Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Summary

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Abstract

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

Introduction

Mitochondria and lysosomes are the principal membrane-bound organelles that are essential for the maintenance of cellular homeostasis. Mitochondria are highly dynamic cellular organelles modulated by fission and fusion events¹. In general, mitochondria are referred to as the powerhouse of the cell and are known for their important role in oxidative phosphorylation, ATP production, and metabolite storage such as calcium². In addition to their role in energy metabolism, mitochondria are involved in a number of other cellular signaling processes, including apoptosis^3,

Protocol

The step-by-step workflow for sample preparation is shown in Figure 1.

1. Cell culture and transfection

1. Culture HeLa cells at a density of 1 × 10⁵ cells in 35 mm glass grid-bottomed culture dishes containing DMEM with 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin at 37 °C.
2. The day after seeding the cells, co-transfect the cells at 30%-40% confluency with TMEM192-V5-APEX2 and Mito-mEosEM plasmids using the transfection reagent according to the manufacturer's instructions(1 µg each of TMEM192-V5-APEX2 and Mito-mEosEM plasmid....

Results

To visualize the interactions between mitochondria and lysosomes using this dual-color CLEM protocol, we employed Mito-mEosEM and TMEM192-V5-APEX2. Fixed cells were first imaged using a light microscope, followed by EM sampling and imaging. The results of the correlated images are shown in Figure 3A,B and Figure 4A. Following the protocol described above, we checked whether we could observe the changes in the interaction between mitochondria and.......

Discussion

This protocol describes dual-color CLEM to observe the interaction of mitochondria and lysosomes in fixed cells using genetically encoded mEosEM and APEX2 tags. Over the past few decades, advances in microscopy have greatly improved the ability to observe changes in organelle networks^32,33, and the study of organelle interactions has become increasingly interesting. Super-resolution microscopy has been used to observe changes in highly dynamic contacts between mi.......

Materials

Name	Company	Catalog Number	Comments
Chemicals and reagents
25% Glutaraldehyde	Electron Microscopy Sciences	16200	Aldehyde fumes are extremely toxic. Use only in fume hood.
30% Hydrogen peroxide solution	Merck	107210
4% Paraformaldehyde solution	Biosesang	PC2031-100-00	ldehyde fumes are extremely toxic. Use only in fume hood.
Amplex Red	ThermoFisher	A12222
Epon 812	Electron Microscopy Sciences	14120
Ethanol	Merck	100983
Fugene HD	Promega	E2311	Plasmid transfection
Glycine	SIGMA	G8898
Lead Citrate 3%	Electron Microscopy Sciences	22410
Osmium tetroxide 4 % aqueous solution	Electron Microscopy Sciences	16320	Very toxic. Use only in fume hood.
Potassium hexacyanoferrate(II) trihydrate	SIGMA	P3289
Sodium cacodylate trihydrate	Sigma-Aldrich	C0250-500G
Uranyl acetate	Electron Microscopy Sciences	22400
UranyLess EM stain	Electron Microscopy Sciences	22409
Plasmid construction
Mito-mEosEM	addgene	132706	DOI: 10.1016/j.chembiol.2024.02.007
TMEM192-V5-APEX2	Sharma N.  et al.	N/A	DOI: 10.1016/j.chembiol.2024.02.007
Tools
0.22 um syringe filter	Sartorius	16534
35 mm Gridded coverslip dish	Mattek	P35G-1.5-14-CGRD
BEEM Embedding Capsules	Ted Pella	130
Formvar Supported Single slot copper grid	Ted Pella	01705
Diamond knife	DiATOME	DU4530
Ultra-microtome	Leica	ARTOS 3D
Microscopes
Inverted confocal microscopy	Nikon	A1 Rsi/Ti-E
Transmission electron microscopy	FEI	Tecnai G2
Software and Algorithms
Fiji/Image J	NIH / open source		https://imagej.nih.gov/ij/
ImageJ BigWarp software package	NIH / open source		https://imagej.net/plugins/bigwarp
Photozoom Pro 8	BenVista		https://www.benvista.com/photozoompro Alternative software Image Resizer : https://imageresizer.com/ Gigapixel AI : https://www.topazlabs.com/gigapixel ON1 resize : https://www.on1.com/products/resize-ai/