Understanding the Changes in Mitochondrial Morphology through Dynamic and Three-dimensional Fluorescence Micrographs

Summary

Here we describe the mitochondrial event localizer (MEL), an ImageJ plugin useful in the quantification of the 3-dimensional changes in mitochondrial fission and fusion activity over time. We also describe an image processing pipeline useful for the cleanup of micrographs prior to analysis in ImageJ.

Abstract

Mitochondria are highly dynamic organelles that are vital to the survival of any animal, undergoing regular fission and fusion events in response to the needs or stresses of the host, leading to the constant remodeling of the mitochondrial network. Because of this, being able to evaluate the mitochondrial network in three dimensions, as well as over time, offers a benefit in understanding how the system responds to factors such as stress or pharmaceutical intervention. Fluorescence imaging of the mitochondrial networks of cells enables the ability to visualize and monitor these changes. However, the mitochondrial network is often described as a two-dimensional and static structure that is defined by unstandardized metrics. Therefore, we set out to describe a pipeline that enables the user to prepare their images for the mitochondrial event localizer (MEL), an ImageJ plugin tool that detects fission and fusion events in the mitochondrial network over time and in a 3-dimensional manner, thus, offering insight into the dynamic changes that this network undergoes. Additionally, we describe the benefits of understanding fission and fusion in light of the changes in the mitochondrial count and morphological changes.

Introduction

Mitochondria are highly dynamic organelles present in all eukaryotic cells, providing them with energy and regulate their metabolism. Thus, mitochondria are at the crossroads of cellular death and survival. Mitochondria have been shown to be essential for a variety of processes, ranging from lysosomal acidification and molecular motor action to muscle contraction and synapse firing^1,2.

Mitochondria undergo regular fission and fusion events to maintain a mitochondrial network that efficiently produces ATP in response to the cell's metabolic demand and stress. Indeed, mitochondria....

Protocol

1. Cell treatment and microscopy acquisition

1. Culture GT1-7 cells in 8 chamber dishes in DMEM supplemented with 10% FBS and 1% Penstrep (complete media). Allow cells to attach overnight and then treat with 2.5 mM metformin hydrochloride made in DMEM with 10% FBS for 72 h, being sure to replace the media every 24 h. Co-treat the cells with 10 µM CCCP 6 h before imaging and 400 nM of Bafilomycin A1 (Baf) 4 h before imaging.
   NOTE: This can be done with any eukaryotic cell line of choice.
2. Prior to imaging, prepare a cocktail of prewarmed complete media containing 5 nM Hoechst and 100 nM TMRE.
3. Replace the cell tre....

Results

Selecting appropriate cells
Users should be aware that the mitochondrial network changes depending on the mitotic state of the cell. Should the nucleus appear dumbbell- or U-shaped, or if there is a space near the nucleus with a lack of fluorescence signal, then this may indicate that the cell is nearing mitosis. In this state, mitochondria are likely undergoing fission due to cell division and not because of the treatment intervention and its effect on the network (Supplemental Figure S8

Discussion

Although a growing number of approaches exist to describe mitochondrial morphology, limited techniques are available to adequately capture mitochondrial dynamics in a quantitative manner. Additionally, it should be noted that mitochondrial network morphology as well as the mechanisms governing this morphology are varied in nature. This results in networks that are linked to the needs of the cell, ranging from a branched formation for enhanced energy output to temporally distinct regions of fission to facilitate mitophagy.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8 chamber dishes	ThermoFisher	#Z734853
Adjusted thresholding			https://sites.google.com/site/qingzongtseng/adaptivethreshold
Bafilomycin A1	LKT labs	#B0026
Carbonyl cyanide chlorophenylhydrazone (CCCP)	Merck	#C2759
Confocal microscope	Carl Zeiss AG		LSM780 ELYRA PS.1 super-resolution platform
Dulbecco's Modified Eagle Medium (DMEM)	ThermoFisher	#341956062
Fetal Bovine Serum (FBS)	Sigma-Aldrich	#F0679
Github link			https://github.com/rensutheart/MEL-Fiji-Plugin
GraphPad Prism v7.06
GT1-7 cells	ATCC	SCC116
Hoecsht	Sigma-Aldrich	H6024
ImageJ v1.53t	Fiji
Macros			https://github.com/rensutheart/FMPP/tree/master/Sections
Metformin	European Pharmacopoeia	M06050000
Penicillin/streptomycin (PenStrep)	Sigma-Aldrich	#P4333
T25s	Bio-Smart Scientific	#70025
TMRE	ThermoFisher	#T669
Trypsin	Sigma-Aldrich	#T4049