Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Summary

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Abstract

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.

Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.

Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Introduction

Autophagy is a highly dynamic process, which requires the coordination of a large number of proteins for the final outcome of autophagosome formation^1-3. Microscopy is probably the technique most commonly applied for studying autophagy⁴. The localization of most autophagy proteins has been extensively studied in fixed cells, both by immuno-staining the endogenous proteins and by expression of fluorescently tagged exogenous protein. In addition, Electron Microscopy (EM), alone and in combination with immuno-gold labeling, has described the fine details of these structures^5,6 . Despite the fact that these techniques have establish....

Protocol

1. Cell Preparation

1. Seed low passage number of HEK-293T cells stably expressing GFP-DFCP1 on 22 mm round coverslips; culture cells overnight in Dulbecco Modified Eagles's Medium (DMEM), to a confluency of 30-40% (aim for a confluency of 80% after 2 days - day of live cell imaging).

2. Cell Transfection

1. Prepare the transfection complex mix for each plate, containing 100 μl OptiMEM I reduced serum medium, 3 μl X-tremeGENE 9 DNA Transfection Reagent, and 0.5 μg of pECFP-LC3 plasmid DNA. Mix gently by pipetting up and down and incubate 15 min at room temperature. (Note: we have repeatedly....

Results

In the protocol described, we have used time-lapse microscopy to follow the localization of CFP-tagged LC3 in a cell line stably expressing GFP-tagged DFCP1, under autophagy inducing conditions. The outcome of this experiment is the capture of 2 series or stacks of images, one from the green and one from the blue channel, corresponding to GFP-DFCP1 and CFP-LC3. We have further analyzed these videos using ImageJ, in order to create montages corresponding to single autophagosome formation events, as described in the.......

Discussion

The method described in this protocol allows the visualization of a protein's localization during autophagosome formation. We have tried various methods of visualizing the events described including point-scanning confocal, spinning disk confocal and Total Internal Reflection Fluorescence (TIRF) microscopy. We have found that for general purposes standard wide-field epi-fluorescence provides the best compromise between sensitivity and resolution. This ensures good signal to noise, minimal photo-bleaching/photo-to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	41965
OptiMEM I	Invitrogen	31985-062
MitoTracker Red FM	Invitrogen	M22425
LysoTracker Red DND-99	Invitrogen	L-7528
X-tremeGENE 9 DNA Transfection Reagent	Roche Applied Science	6365787001
22 mm coverslips	VWR	631-0159
35 mm plates	Fisher NUNC	153066
Silicon grease	RS Components Ltd.	RS 494-124
O-rings			Custom made
Attofluor Cell Chamber	Invitrogen	A-7816	Suggested alternative to custom-made O-rings
Microscope	Olympus	IX81	Inverted microscope
Objective	Olympus	UPLSAPO 100XO	N.A. 1.4, W.D. 0.13, FN 26.5
Camera	Hamamatsu	ORCA-R2 C10600 10B	Progressive scan interline CCD
Illuminator	TILL Photonics	Polychrome V	Ultrafast monochromator
Incubation chamber	Solent Scientific	Cell^R IX81
Software	Olympus	SIS xcellence