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.

1. Preparation of the cell, P1, and P2 EV fraction from the cultured medium of HeLa cells
<ol>
	<li>Preparation of the P1 and P2 EV fraction from the cultured medium of HeLa cells
	<ol>
		<li>Day 1
		<ol>
			<li>Count the cells using a cell counter and seed 1 &#215; 106 cells on a 6 cm plate containing a culture medium composed of DMEM High Glucose, 10% FBS, and 1% penicillin/streptomycin. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 2 (optional)
		<ol>
			<li>Prepare 20 &#181;M siRNA solution.</li>
			<li>Mix 100 &#181;L of reduced serum medium and 3 &#181;L of siRNA in a low-retention tube to prepare solution A.</li>
			<li>Mix 100 &#181;L of reduced serum medium and 5 &#181;L of transfection reagent in a low-retention tube to prepare solution B.</li>
			<li>Mix solution A and solution B, then incubate the mixture at room temperature (RT) for 5 min.</li>
			<li>Add the mixture of solutions A and B to the medium used for culturing HeLa cells. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 3
		<ol>
			<li>Wash the cells with PBS and expose them to 500 &#181;L of 0.25% trypsin and 1 mM EDTA solution at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
			<li>Add 2.5 mL of the culture medium to the cells, and use a Pasteur transfer pipette with a 200 &#181;L pipette tip attached to prepare a single-cell suspension.</li>
			<li>Count the cells using a cell counter, and seed 1 &#215; 106 cells on a 10 cm plate. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h. 
			NOTE: It is necessary to prepare at least 1 &#215; 106 cells to collect EVs from the culture medium required for immunoblotting analysis.</li>
		</ol>
		</li>
		<li>Day 4
		<ol>
			<li>Prepare culture medium (see step 1.1.1.1) containing either vehicle (DMSO) or 100 nM Bafilomycin A1 (Baf).</li>
			<li>Expose the cells to the medium containing either vehicle or 100 nM Baf and incubate them at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
		</ol>
		</li>
		<li>Day 5
		<ol>
			<li>Collect all the culture medium from the 10 cm plate, transfer it into a 15 mL tube, and centrifuge it at 3,000 &#215; g for 10 min at 4 &#176;C to remove cell debris. 
			NOTE: The remaining cells in the 10 cm plate will be used in section 1.2.1.</li>
			<li>For the isolation of the P1 EV fraction, transfer the supernatant after centrifugation at 3,000 &#215; g into a centrifugation tube, and then centrifuge it at 20,000 &#215; g at 4 &#176;C for 1 h.</li>
			<li>For the isolation of the P2 EV fraction, transfer the supernatant after centrifugation at 20,000 &#215; g into an ultracentrifuge tube, and then centrifuge it at 110,000 &#215; g at 4 &#176;C for 1 h.</li>
			<li>After wiping off the excess moisture inside the tube with a laboratory wipe, resuspend the remaining pellets in the centrifugation tube in 50 &#181;L of 2x sample buffer [2.5% SDS, 125mM Tris-HCl buffer (pH 6.8), 30% glycerol, 10% 2-mercaptoethanol, 0.4% Bromophenol Blue] to prepare the P1 EV fraction.</li>
			<li>Remove the supernatant by decanting, wipe off the excess moisture inside the tube with a laboratory wipe, and then resuspend the remaining pellets in the ultracentrifugation tube in 50 &#181;L of 2x sample buffer to prepare the P2 EV fraction.</li>
			<li>Incubate the P1 and P2 EV fractions at 100 &#176;C on a heat block for 10 min. 
			NOTE: Do not shake the tubes after centrifugation at 20,000 &#215; g and 110,000 &#215; g to avoid accidentally discarding the remaining pellets. After tilting the tube to transfer the supernatant, maintain the tube&#39;s position to prevent the pellet from being immersed in the remaining supernatant again.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of the cell fraction from cultured HeLa cells
	<ol>
		<li>Day 5
		<ol>
			<li>After transfer of the culture medium from the 10 cm plate into a 15 mL tube, wash the cells in the 10 cm dish with PBS and expose them to 2 mL of 0.25% trypsin and 1 mM EDTA solution at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
			<li>Add 8 mL of growth medium to the cells, then use a Pasteur transfer pipette with a 200 &#181;L pipette tip to pipette the cells and prepare a single-cell suspension.</li>
			<li>Transfer the cell suspension into a 15 mL tube and centrifuge it at 1,000 &#215; g for 10 min at 4 &#176;C.</li>
			<li>Remove the supernatant by an aspirator, add PBS to the cell pellet, and then centrifuge at 1,000 &#215; g for 5 min at 4 &#176;C.</li>
			<li>Remove the PBS using an aspirator and sonicate the cell pellet in 1 mL of A68 buffer [10 mM Tris-HCl buffer, pH 7.5, 0.8 M NaCl, 1 mM ethylene glycol bis(&#946;-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)] containing 1% sarkosyl.</li>
			<li>Measure the protein concentration of the cell lysate using a BCA protein assay kit.</li>
			<li>Mix 300 &#181;L of the cell lysate with 100 &#181;L of 4x sample buffer and incubate the mixture at 100 &#176;C on a heat block for 10 min to prepare the cell fraction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Immunoblotting analysis
<ol>
	<li>Separate equivalent amounts of proteins in the samples by SDS-PAGE18.</li>
	<li>Transfer the separated proteins onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>After transfer, block the membranes with the blocking agent for 20 min.</li>
	<li>Incubate the blocked membrane overnight with the indicated primary antibody in Tris buffer containing 10% (v/v) calf serum at RT.</li>
	<li>Wash the membrane with Tris buffer for 5 s and then incubate it with a biotin-conjugated secondary antibody at RT for 2 h.</li>
	<li>Wash the membrane with Tris buffer for 5 s and then incubate it with Tris buffer containing 0.4% (v/v) solutions A and solution B from the kit at RT for 1 h.</li>
	<li>Wash the membrane with PBS, and then incubate it with PBS containing 0.04% (w/v) diaminobenzidine, 0.8% (w/v) NiCl2&#183;6H2O, and 0.3% (v/v) H2O2 for colorimetric detection of bands. 
	NOTE: For the detection of signals, a chemiluminescence method is also acceptable.</li>
	<li>Digitize the image of the membrane by a scanner and subject it to densitometry analysis using Fiji.
