehicle-treated condition (Figure 2B,C). However, STX6 knockdown did not affect autophagosome and autolysosome numbers under the Baf-treated condition (Figure 2B,C).
To determine whether the increase in intracellular autophagosome numbers due to STX6 knockdown resulted from enhanced autophagosome formation, omegasome formation, a process required for autophagosome formation, was examined using the omegasome marker GFP-ATG13 signal intensity. Confocal live-cell imaging provided an assembled image of GFP-ATG13, indicating omegasome formation (Figure 3A). STX6 knockdown decreased GFP-ATG13 signal intensity (P = 0.023, as determined by two-way ANOVA in EZR19). While STX6 knockdown did not significantly influence GFP-ATG13 signal intensity under the vehicle-treated condition, it significantly decreased the signal under Baf-treated conditions (Figure 3B). Collectively, these findings indicate that STX6 knockdown increases the release of LC3-II positive vesicles linked to the accumulation of autophagosome-associated vesicles.
Figure 1: Immunoblotting results of the cell, P1, and P2 fractions from HeLa cells transfected with either control or STX6 siRNA under vehicle or Baf treatment. HeLa cells were treated with vehicle (DMSO) or 100 nM Baf for 24 h after transfection with control or STX6 siRNA. The cell, P1 (20,000 × g pellet), and P2 (110,000 × g pellet) fractions were isolated. (A) Representative immunoblotting images of each fraction showing STX6, TSG101, LC3, and PCNA. (B-D) Densitometric analysis for (B) STX6, (C) TSG101, and (D) LC3-II was performed from the immunoblot images. Signal intensities were normalized to the control or STX6 siRNA-transfected cells treated with vehicle or Baf. The bar graphs represent mean values ± S.E.M. (N = 3). * indicates P < 0.05 by a two-tailed unpaired t-test in Excel. Abbreviations: STX6 = syntaxin 6; Baf = bafilomycin A1; DMSO = dimethyl sulfoxide; TSG101 = tumor susceptibility gene 101; LC3 = microtubule-associated proteins 1A/1B light chain 3B; PCNA = proliferating cell nuclear antigen; S.E.M. = standard error of the mean. Please click here to view a larger version of this figure.
Figure 2: Confocal image analysis of HeLa cells co-expressing Lamp1-GFP and mCherry-LC3 transfected with control or STX6 siRNA under the vehicle- and Baf-treated condition. (A) Confocal images of HeLa cells stably expressing LAMP1-GFP and mCherry-LC3 were transfected with control or STX6 siRNA for 48 h, followed by exposure to DMSO or 400 nM Baf for 4 h. Scale bar = 20 µm. (B,C) Qunatification of (B) autophagosomes (LAMP1-GFP negative and mCherry-LC3 positive dots) and (C) autolysosomes (LAMP1-GFP and mCherry-LC3 double-positive dots) was performed using Fiji software. Data in the bar graphs are presented as mean values ± S.E.M. (N = 3). * indicates P < 0.05, based on a two-tailed unpaired t-test in Excel. Abbreviations: Lamp1 = lysosomal-associated membrane protein 1; LC3 = microtubule-associated proteins 1A/1B light chain 3B; STX6 = syntaxin 6; Baf = bafilomycin A1; GFP = green fluorescent protein; S.E.M. = standard error of the mean. Please click here to view a larger version of this figure.
Figure 3: Confocal imaging of HeLa cells expressing GFP-ATG13, transfected with either control or STX6 siRNA, and subjected to vehicle or Baf treatment. (A) HeLa cells were transfected with control or STX6 siRNA, followed by pEGFP-C1-hAtg13 transfection, and treated with DMSO or 400 nM Baf for 4 h. Scale bar = 20 µm. (B) Relative signal intensities were determined by normalizing the signal in each condition to that of control siRNA-transfected cells treated with Baf. The Bar graph shows the data as means ± S.E.M. (N = 3). * indicates P < 0.05 by the two-tailed unpaired t-test in Excel. Abbreviations: hAtg13 = human autophagy-related gene 13; STX6 = syntaxin 6; Baf = bafilomycin A1; DMSO = dimethyl sulfoxide; S.E.M. = standard error of the mean. Please click here to view a larger version of this figure.
