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 re-seed 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, eGFPc terminally tagged ATG9A is more prone to aggregate within the cell. Fusing a 3x-FLAG sequence between an N-terminal fluorophore and ATG9A helped the overexpressed protein behave similarly to the endogenous one. The overexpressed mCherry 3x-FLAG ATG9A colocalizes with the Golgi marker GM130 in fed conditions.
This localization and the AG9A vesicular compartment were preserved over time, allowing the spatiotemporal study of the trafficking of ATG9A.