	<ol>
		<li>Open the digitized image using Fiji, and convert the RGB color image to an 8-bit image by clicking Image | Type | 8-bit.</li>
		<li>Click on the rectangle tool and adjust the size of the rectangle by dragging it to surround the bands. Identify the band to be analyzed by clicking Analyze | Gels | Select First lane.</li>
		<li>Position the rectangle on the second and the subsequent bands, and identify the bands for analysis by clicking Analyze | Gels | Select Next Lane.</li>
		<li>After recognizing the final band, measure the densitometry across the rectangle by clicking Analyze | Gels | Plot Lanes.</li>
		<li>Surround the signal area of the band with a straight line, click the wand tool, and click in the area to calculate the signal intensity of the band.</li>
	</ol>
	</li>
</ol>
3. Analysis of the number of autophagosomes and autolysosomes
<ol>
	<li>Day 1
	<ol>
		<li>Count the number of HeLa cells using a cell counter, and seed 1 &#215; 105 HeLa cells expressing LAMP1-GFP and mCherry-LC3 on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 2 (optional)
	<ol>
		<li>Perform the steps described in section 1.1.2 in 3.5 cm dishes.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Discard the culture medium from the 3.5 cm dishes.</li>
		<li>Wash cells with PBS, expose them to 400 &#181;L of 0.25% trypsin and 1 mM EDTA solution, and incubate at 37 &#176;C with 5% CO2 and controlled humidity for 5 min.</li>
		<li>Add 1.6 mL of the growth medium to the cells, and pipette them with a Pasteur transfer pipette to prepare a single-cell suspension.</li>
		<li>Count the number of cells using the cell counter, and seed 1 &#215; 104 cells on an 8-chambered coverslip. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h. 
		NOTE: Two wells of the chambered coverslip are prepared for control and STX6 knockdown cells, respectively, following DMSO or Baf treatment as described in section 3.4.2.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Prepare the growth medium without phenol red (DMEM without Phenol Red, 10% FBS, 1% penicillin/streptomycin), containing either vehicle (DMSO) or 100 nM Baf dissolved in DMSO.</li>
		<li>Replace the culture medium with the medium without phenol red, containing either vehicle or 100 nM Baf, and incubate for 4 h. 
		NOTE: To assess the effect of Baf on autophagosome and autolysosome numbers, prepare vehicle-treated cells as controls.</li>
		<li>Stain the nuclei with 0.33 &#181;g/mL Hoechst 33342 for 30 min before observation.</li>
		<li>Conduct Live cell imaging using a 60x objective lens on a confocal laser microscope and capture ten different frames.
		<ol>
			<li>Open the RGB merged image in Fiji, and split it into the respective red, green, and blue image components by clicking on Image | Color | Split Channels.</li>
			<li>Set a constant threshold for the red and green signals in each image by clicking Image | Adjust | Threshold for each experiment.</li>
			<li>Count the number of areas positive for red or green signals by clicking Analyze | Analyze Particles. Check the Summarize box, then click OK. Record the values from the Count to identify autophagosome- and lysosome-associated vesicles.</li>
			<li>To overlay the green image on the red image, set the transfer mode to AND by clicking Edit | Paste Control. Copy the green image and then paste it onto the red image to merge them, highlighting areas that are positive for both red and green signals.</li>
			<li>To identify autolysosome numbers, count the number of areas positive for both red and green signals by clicking Analyze | Analyze Particles. Check the Summarize box, click OK, and record the values in the Count section to identify vesicles associated with autophagosomes and lysosomes.</li>
		</ol>
		</li>
		<li>Divide the total number of autolysosomes and autophagosomes by the total number of cells to calculate the number of autolysosomes and autophagosomes per cell. 
		NOTE: Count at least 35 cells in each of the three independent experiments.</li>
	</ol>
	</li>
</ol>
4. Analysis for omegasome formation
<ol>
	<li>Day 1
	<ol>
		<li>Count the number of HeLa cells using a cell counter, and seed 1 &#215; 105 cells on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 2 (optional)
	<ol>
		<li>Perform the steps described in section 1.1.2.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Prepare a single-cell suspension as described in steps 3.3.1-3.3.2.</li>
		<li>Count the cells using the cell counter, and seed 1 &#215; 105 cells on a 3.5 cm dish. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Mix 1 &#181;g of pEGFP-C1-hAtg13, 3 &#181;L of the DNA transfection reagent, and 100 &#181;L of reduced serum medium in a low retention tube. Incubate the mixture for 20 min, then add it to the cultured medium.</li>
		<li>Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 5
	<ol>
		<li>Trypsinize the cells as described in steps 3.3.1-3.3.2.</li>
		<li>Add 2.5 mL of the growth medium to the cells. Pipette the mixture using a Pasteur transfer pipette to prepare a single-cell suspension.</li>
		<li>Count the cells using the cell counter, and seed 1 &#215; 104 cells on&#160;an 8-chambered coverslip. Incubate the cells at 37 &#176;C with 5% CO2 and controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 6
	<ol>
		<li>Follow steps 3.4.1-3.4.4 to conduct live cell imaging of the cells from step 4.5.3 using a confocal laser microscope. Capture ten different frames of images.
		<ol>
			<li>Follow steps 3.4.4.1-3.4.4.3 to split the RGB merged images into the respective red, green, and blue image components, set a constant threshold for the green signals in each image, and determine GFP signal intensity. Record the values from the Total Area section to determine the signal intensity of GFP-ATG13.</li>
			<li>Divide the total signal intensity by the number of cells examined to calculate the signal intensity per cell. 
			NOTE: Count at least 34 cells in each of the three independent experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Assessing LC3-II-Positive EV Release in Autophagy with Impaired Lysosome Fusion

