In our lab, we're interested in understanding which lipids and protein machineries are involved in ATG9A vesicle trafficking. ATG9A is crucial in the autophagy pathway for phagophore formation and maturation. However, we've recently been studying the impact of ATG9A in organelle's homeostasis.
The biggest challenge is to uncover the molecular details of how proteins and protein machineries function. Technologies such as protein engineering based on alpha-fold and high resolution in situ cryo-EM are currently providing the most advances. My lab's work has established fundamental insight into how autophagosomes are formed.
These insights have been supported by advanced molecular cell biology techniques such as the one described here. We describe some drawbacks of using certain protein tags and how ATG9A's behavior can change depending on the localization of the tag, so either N or C-terminus. We also suggest ways of detecting these drawbacks and how to avoid them.
Besides this, we also provide a workflow of quantifying ATG9A's dispersal in a reproducible manner. Here, we provide with a workflow from immunofluorescence and live imaging to image acquisition and analysis for the analysis of the dispersal of ATG9A among different cellular compartments. We do think that standardization of pipelines for image analysis is a priority for cell biologists to be able to increase their reliability and reproducibility of our results.
To begin, seed HEK293A cells on glass cover slips placed into a 24-well plate containing high glucose DMEM until they become 80%confluent. The next day, starve the cells for two hours in Earle's balanced salt solution. After treatment, aspirate the medium and add 500 microliters of 4%formaldehyde solution in PBS and incubate for 20 minutes at room temperature to fix the cells.
After fixation, replace the formaldehyde solution with 500 microliters of PBS. Next, quench the free aldehyde group by adding 500 microliters of 50 millimolar ammonium chloride solution in PBS for 10 minutes at room temperature. Replace the ammonium chloride solution with 500 microliters of a 50 microgram per milliliter digitonin solution in PBS for five minutes to permeabilize the cells.
After permeabilization, replace the digitonin solution with 500 microliters of PBS, repeating PBS washing three times. After the last wash, incubate the cells in 500 microliters of blocking solution for 30 minutes at room temperature. After incubation, replace the blocking solution with 500 microliters of PBS.
Next, using tweezers, collect the cover slips from the well and remove the excess PBS using thin tissue wipes. In the humidified chamber, gently lay down the cover slip with cell side down onto a 50-microliter drop of primary antibody solution. Incubate the cells in a humidified chamber for one hour at room temperature.
After incubation, collect the cover slips and drain off the excess primary antibody solution. Place the cover slips back in the 24-well plate with the cell side up and wash them three times with PBS. After the last wash, collect the cover slips and drain off the excess PBS.
Before placing each cover slip with cell side down onto a 50-microliter drop of secondary antibody solution, incubate in a humidified chamber for one hour. After incubation, drain off the excess secondary antibody solution and wash the cover slip three times with 500 microliters of PBS in the 24-well plate. After draining excess PBS, place each cover slip with cell side down onto a 50-microliter drop of Hooke's solution diluted one to 4, 000 in PBS in a humidified chamber.
Incubate in a humidified chamber for five minutes. Next, remove the excess Hooke's solution and wash the cover slips three times with PBS and once with deionized water in a 24-well plate. After draining excess deionized water, place each cover slip onto a 10 to 20-microliter drop of mounting solution spotted onto a microscope slide, avoiding the formation of air bubbles.
For live cell imaging of ATG9A constructs, seed HEK293A cells in two milliliters of high glucose DMEM into a 60-millimeter tissue culture dish until they reach 65 to 70%confluency. The next day, prepare and add the lipofectamine DNA mixture to the cell culture plate containing four milliliters of growth medium, and gently rock the plate back and forth to distribute the mix evenly. Incubate the cells at 37 degrees Celsius and 10%carbon dioxide.
After four hours of transfection, replace the growth medium with a fresh medium and incubate the cells overnight at 37 degrees Celsius with 10%carbon dioxide. The next day, trypsinize the cells by adding trypsin EDTA. Then count the detached cells and reseed them on a culture dish suitable for live microscopy overnight.
The next day, image the cells using a confocal microscope. In basal conditions, ATG9A is mainly localized at the golgi network as indicated by the immunofluorescence of the endogenous protein and overlaps with GM130, a cis-golgi marker. eGFP-N terminally tagged ATG9A and mRFP-N terminally tagged ATG9A were less localized at the golgi and primarily resided in vesicles.
In contrast, eGFP-C terminally tagged ATG9A is more prone to aggregate within the cell. Fusing a 3xFLAG sequence between an N-terminal fluorophore and ATG9A helped the overexpressed protein behave similarly to the endogenous one. The overexpressed mCherry3xFLAG-ATG9A co-localizes with the golgi marker GM130 in fed conditions.
This localization and the ATG9A vesicular compartment were preserved over time, allowing the spatial temporal study of the trafficking of ATG9A.
