Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Summary

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Abstract

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Introduction

Mitochondria are the powerhouses of nearly all eukaryotic cells^1,2. In addition to ATP production through oxidative phosphorylation, mitochondria play a vital role in other processes such as bioenergetics, calcium homeostasis, free radical generation, apoptosis, and cellular homeostasis^3,4,5. As mitochondria generate reactive oxygen species (ROS) from multiple complexes in the electron transport chain, they are constantly stimulated by potential oxidative stress, which can eventually lead to structural damage and dysfunction when the antioxidant defense system collapses^6,7. Mitochondrial dysfunction has been found to contribute to many diseases, including metabolic disorders, neurodegeneration, and cardiovascular disease⁸. Therefore, it is crucial to maintain healthy mitochondrial populations and their proper function. Mitochondria are highly plastic and dynamic organelles; their morphology and function are controlled by mitochondrial quality control mechanisms, including post-translational modifications (PTM) of mitochondrial proteins, mitochondrial biogenesis, fusion, fission, and mitophagy^9,10. Mitochondrial fission mediated by dynamin-related protein 1 (DRP1), a GTPase of the dynamin superfamily of proteins, results in small and round mitochondria and isolates the dysfunctional mitochondria, which can be cleared and degraded by mitophagy^11,12.

Mitophagy is a cellular process that selectively degrades mitochondria by autophagy, usually occurring in damaged mitochondria following injury, aging, or stress. Subsequently, these mitochondria are delivered to lysosomes for degradation¹⁰. Thus, mitophagy is a catabolic process that helps maintain the quantity and quality of mitochondria in a healthy state in a wide range of cell types. It plays a crucial role in the restoration of cellular homeostasis under normal physiological and stress conditions^13,14. Cells are characterized by a complex mitophagy mechanism, which is induced by different signals of cellular stress and developmental changes. Mitophagy regulatory pathways are classified as ubiquitin-dependent or receptor-dependent^15,16; the ubiquitin-dependent autophagy is mediated by the kinase PINK1 and the recruitment of ubiquitin ligase Parkin E3 to the mitochondria^17,18, while receptor-dependent autophagy involves the binding of autophagy receptors to the microtubule-associated protein light chain LC3 that mediates mitophagy in response to mitochondrial damage¹⁹.

Transmission electron microscopy (TEM) is the most commonly used method, and still one of the best methods, to observe and detect mitophagy²⁰. The morphological features of mitophagy are autophagosomes or autolysosomes formed by the fusion of autophagosomes with lysosomes, which can be observed from electron microscopy images²¹. The weakness of electron microscopy (EM), however, is the inability to monitor the dynamic processes of mitophagy, such as mitochondrial depolarization, mitochondrial fission, and fusion of autophagosomes and lysosomes, in the living cell²⁰. Thus, evaluating mitophagy through imaging living organelles is an attractive alternative method for mitochondrial research. The live cell imaging technique described here uses two fluorescent dyes to stain mitochondria and lysosomes. When mitophagy occurs, damaged or superfluous mitochondria engulfed by autophagosomes are stained green by the mitochondrial dye, while the red dye stains the lysosomes. The fusion of these autophagosomes and lysosomes, referred to as autolysosomes, causes the green and red fluorescence to overlap and manifest as yellow dots, thus indicating the occurrence of mitophagy²². The cell-permeant mitochondria dye (MitoTracker Green) contains a mildly thiol-reactive chloromethyl moiety to label mitochondria²³. To label mitochondria, cells are simply incubated with the dye, which diffuses passively across the plasma membrane and accumulates in active mitochondria. This mitochondria dye can easily stain live cells, and is less effective in staining aldehyde-fixed or dead cells. The lysosome dye (LysoTracker Red) is a fluorescent acidotropic probe used for labeling and tracking acidic organelles in live cells. This dye exhibits a high selectivity for acidic organelles and can effectively label live cells at nanomolar concentrations²⁴.

The procedures for using these fluorescent dyes in living cells, including loading the dyes and the visualization of mitochondria and lysosomes, are presented here. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. It can also be used to quantify mitochondria and lysosomes, and assess mitochondrial morphology.

Protocol

1. Cell culture and passaging

NOTE: The protocol is described using routinely cultured mouse embryonic fibroblasts (MEFs) as an example.

1. Culture MEF cells in 10 cm cell culture dishes with 10 mL of Dulbecco's Modified Eagle Medium (DMEM). Incubate at 37 °C and 5% CO₂ and monitor the cells under a microscope at 100x magnification.
2. Perform routine cell passaging.
   1. When the cells reach 80%-90% confluency (every 3 days), wash the cells with 2 mL of Dulbecco's phosphate buffered saline (DPBS). Then add 2 mL of 0.05% trypsin-EDTA for 1 min to dissociate the cells, followed by 2 mL of DMEM to stop the action of trypsin-EDTA. Centrifuge the cell suspension at 100 x g for 3 min and resuspend the cell pellet in 1 mL of DMEM.
   2. Count the cells using an automated cell counter and cell counting chamber slides (see Table of Materials), and then inoculate 1.5 x 10⁶ cells into a new 10 cm cell culture dish containing 10 mL of DMEM.
3. For the mitophagy assay, prepare a cell suspension as in step 1.2.1. Dilute the cell suspension to 1 x 10⁵ cells/mL in fresh DMEM.
4. Add 2 mL of the diluted cell suspension to a 20 mm confocal dish (see Table of Materials) and shake the culture dish in a "cross". Incubate the cell culture dish in a 37 °C, 5% CO₂ incubator for 24 h.

