This method can help answer key questions in the autophagy field, such as how autophagosome formation is regulated and how various cell treatments and conditions affect bulk autophogy. The main advantage for this technique is that it measures endogenous cargo sequestration. Therefore, the method is broadly applicable and independent of artificial overexpression or introduction of cargo probes.
Demonstrating the procedure, will be Paula Szalai and Morten Luhr, PhD students from my laboratory. To begin, culture adherent cells in 75 square centimeter tissue culture flasks in a humidified incubator with 5%carbon dioxide at 37 degrees celsius. Allow the cells to grow until they reach a near confluent cell layer.
After this, wash the cells with three milliliters of PBS at 37 degrees celsius in pH 7.4. Remove the PBS and replace it with three milliliters of 0.25%trypsin EDTA. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius, until the cells detach.
Next, resuspended the cells in seven milliliters of culture medium containing 10%FBS, and transfer to a sterile 50 milliliter tube. Using a 0.5 to 20 microliter pipette tip, transfer 10 microliters of cell suspension to a microcentrifuge tube containing 10 microliters of trypan blue. Use the same pipette tip to immediately fill a counting chamber slide and count the cells in an automated cell counter.
Then use culture medium containing 10%FBS to prepare suitable dilution of the cell suspension. Using aseptic techniques, seed one milliliter of the dilute cell suspension into each well of a 12 well tissue culture plate. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius until the desired cell density has been reached.
At the desired experimental treatments leaving one set of wells untreated in order to define the background levels of sedimentable LDH. Three to four hours before the harvest, add a saturating amount of post sequestration inhibitor Bafilomycin A1 to each well. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius.
At the end of the treatment period, you suction to aspirate the medium. Add 200 microliters of cell detachment solution preheated to 37 degrees celsius to each well. Incubate at 37 degrees celsius until the cells detach.
Add 500 microliters of room temperature PBS at pH 7.4 containing 2%BSA to each well. Using a pipette, resuspended each well until no cell clumps are visible. After this, immediately transfer the cell suspension in each well to a separate 1.5 milliliter microcentrifuge tube on ice.
Centrifuge the tubes at 400 times g and four degrees celsius for five minutes to sediment the cells. Use suction to thoroughly aspirate the supernatant, leaving the cell pellets as dry as possible. Then, add 400 microliters of solution containing 10%sucrose in ultra pure water to each tube.
Using a pipette, resuspended the cell pellet to obtain a near single cell suspension. Transfer this suspension to a four millimeter electroporation cuvette. Place the cuvette in an exponential decay wave electroporator and discharge a single electric pulse at 800 volts, 25 microfarads and 400 ohms.
Use a new pipette tip to transfer the cell disruptate into a 1.5 milliliter microcentrifuge tube containing 400 microliters of ice-cold phosphate buffered sucrose solution and mix briefly by pipetting. Repeat this resuspension, electro-disruption and mixing process for each sample. If performing the procedure for the first time, make sure to verify efficient and selective plasma membrane electro-disruption as described in the text.
To obtain sedimented LDH fractions, remove 550 microliters from each diluted cell-disruptate solution and transfer it to its own two milliliter microcentrifuge tube containing 900 microliters of ice-cold resuspension buffer with 0.5%BSA and 0.01%Tween 20. Pipette briefly to mix. Centrifuge at 18, 000 times g and four degrees celsius for 45 minutes to produce pellets containing sedimented LDH.
Using suction, thoroughly aspirate the supernatant to leave the pellets as dry as possible. Store the sedimented LDH samples at minus 80 degrees celsius until ready to analyze. For total LDH fractions, transfer 150 microliters from each of the diluted cell disruptate solutions to its own new tube.
Store these in a minus 80 degrees Celsius freezer until ready to analyze. Thaw both the sedimented LDH and the total LDH samples on ice. Once thawed, add 300 microliters of ice-cold resuspension buffer containing 1.5%Triton X-405 to the total LDH samples.
Rotate the samples on a roller in a cold room for 30 minutes. Next, add 750 microliters of ice-cold resuspension buffer containing 1%Triton X-405 to the sedimented LDH samples. Use a pipette to resuspended the pellets until the solution is homogenous.
Centrifuge all of the samples at 18, 000 times g and four degrees celsius for five minutes to sediment undissolved cellular debris. After centrifugation is complete, put the samples on ice. Determine the levels of LDH in the supernatants using either a classic enzymatic assay as described in the text or any of the wide variety of commercially available kits.
In this study, the bulk autophagic sequestration activity is monitored in a number of different mammalian cell lines. As shown here, both the basal and starvation-induced LDH sequestration and LNCaP cells are completely abolished by the pan-PI3-kinase inhibitor, 3MA. The selective PI3-kinase Class three inhibitor, SAR-405 and the ER calcium pump inhibitor, Thapsigargin.
In HEK293 cells, LDH sequestration under starvation conditions is strongly reduced by both Thapsigargin and by the ULK inhibitor, MRT 67307. Moreover, starvation-induced LDH sequestration is shown to be consistently inhibited by 3MA in the other observed cell lines. Next, various ATG gene knockout MEFs are employed to test if autophagy related genes reported to be essential for autophagosome formation are required for LDH sequestration.
As can be seen, starvation-induced LDH sequestration is abolished in ATG5 knockout MEFS. Moreover, it is also blunted in ATG7 and ATG9A knockout MEFs. Finally, the effects of RNAi mediated silencing of key ATG gene transcripts are determined.
Transfection with an ATG9A targeting siRNA or with combined targeting of ULK1 and ULK2 is seen to strongly reduce LDH sequestration in LNCaP cells. While attempting this procedure, it's important to be accurate in all pipetting steps. Following this procedure other methods like the long-lived protein degradation assay can be performed in order to answer additional questions like were there observed alterations in sequestration activity resulting comparable changes in autophagic degradation activity.
After establishing this method in mammalian cell lines, we have used it to demonstrate that bulk autophagy is independent of the LC3 protein family and instead requires GABARAPs. This highlights the importance of employing LC3 independent methods to monitor autophagy activity.