Supplementary Figure S1: Immunoblot analysis of the cell, P1, and P2 fractions from HeLa cells transfected with control siRNA and treated with Baf. HeLa cells were transfected with control siRNA and exposed to 100 nM Baf for 24 h. Fractions were then prepared: cell, P1 (20,000 × g pellet), and P2 (110,000 × g pellet). Representative immunoblot images of ULK1 and PCNA, showing their levels in each fraction for comparison. Abbreviations: Baf = bafilomycin A1; ULK1 = unc-51 like autophagy activating kinase 1; PCNA = proliferating cell nuclear antigen. Please click here to download this File.
The immunoblotting study revealed LC3-II and TSG101 levels in the cellular fraction, the microvesicle-rich P1 EV fraction, and the exosome-rich P2 EV fraction. Live-cell imaging was used to examine autophagosomes, autolysosomes, and omegasomes, providing insight into whether STX6 knockdown influences autophagy. These combined results suggest that STX6 knockdown affects the release of LC3-II positive EVs, potentially linked to dysregulation of the autophagy pathway. A key advantage of this method is its ability to distinguish between large and small EVs originating from different sources. Flow cytometry analysis using specific markers for microvesicles, exosomes, or the Golgi apparatus may further clarify how STX6 knockdown influences the release of LC3-II positive vesicles.
However, a limitation of the immunoblotting study is that it remains unclear whether the observed increase in LC3-II levels in the EV fraction following STX6 knockdown under Baf treatment is due to an increased number of LC3-II positive vesicles being released or an increase in LC3-II levels per EV. To distinguish between these possibilities, counting vesicles using nanoparticle tracking analysis or flow cytometry analysis might help determine whether the number of LC3-II positive vesicles is increased or if LC3-II levels per vesicle are elevated.
A critical step in the protocol is the fractionation of the cultured medium to prepare the EV fraction. EVs are observed as tiny pellets in the centrifugation tube. To prepare a comparable EV fraction, it is important to carefully transfer or discard supernatant after centrifugation to avoid diluting the protein concentration of the EV pellets. To examine contamination from the cell fraction into the EV fraction, the levels of a cytoplasmic protein, unc-51-like autophagy activating kinase 1 (ULK1), and a nuclear protein, PCNA were compared between the fractions in this study (Supplementary Figure S1). The protein concentration in the cell fraction generally reflects cell numbers, but cellular protein concentration is influenced by cell-cycle stages20 or disturbances in proteostasis21. Autophagy is crucial for maintaining proteostasis22. Dysregulation of autophagy by STX6 knockdown or Baf treatment might alter protein concentration regardless of cell number.
STX6 knockdown is achieved through siRNA transfection, and overexpression of GFP-ATG13 is accomplished via DNA transfection. Transfection efficiency is influenced by cell type, density, and numbers. Efficient alignment of siRNA sequences might also affect knockdown efficiency. Adjustments are necessary when transfection efficiency is low.
The present results indicate that STX6 knockdown increased levels of the autophagosome marker LC3-II in the EV fraction under Baf-treated conditions but did not affect levels of TSG101. This suggests that STX6 suppresses the release of vesicles derived from autophagosomes, but not from MVBs, when autophagosome-lysosome fusion is inhibited.
STX6 knockdown also increased cellular LC3-II levels under growth conditions, suggesting that STX6 regulates autophagy. Indeed, STX6 knockdown increased the number of autophagosomes and autolysosomes, while it did not enhance omegasome formation. Maturation of autolysosomes causes autophagic lysosome reformation23. It is possible that STX6 mediates the maturation process of autolysosomes, and its loss of function might increase the number of immature autolysosomes, leading to dysregulation of autophagosome-lysosome fusion due to suppression of autophagic lysosome reformation.