2. Mitochondrial staining

1. Remove the stock solution aliquots of the green-fluorescent mitochondrial dye and red-fluorescent lysosome dye (see Table of Materials) from the -20 °C freezer.
2. Prepare working solutions of the dyes by diluting the stock solutions 1:1,000 in DMEM and mix well. For example, add 2 µL each of 1 mM mitochondrial dye and lysosome dye to 2 mL of DMEM to obtain a working concentration of 1 µM for both dyes.
3. Remove the medium from the confocal culture dish (step 1.4). Add 1 mL of the staining solution (prepared in step 2.2) to cover the cells. Place the cell culture dish in an incubator at 37 °C, 5% CO₂ for 20-30 min.

3. Confocal imaging

1. Prepare 1 L of Krebs-Henseleit (KH) buffer (138.2 mM NaCl, 3.7 mM KCl, 0.25 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄.7H₂O, 15 mM glucose, and 21.85 mM HEPES; final pH 7.4) and store at 4 °C (for up to 1 month).
2. On the day of confocal imaging, remove the KH buffer from the refrigerator in advance and pre-warm it to room temperature (20 to 25 °C).
3. Set the parameters of the confocal microscopy imaging software (see Table of Materials): For dual excitation images, use sequential excitation at 488 nm and 543 nm, and collect emission at 505-545 nm and >560 nm, respectively.
   NOTE: Set the imaging settings as follows. Scan Mode: frame; Speed: 9; Average: number, 1; Gain: 450 to 600; Pinhole: 30 to 200; laser: <10%. It is best to start the imaging software first and then completely turn on the 488 nm laser. The 543 nm laser needs to be turned on and stabilized for 3-5 min before use (Figure 1A).
4. Remove the culture medium containing the dye from the incubator (step 2.3) and add 1 mL of KH buffer to the dish.
5. To induce mitophagy, treat the cells with carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) at 1 µM (final concentration) in KH buffer for 10 min at room temperature, and immediately proceed to image the cells using the confocal microscope.
6. Apply an appropriate amount of oil to the top of the 63x oil lens (see Table of Materials). Place the cell sample on the sample stage of the confocal microscope and move it directly above the objective lens.
7. Use the imaging software to find the sample by clicking on the Locate tab in the top left corner of the software interface (Figure 1B). Select a green filter set for the experiment.
8. Use the coarse adjustment knob to quickly focus by moving the objective lens up and down. After the cell sample is clearly visible through the eyepiece, search and focus the area of single cells and move it to the center of the field of view.
9. Click on the Acquisition tab in the top left corner in the software interface to acquire images. Select only the 488 nm channel and the frame resolution 1024 x 1024 for preview.
10. Click on the Live tab in the top left corner to start a live scan. Adjust the field of view to the sharpest and adjust the laser power by moving the slider left or right (Figure 1A). Keep the gain setting below 600 to avoid overexposure.
11. Adjust the pinhole value to 156, gain value to 545, and digital offset value to 0.
12. Select the best field of view, check the two channels (488 nm and 543 nm), and choose the frame resolution 1024 x 1024. Click Snap to acquire 2D images. Save the acquired images.
    ​NOTE: The green mitochondria dye has an excitation peak at 490 nm and an emission peak at 516 nm; it can be excited using a 488 nm laser. The red lysosome dye has an excitation peak at 576 nm and an emission peak at 590 nm; it can be excited using a 543 nm laser.

4. Image analysis

1. Open the saved image with ImageJ and import the merged image into it.
2. Manually count the number of yellow dots in each cell, which indicate that the lysosome is engulfing mitochondria.

Results

MitoTracker Green is a green-fluorescent mitochondrial stain that is able to accurately localize to mitochondria. The dye can easily stain live cells and is less effective in staining aldehyde-fixed or dead cells (Figure 2). The red-fluorescent lysosome dye LysoTracker Red is capable of labeling and tracking acidic lysosomal organelles and can only stain live cells (Figure 2). Confocal microscope imaging allows the visualization of mitochondria and lysosomes sta...

Discussion

The protocol described here provides a method for evaluating and monitoring the dynamic process of mitophagy in living cells, involving autophagosomes, lysosomes, and mitochondrial fission, through co-staining with cell-permeant mitochondria and lysosome dyes. The method can also be used to identify mitochondria and assess mitochondrial morphology. Both dyes used in this study should be protected from light, multiple freeze-thaw cycles should be avoided, and the dyes should be stored in single-use aliquots as much as pos...

Materials

Name	Company	Catalog Number	Comments
Automated cell counter	Countstar	IC1000
Cell counting chamber slides	Countstar	12-0005-50
Dulbecco's modified Eagle medium (DMEM)	Corning	10-013-CV
Dulbecco's phosphate-buffered saline (DPBS)	Corning	21-031-CVC
Glass bottom cell culture dish (confocal dish)	NEST	801002
Image J (Rasband, NIH)	NIH		https://imagej.nih.gov/ij/download.html
Krebs–Henseleit(KHB) buffer			Self-prepared
LysoTracker Red	Invitrogen	1818430	100 µmol/L, red-fluorescent lysosome dye
MitoTracker Green	Invitrogen	1842298	200 µmol/L stock, green-fluorescent mitochondria dye
Mouse Embryonic Fibroblasts			Self-prepared
Objective (63x oil lens)	ZEISS	ZEISS LSM 880
Trypsin-EDTA 0.25%	Gibico	Cat# 25200056
ZEISS LSM 880 Confocal Laser Scanning Microscope	ZEISS	ZEISS LSM 880
ZEN Microscopy Software 2.1 (confocal microscope imaging software)	ZEISS	ZEN 2.1