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The authors declare that they have no conflicts of interest with the contents of this article.

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 ...

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 &#215; g and 110,000 &#215; g, respectively. The P1 EV fraction (20,000 &#215; g pellets) is prepared to collect large EVs, and the P2 EV fraction (110,000 &#215; 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.

<table><tbody><tr><td>0.25 % Trypsin EDTA</td><td>Fujifilm Wako</td><td>201-16945</td><td></td></tr><tr><td>10 cm Dish</td><td>Thermo Fisher Scientific</td><td>150464</td><td></td></tr><tr><td>15 mL Tube</td><td>Thermo Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>200 &amp;mu;L Pipette Tip</td><td>Nippon Genetics</td><td>FG-301</td><td>pipetting</td></tr><tr><td>2-Mercaptoethanol</td><td>Nacalai Tesque</td><td>21417-52</td><td>a material for&lt;br/&gt; sample buffer solution</td></tr><tr><td>3,3&#039;-Diaminobenzidine Tetrahydrochloride</td><td>Nacalai Tesque</td><td>11009-41</td><td>a material for DAB solution</td></tr><tr><td>3.5 cm Dish&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>150460</td><td></td></tr><tr><td>6 cm Dish&amp;nbsp;</td><td>TrueLine</td><td>TR4001</td><td></td></tr><tr><td>Aluminium Block Thermostatic Baths (dry thermobaths)</td><td>EYELA</td><td>273860</td><td></td></tr><tr><td>Aspirator</td><td>SANSYO</td><td>SAP-102</td><td>inhaling solution</td></tr><tr><td>Avanti JXN-30</td><td>Beckman Coulter</td><td>B34193</td><td></td></tr><tr><td>Bafilomycin A1</td><td>Adipogen</td><td>BVT-0252</td><td></td></tr><tr><td>Biotin-conjugated Goat Anti-rabbit IgG Antibody</td><td>Vector Laboratories&amp;nbsp;</td><td>BA-1000</td><td>2nd antibody for immunoblotting</td></tr><tr><td>Biotin-conjugated Horse Antimouse IgG Antibody</td><td>Vector Laboratories&amp;nbsp;</td><td>BA-2000</td><td>2nd antibody for immunoblotting</td></tr><tr><td>Blocking One</td><td>Nacalai Tesque</td><td>03953-95</td><td>a material for immunoblotting</td></tr><tr><td>Bromophenol Blue</td><td>Nacalai Tesque</td><td>05808-61</td><td>a material for&lt;br/&gt; sample buffer solution</td></tr><tr><td>Calf Serum</td><td>cytiva</td><td>SH30073.03</td><td></td></tr><tr><td>CanoScan LiDE 220</td><td>Canon</td><td>CSLIDE220</td><td>Scanner</td></tr><tr><td>Centrifuge 5702 R</td><td>eppendolf</td><td>5703000039</td><td></td></tr><tr><td>Counting Slides Dual Chamber</td><td>Bio-Rad</td><td>1450015J</td><td>cell counting</td></tr><tr><td>Digital Sonifier 450</td><td>BRANSON</td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>nacalai tasque</td><td>09659-14</td><td>vehicle</td></tr><tr><td>DMEM High Glucose</td><td>Nacalai Tesque</td><td>08458-45</td><td>culture medium</td></tr><tr><td>DMEM without Phenol Red</td><td>Nacalai Tesque</td><td>08489-45</td><td>culture medium</td></tr><tr><td>EGTA</td><td>Dojindo&amp;nbsp;</td><td>348-01311</td><td>a material for A68 solution</td></tr><tr><td>Excel</td><td>Microsoft</td><td>version 16.16.27</td><td>satistical analysis</td></tr><tr><td>EZR</td><td>Reference No. 24</td><td>version 1.68</td><td>satistical analysis</td></tr><tr><td>FBS</td><td>Sigma</td><td>173012</td><td>Culture medium</td></tr><tr><td>Fiji</td><td>NIH</td><td></td><td>Image analysis tool</td></tr><tr><td>Glycerol</td><td>Nacalai Tesque</td><td>09886-05</td><td>a material for&lt;br/&gt; sample buffer solution</td></tr><tr><td>Hoehst33342</td><td>Dojindo&amp;nbsp;</td><td>H342</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fujifilm Wako</td><td>080-0186</td><td>a material for DAB solution</td></tr><tr><td>Kimwipe S-200</td><td>NIPPON PAPER CRECIA</td><td>62011</td><td>cleaning