Baf treatment increased STX6 levels in the EV fraction, even when its expression was downregulated. Previous studies suggested that autophagosome-associated vesicles are released as EVs when lysosomal function is inhibited13,14. STX6 is known to localize to autophagosome-associated vesicles, such as Group A Streptococcus (GAS)-containing autophagosome-like vacuoles24. Therefore, STX6 might be localized to autophagosome-associated vesicles that are released as EVs when autophagosome-lysosome fusion is inhibited.
The spread of TDP-43 pathology is closely linked to the progression of ALS and FTLD-TDP3. Although it remains unclear how TDP-43 pathology spreads through the brain and spinal cord, the EV pathway may mediate its spread. Previous studies have indicated that dysregulation of the autophagy-lysosome pathway results in the release of LC3-II positive EVs containing TDP-4313. The present study suggests a role for STX6 in regulating EV release when autophagosome-lysosome fusion is inhibited. The methodology for assessing LC3-II levels in the cell and EV fractions by immunoblotting should prove helpful in future studies.
The authors have nothing to disclose.
This work was supported by funding to Y.T. from Japan Society for the Promotion of Science KAKENHI [Grant Number 23K06837] (Tokyo, Japan) and Takeda Science Foundation (Osaka, Japan). The authors appreciate Dr. David C. Rubinsztein (Cambridge Institute for Medical Research, Cambridge, UK) for supplying HeLa cells.
0.25 % Trypsin EDTA | Fujifilm Wako | 201-16945 | |
10 cm Dish | Thermo Fisher Scientific | 150464 | |
15 mL Tube | Thermo Fisher Scientific | 339650 | |
200 μL Pipette Tip | Nippon Genetics | FG-301 | pipetting |
2-Mercaptoethanol | Nacalai Tesque | 21417-52 | a material for sample buffer solution |
3,3'-Diaminobenzidine Tetrahydrochloride | Nacalai Tesque | 11009-41 | a material for DAB solution |
3.5 cm Dish | Thermo Fisher Scientific | 150460 | |
6 cm Dish | TrueLine | TR4001 | |
Aluminium Block Thermostatic Baths (dry thermobaths) | EYELA | 273860 | |
Aspirator | SANSYO | SAP-102 | inhaling solution |
Avanti JXN-30 | Beckman Coulter | B34193 | |
Bafilomycin A1 | Adipogen | BVT-0252 | |
Biotin-conjugated Goat Anti-rabbit IgG Antibody | Vector Laboratories | BA-1000 | 2nd antibody for immunoblotting |
Biotin-conjugated Horse Antimouse IgG Antibody | Vector Laboratories | BA-2000 | 2nd antibody for immunoblotting |
Blocking One | Nacalai Tesque | 03953-95 | a material for immunoblotting |
Bromophenol Blue | Nacalai Tesque | 05808-61 | a material for sample buffer solution |
Calf Serum | cytiva | SH30073.03 | |
CanoScan LiDE 220 | Canon | CSLIDE220 | Scanner |
Centrifuge 5702 R | eppendolf | 5703000039 | |
Counting Slides Dual Chamber | Bio-Rad | 1450015J | cell counting |
Digital Sonifier 450 | BRANSON | ||
Dimethyl Sulfoxide | nacalai tasque | 09659-14 | vehicle |
DMEM High Glucose | Nacalai Tesque | 08458-45 | culture medium |
DMEM without Phenol Red | Nacalai Tesque | 08489-45 | culture medium |
EGTA | Dojindo | 348-01311 | a material for A68 solution |
Excel | Microsoft | version 16.16.27 | satistical analysis |
