focus on Microscopy & Microtechniques New Method for Real-Time Autophagy Studies


Cindy Chen – R&D Scientist, Terry Gaige – Science and Technology, Engineer, Ivana Zubonja – R&D Associate, Paul Hung* – Senior R&D Manager EMD Millipore, 290 Concord Rd, Billerica, MA 01821 *Corresponding author: Paul.hung@emdmillipore.com


Autophagy is an intracellular process leading to the lysosomal degradation of cytosolic components and organelles. Its best understood role is in cellular housekeeping; this activity directs the removal of damaged or unwanted products [1]. However, autophagy can also be induced in response to cancer therapies, when autophagy functions as a survival mechanism and thus potentially limits drug effi cacy [2,3]. In established tumours, malignant progression and tumour maintenance have been linked to physiological adaptations resulting in upregulated or constitutively active autophagic pathways [2]. In addition, there are many stimuli that have been shown to activate autophagy, including nutrient starvation, reactive oxygen species [4], stress on the endoplasmic reticulum, and ammonia [5].


Once stimulated, unwanted cytosolic proteins and aging organelles are sequestered by a double-membrane vesicle known as an autophagosome (Figure 1). Protein complexes coordinate vesicle formation and enable the recruitment of LC3 into the inner and outer membranes of the autophagosome. LC3-labelled vesicles are traffi cked to the lysosome. During this last phase, autophagosomes fuse with lysosomes to form autolysosomes, where unwanted nutrients are reduced to basic molecular building blocks and ultimately released back into the cytoplasm.


Measurement and tracking of autophagy are essential for elucidating this process. Many newer autophagy assays rely on the expression of stably transfected green fl uorescence protein (GFP)-LC3 fusion proteins; in this case, autophagosome activity is visually identifi ed by changes in GFP puncta [6]. Lysosomal inhibitors, such as chloroquine (CQ), have also been invaluable in determining the relative autophagic response to cellular stress. CQ blocks the last step of autophagy, lysosomal degradation; the resulting buildup of intermediates can serve as a quantifi able marker of autophagic activity [7]. By combining the use of live cell imaging with transduction of a GFP-tagged autophagosome marker (LC-3) in the presence of CQ, researchers can monitor the autophagosome formation process on a fl uorescent microscope in real time. However, little is known about the latter stages of autophagy and the dynamics of lysosomal degradation.


In this article, we demonstrate the use of a microfl uidic live cell imaging platform (the CellASIC® ONIX Microfl uidic Platform,


EMD Millipore) to develop a dynamic cell-based assay for monitoring the whole autophagy process. This platform offers temperature and gas control as well as media perfusion for precise environmental control. Using this system, LC3-GFP CHO reporter cells were subjected to nutrient starvation or hypoxic stresses for a designated time period followed by reintroduction of normal growth conditions. The time course of autophagy was visualised in real time under a fl uorescent microscope, providing quantitative information on both autophagosome formation and lysosomal degradation machinery.


Assay Validation To validate the media exchange capability of the CellASIC® ONIX platform as well as the


ability to monitor and quantify autophagy through autophagosome counting, LC3- GFP CHO cells (70% confl uent) were perfused with EBSS + 50 µM CQ for 100 minutes followed by regular culture medium for 200 minutes. As shown in Figures 2 and 3, the dynamic changes of autophagy in both the stress and recovery phase could be quantifi ed through autophagosome counting.


Assessment of CQ Dose Response


Once the assay was validated, profi ling of the CQ dose response in CHO cell lines was conducted. Once established in the microchamber, exposure conditions involved three phases: standard culture medium for 135 minutes, continuous CQ (10 µM, 100 µM, or 1 mM) perfusion for 255 minutes, and culture medium for the fi nal 240 minutes to permit visual capture of the lysosomal degradation process. Images were taken every 15 minutes. Overall, the rate of autophagosome formation was proportional to the CQ concentration applied. However, at 1 mM, cells ceased committing to the autophagy pathway, and the number of autophagosomes stayed constant for the rest of the experiment. We also observed more dead cells in this treated group, indicating either that the maximal levels of autophagy in this cell line had been achieved, or that the cells committed to apoptosis or necrosis at the high CQ dose. Furthermore, degradation of autophagosomes occurred at a faster rate than the accumulation (Figure 4).


Figure 1. Four stages of autophagy. Autophagy can be induced by nutrient depletion or inhibition of the mTOR pathway. During autophagy, cytosolic proteins and aging organelles are sequestered by a double-membraned autophagosome. One of the hallmarks of autophagy is translocation of LC3 from the cytoplasm to the autophagosome. Autophagosomes then fuse with lysosomes to promote breakdown of the vesicle and all contents, including LC3. This process can be visualised using either a LC3-GFP fusion protein or an anti-LC3 antibody.


Figure 2. Schematic of live cell imaging for autophagy of LC3-GFP expressing CHO cells. First, medium was perfused to establish fl uorescent baseline. A stressor (serum starvation) and the lysosome inhibitor CQ1 were then introduced to trigger autophagosome accumulation within cells. When cells were returned to standard growth medium, autophagosomes underwent lysosomal degradation.


INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2014


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