live cell imaging of atg9a constructs

Studio della localizzazione di ATG9A nelle cellule

ATG9A is the only transmembrane protein of the core autophagy machinery and is trafficked between the Golgi and a cytosolic ATG9A vesicle compartment, transiting through the endosomal compartment1. Having long been enigmatic, ATG9A has recently been described to function as a lipid scramblase, as it equilibrates lipids across membrane bilayers2,3. It is now clear that ATG9A resides at the top of the hierarchy in autophagosome formation, and its study is, thus, vital for understanding autophagy4,5. As such, ATG9A vesicles have been recently proposed as the &#34;seed&#34; of the autophagosome6,7. However, previous studies have demonstrated that ATG9A only transiently interacts with the forming autophagosome at different steps of its maturation and does not integrate into the autophagic membrane6,8,9,10,11. Thus, further investigations are needed to completely unravel the role and potential multiple functions of ATG9A in autophagosome formation. However, the discrepancy between the current models and the previous data can only be resolved through targeted experiments addressing the trafficking of ATG9A using validated quantitative approaches and intracellular markers.
There are various tools in use to study ATG9A, each with advantages and disadvantages, and the use of these tools is complicated by the structure of ATG9A, its molecular function, and cellular trafficking2,8,12. ATG9A forms a homotrimer, is glycosylated, and is trafficked throughout the cell to compartments such as the Golgi, the endosomes, and the plasma membrane13,14. Given its complex itinerary, there are several challenges in interpreting readouts such as ATG9A dispersal from the Golgi upon specific treatments or stimuli (such as nutrient and serum starvation). ATG9A is extremely dynamic in terms of vesicular trafficking; indeed, ATG9A-containing vesicles have been defined as the ATG9A compartment in the context of starvation-induced autophagy. The ATG9A compartment, formed by these dynamic vesicles, transiently interacts with several intracellular organelles8,15,16,17. The techniques described here, including immunofluorescence, live imaging, and glycosylation assays, should aid in the detection and understanding of ATG9A biology. In particular, the approaches described in this article will help to address questions about localization to specific cellular compartments and interactions with specific protein partners and/or membrane compartments. As the ATG9A hydrophobic conserved core domain (PFAM domain PF04109) has a unique topology and ATG9A cycles between several membrane compartments, researchers should be aware of certain pitfalls and artifacts when transiently overexpressing ATG9A, including, but not restricted to, endoplasmic reticulum (ER) retention. Other possible issues may arise due to misfolding of the protein, artifactual aggregation in normal growing conditions, or insufficient detection of the vesicular compartment due to suboptimal permeabilization protocols for immunofluorescence.
When imaging endogenous ATG9A, care must be taken in the sample preparation and image acquisition to ensure the quality of the subsequent quantitative analysis and the correct interpretation of the data. Combining the techniques described in this article with standard biochemical approaches (such as immunoprecipitation or pull-down experiments not described here) should improve our understanding of ATG9A function. This experimental toolkit is intended to help new researchers navigate some of the assays required to determine the function of ATG9A in their biological system.

Autore in primo piano: Imaging ATG9A, una proteina di membrana multi-spanning  

Imaging di ATG9A, una proteina di membrana multi-spanning

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.

Watch this Scientific Journal Video about Live Cell Imaging of ATG9A Constructs at JoVE.com

Live Cell Imaging of ATG9A Constructs  

Imaging di cellule vive di costrutti ATG9A

Per l'imaging di cellule vive di costrutti ATG9A, seminare le cellule HEK293A in due millilitri di DMEM ad alto contenuto di glucosio in un piatto di coltura tissutale da 60 millimetri fino a quando non raggiungono il 65-70% di confluenza. Il giorno successivo, preparare e aggiungere la miscela di DNA di lipofectamina alla piastra di coltura cellulare contenente quattro millilitri di terreno di coltura e far oscillare delicatamente la piastra avanti e indietro per distribuire uniformemente la miscela. Incubare le cellule a 37 gradi Celsius e al 10% di anidride carbonica.Dopo quattro ore di trasfezione, sostituire il terreno di coltura con un terreno fresco e incubare le cellule per una notte a 37 gradi Celsius con il 10% di anidride carbonica. Il giorno successivo, tripsinizzare le cellule aggiungendo tripsina EDTA. Quindi, contare le cellule staccate e riseminarle su una piastra di coltura adatta per la microscopia dal vivo durante la notte.Il giorno successivo, riprendi l'immagine delle cellule usando un microscopio confocale. In condizioni basali, ATG9A è localizzato principalmente nella rete di Golgi come indicato dall'immunofluorescenza della proteina endogena e si sovrappone a GM130, un marcatore cis-Golgi. ATG9A marcato con eGFP N-terminale e ATG9A marcato con mRFP N-terminale erano meno localizzati al Golgi e risiedevano principalmente nelle vescicole.Al contrario, l'ATG9A marcato terminalmente con eGFPc è più incline ad aggregarsi all'interno della cellula. La fusione di una sequenza 3x-FLAG tra un fluoroforo N-terminale e ATG9A ha aiutato la proteina sovraespressa a comportarsi in modo simile a quella endogena. Il sovraespresso mCherry 3x-FLAG ATG9A colocalizza con il marcatore di Golgi GM130 in condizioni di alimentazione.Questa localizzazione e il compartimento vescicolare di AG9A sono stati preservati nel tempo, permettendo lo studio spazio-temporale del traffico di ATG9A.

Research

JoVE Journal

Biology

Live Cell Imaging of ATG9A Constructs

396 Views.  The Francis Crick Institute. Per l'imaging di cellule vive di costrutti ATG9A, seminare le cellule HEK293A in due millilitri di DMEM ad alto contenuto di glucosio in un piatto di coltura tissutale da 60 millimetri fino a quando non raggiungono il 65-70% di confluenza. Il giorno successivo, preparare e aggiungere la miscela di DNA di lipofectamina alla piastra di coltura cellulare contenente quattro millilitri di terreno di coltura e far oscillare delicatamente la piastra avanti e indietro per distribuire uniformemente la...

Video: Live Cell Imaging of ATG9A Constructs  

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

Ricerca

Biologia

﻿Endogenous Staining of ATG9A from HEK293A Cells

Endogenous Staining of ATG9A from HEK293A Cells  

To begin, seed HEK293A cells on gloss cover slips placed into a 24 well plate containing high glucose DMEM until they become 80%confluent. The next day, starved the cells for two hours in Earl's balanced salt solution. After treatment, aspirate the medium and add 500 microliters or 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 hooks solution diluted one to 4, 000 in PBS in a humidified chamber.Incubate in a humidified chamber for five minutes. Next, remove the excess hooks 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.

Colorazione endogena di ATG9A da cellule HEK293A

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.