EZR | Reference No. 24 | version 1.68 | satistical analysis |
FBS | Sigma | 173012 | Culture medium |
Fiji | NIH | Image analysis tool | |
Glycerol | Nacalai Tesque | 09886-05 | a material for sample buffer solution |
Hoehst33342 | Dojindo | H342 | |
Hydrogen Peroxide | Fujifilm Wako | 080-0186 | a material for DAB solution |
Kimwipe S-200 | NIPPON PAPER CRECIA | 62011 | cleaning wipe |
Low Retention Tube | Nippon Genetics | FG-MCT015CLB | siRNA and DNA transfection |
LSM780 Confocal Laser Microscope | Carl Zeiss | ||
Monoclonal Mouse Anti-LC3 Antibody | MBL | M186-3 | 1st antibody for immunoblotting |
Nickel(II) Chloride Hexahydrate | Fujifilm Wako | 149-01041 | a material for DAB solution |
N-Lauroylsarcosine Sodium Salt | Nacalai Tesque | 20117-12 | |
Optima XE-90 Ultracentrifuge | Beckman Coulter | A94471 | |
Opti-MEM I Reduced Serum Medium | Thermo Fisher Scientific | 31985-070 | siRNA and DNA transfection |
pEGFP-C1-hAtg13 | Addgene | 22875 | |
Penicillin/Streptomycin | Nacalai Tesque | 26253-84 | Culture medium |
Pierce BCA Protein Assay Kits | Thermo Fisher Scientific | 23225 | |
Polyclonal Rabbit Anti-PCNA Antibody | BioAcademia | 70-080 | 1st antibody for immunoblotting |
Polyclonal Rabbit Anti-syntaxin 6 Antibody | ProteinTech | 10841-1-AP | 1st antibody for immunoblotting |
Polyclonal Rabbit Anti-TSG101 Antibody | ProteinTech | 28283-1-AP | 1st antibody for immunoblotting |
Polyclonal Rabbit Anti-ULK1 Antibody | ProteinTech | 20986-1-AP | 1st antibody for immunoblotting |
Polyvinylidene Difluoride Membrane | Milliore | IPVH00010 | a material for immunoblotting |
R | R development core team | version 4.4.1 | satistical analysis |
RNAiMAX | Thermo Fisher Scientific | 13778 | siRNA transfection reagent |
siRNA STX6 | Thermo Fisher Scientific | HSS115604 | siRNA for transfection |
Sodium Chloride | Nacalai Tesque | 31320-05 | a material for Tris buffer and A68 solution |
Sodium Dodecyl Sulfate | Fujifilm Wako | 192-13981 | a material for sample buffer solution |
SPARK Microplate Reader | TECAN | ||
Stealth RNAi Negative Control Duplexes, Med GC | Thermo Fisher Scientific | 12935300 | siRNA transfection |
Sucrose | Fujifilm Wako | 193-00025 | a material for A68 solution |
TC20 Automated Cell Counter with Thermal Printer | Bio-Rad | 1450109J1 | cell counting |
Thermobath | TOKYO RIKAKIKAI | MG-3100 | incubation |
TransIT-293 | Mirus Bio | MIR 2700 | DNA transfection reagent |
TransIT-LT1 | Mirus Bio | MIR2300 | DNA transfection reagent |
Tris(hydroxymethyl)aminomethane | Nacalai Tesque | 35406-75 | a material for Tris buffer, sample buffer and A68 solution |
Trypan Blue Dye 0.40% | Bio-Rad | 1450021 | cell counting |
Ultra-Clear Open-Top Tube, 16 x 96mm | Beckman Coulter | 361706 | collecting for the P1 EV fraction |
Ultra-Clear Tube, 14 x 89mm | Beckman Coulter | 344059 | collecting for the P2 EV fraction |
Vectastain ABC Standard Kit | Vector Laboratories | PK-4000 | immunoblotting |
Wash Bottle | As One | 1-4640-02 | washing membrane |
μ-Slide 8 Well High | ibidi | 80806 |
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