wipe</td></tr><tr><td>Low Retention Tube</td><td>Nippon Genetics</td><td>FG-MCT015CLB</td><td>siRNA and DNA transfection</td></tr><tr><td>LSM780 Confocal Laser Microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Monoclonal Mouse Anti-LC3 Antibody</td><td>MBL</td><td>M186-3</td><td>1st antibody for immunoblotting</td></tr><tr><td>Nickel(II) Chloride Hexahydrate</td><td>Fujifilm Wako</td><td>149-01041</td><td>a material for DAB solution</td></tr><tr><td>N-Lauroylsarcosine Sodium Salt</td><td>Nacalai Tesque</td><td>20117-12</td><td></td></tr><tr><td>Optima XE-90 Ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985-070</td><td>siRNA and DNA transfection</td></tr><tr><td>pEGFP-C1-hAtg13</td><td>Addgene</td><td>22875</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Nacalai Tesque</td><td>26253-84</td><td>Culture medium</td></tr><tr><td>Pierce BCA Protein Assay Kits</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Polyclonal Rabbit Anti-PCNA Antibody</td><td>BioAcademia</td><td>70-080</td><td>1st antibody for immunoblotting</td></tr><tr><td>Polyclonal Rabbit Anti-syntaxin 6 Antibody</td><td>ProteinTech</td><td>10841-1-AP</td><td>1st antibody for immunoblotting</td></tr><tr><td>Polyclonal Rabbit Anti-TSG101 Antibody</td><td>ProteinTech</td><td>28283-1-AP</td><td>1st antibody for immunoblotting</td></tr><tr><td>Polyclonal Rabbit Anti-ULK1 Antibody</td><td>ProteinTech</td><td>20986-1-AP</td><td>1st antibody for immunoblotting</td></tr><tr><td>Polyvinylidene Difluoride Membrane</td><td>Milliore</td><td>IPVH00010</td><td>a material for immunoblotting</td></tr><tr><td>R</td><td>R development core team</td><td>version 4.4.1</td><td>satistical analysis</td></tr><tr><td>RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778</td><td>siRNA transfection reagent</td></tr><tr><td>siRNA STX6</td><td>Thermo Fisher Scientific</td><td>HSS115604</td><td>siRNA for transfection</td></tr><tr><td>Sodium Chloride</td><td>Nacalai Tesque</td><td>31320-05</td><td>a material for Tris&lt;br/&gt; buffer and A68 solution</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Fujifilm Wako</td><td>192-13981</td><td>a material for&lt;br/&gt; sample buffer solution</td></tr><tr><td>SPARK Microplate Reader</td><td>TECAN</td><td></td><td></td></tr><tr><td>Stealth RNAi Negative Control Duplexes, Med GC</td><td>Thermo Fisher Scientific</td><td>12935300</td><td>siRNA transfection</td></tr><tr><td>Sucrose</td><td>Fujifilm Wako</td><td>193-00025</td><td>a material for A68 solution</td></tr><tr><td>TC20 Automated Cell Counter with Thermal Printer</td><td>Bio-Rad</td><td>1450109J1</td><td>cell counting</td></tr><tr><td>Thermobath</td><td>TOKYO RIKAKIKAI</td><td>MG-3100</td><td>incubation</td></tr><tr><td>TransIT-293</td><td>Mirus Bio</td><td>MIR 2700</td><td>DNA transfection reagent</td></tr><tr><td>TransIT-LT1</td><td>Mirus Bio</td><td>MIR2300</td><td>DNA transfection reagent</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Nacalai Tesque</td><td>35406-75</td><td>a material for Tris buffer, sample buffer and A68 solution</td></tr><tr><td>Trypan Blue Dye 0.40%</td><td>Bio-Rad</td><td>1450021</td><td>cell counting</td></tr><tr><td>Ultra-Clear Open-Top Tube, 16 x 96mm&amp;nbsp;</td><td>Beckman Coulter</td><td>361706</td><td>collecting for the P1 EV fraction</td></tr><tr><td>Ultra-Clear Tube, 14 x 89mm</td><td>Beckman Coulter</td><td>344059</td><td>collecting for the P2 EV fraction</td></tr><tr><td>Vectastain ABC Standard Kit</td><td>Vector Laboratories&amp;nbsp;</td><td>PK-4000</td><td>immunoblotting</td></tr><tr><td>Wash Bottle</td><td>As One</td><td>1-4640-02</td><td>washing membrane</td></tr><tr><td>&amp;mu;-Slide 8 Well High</td><td>ibidi</td><td>80806</td><td></td></tr></tbody></table>

