Imaging ATG9A, a Multi-Spanning Membrane Protein

In our lab, we are 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 organelles homeostasis.

The biggest challenge is to uncover the molecular details of how proteins and protein machineries function. Technologies such as protein engineering, based on AlphaFold 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 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 ATG9 among different cellular compartments.

And we do think that standardization of pipelines for image analysis is a priority for cell biologists to be able to increase the reliability and reproducibility of our results.