Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Summary

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Abstract

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

Introduction

Circular 16.5 kbp DNA molecules constitute the genetic material of mitochondria, encoding 22 tRNAs, 2 rRNAs, and 13 polypeptides needed for mitochondrial oxidative phosphorylation complexes. Mitochondrial DNA bound to mitochondrial transcription factor a (TFAM) and several other proteins form the mitochondrial nucleoids^1,2,3,4. Mitochondrial nucleoids moveand redistribute between the components of the mitochondrial network^5,6 during its morphological remodeling, fission or fusion dep....

Protocol

NOTE: All the cell lines mentioned here were cultured in high glucose Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamine, penicillin/streptomycin, and pyruvate. Equilibrate all media and supplements to be used on the day of labeling and imaging by warming them up to 37 °C in an incubator set to 5% CO₂. All cell culture work including labeling takes place in sterile conditions under a laminar flow hood.

1. L.......

Discussion

There are several critical components to the protocol: To achieve preferential labeling of mitochondrial DNA, the concentration of the DNA binding dye during incubation should be kept very low (e.g., a 1:10,000 dilution of a typical commercial stock), and the incubation time should be 30 min. The incubation time should never exceed 1 h. SYBR Gold dye should be used; other DNA-binding dyes are not bright enough to generate a strong signal upon labeling at a low concentration.

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Materials

Name	Company	Catalog Number	Comments
Elyra PS1	Carl Zeiss		multi-modal super-resolution microscope containing module for super-resolution structured illumination microscopy (SR-SIM)
high glucose DMEM	GIBCO/ThermoFisher	31966021
ibidi 35-mm dish, glass bottom	Ibidi Gmbh	81158
ibidi 8-well microSlide, glass bottom	Ibidi Gmbh	80827
Imaris 8.4.1	Bitplane/Oxford Instruments		image porcessing and visualisation software package
iXon 885	Andor Technologies		EMCCD camera with back-illuminated sensor
LSM880 Airyscan	Carl Zeiss		laser scanning confocal microscope with array detector
Mitotracker CMXRos Red	ThermoFischer	M7512	red live cell mitochondrial stain
Mitotracker Deep Red FM	ThermoFischer	M22426	far red live cell mitochondrial stain
picoGreen	ThermoFischer	P7581	cell permeant DNA stain
Plan Apochromat 100x/1.46 Oil objective	Carl Zeiss
SYBR Gold	ThermoFischer	S11494	cell permeant DNA stain
Zen Black 2012 software	Carl Zeiss		image acquisition and processing software