evaluation of lc3-ii release via extracellular vesicles in relation to the accumulation of intracellular lc3-positive vesicles

(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.

As shown in previous studies, Baf treatment increased the levels of TSG101 (P1: P &#60; 0.01, P2: P = 0.012, as determined by two-way ANOVA in EZR19) and LC3-II (P1: P &#60; 0.01, P2: P &#60; 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 &#60; 0.01, P2: P &#60; 0.01, as determined by two-way ANOVA in EZR19) in the EV fraction (Figure 1A</str...

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

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 ...

evaluation-lc3-ii-release-via-extracellular-vesicles-relation-to

Research

JoVE Journal

Biochemistry

529 Views.  Okayama University of Science. 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.

Video: Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Neuroscience

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicine

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Biology

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways through permeabilization of IV membranes and the formation of endosomes and lysosomes. A method named chromophore-assisted laser inactivation (CALI) is applied to study the formation of IVs and the materials in controlling IV regulation. CALI is an imaging-based photodynamic methodology to study the signaling pathway induced by membrane permeabilization. The method allows spatiotemporal manipulation of the selected organelle to be permeabilized in a cell. The CALI method has been applied to observe and monitor specific molecules through the permeabilization of endosomes and lysosomes. The membrane rupture of IVs is known to selectively recruit glycan-binding proteins, such as galectin-3. Here, the protocol describes the induction of IV rupture by AlPcS2a and the use of galectin-3 as a marker to label impaired lysosomes, which is useful in studying the downstream effects of IV membrane rupture and their downstream effects under various situations.

AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways ...

Immunology and Infection

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins.
Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the &#34;3D Objects Counter&#34; tool.
The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

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Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence