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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

Published: May 5th, 2023

DOI:

10.3791/65236

1Faculty of Biology, Institute of Genetics and Biotechnology, University of Warsaw, 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 3International Institute of Molecular and Cell Biology in Warsaw

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

For most eukaryotic cells mitochondria are essential organelles, as they play a crucial role in numerous cellular processes. First and foremost, mitochondria are the key energy suppliers of cells1. Mitochondria are also involved in regulating cellular homeostasis (for instance, intracellular redox2 and the calcium balance3), cell signaling4,5, apoptosis6, the synthesis of different biochemical compounds7,8, and the innate immune response9

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1. Preparation of the siRNA mixture

  1. One day before the start of the experiment, seed cells (e.g., HeLa) on a 100 mm dish so that they reach 70%-90% confluence the next day.
    NOTE: All operations must be carried out under sterile conditions in a laminar flow chamber.
  2. Prepare the appropriate amount of siRNA diluted to a concentration of 140 nM in Opti-MEM medium (see Table of Materials). A 96-well plate can be used as a reservoir.
  3. Add 5 µL of the .......

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A scheme of the procedure for the high-throughput study of the dynamics of mtDNA synthesis and distribution is shown in Figure 1. The use of a multi-well plate format enables the simultaneous analysis of many different experimental conditions, such as the silencing of different genes using a siRNA library. The conditions used for the labeling of newly synthesized DNA molecules with BrdU allow for the detection of BrdU-labeled DNA in the mitochondria of HeLa cells (Figure.......

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Historically, DNA labeling by BrdU incorporation and antibody detection has been used in nuclear DNA replication and cell cycle research14,27,28. So far, all the protocols for detecting BrdU-labeled DNA have included a DNA denaturation step (acidic or thermal) or enzyme digestion (DNase or proteinase) to enable epitope exposure and facilitate antibody penetration. These protocols were developed for tightly packed nuclear DNA. Ho.......

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This work was supported by the National Science Centre, Poland (Grant/Award Number: 2018/31/D/NZ2/03901).

....

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Name Company Catalog Number Comments
2′,3′-Dideoxycytidine (ddC) Sigma-Aldrich D5782
384  Well Cell Culture Microplates, black Greiner Bio-One #781946
5-Bromo-2′-deoxyuridine (BrdU) Sigma-Aldrich B5002-1G Dissolve BrdU powder in water to 20 mM stock solution and aliquot. Use 20 µM BrdU solution for labeling.
Adhesive sealing film Nerbe Plus 04-095-0060
Alexa Fluor 488 goat anti-mouse IgG1 secondary antibody Thermo Fisher Scientific A-21121
Alexa Fluor 555 goat anti-mouse IgM secondary antibody Thermo Fisher Scientific A-21426
BioTek 405 LS microplate washer Agilent
Bovine Serum Albumin (BSA) Sigma-Aldrich A4503
Cell counting chamber Thoma Heinz Herenz REF:1080339
Dulbecco's Modified Eagle Medium (DMEM) Cytiva SH30243.01
Dulbecco's Modified Eagle Medium (DMEM) Thermo Fisher Scientific 41965-062
Fetal Bovine Serum (FBS) Thermo Fisher Scientific 10270-106
Formaldehyde solution Sigma-Aldrich F1635 Formaldehyde is toxic; please read the safety data sheet carefully.
Hoechst 33342 Thermo Fisher Scientific H3570
IgG1 mouse monoclonal anti-BrdU (IIB5) primary antibody Santa Cruz Biotechnology sc-32323
IgM mouse monoclonal anti-DNA (AC-30-10) primary antibody Progen #61014
LightCycler 480 System Roche
Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific #13778150
MitoTracker Deep Red FM Thermo Fisher Scientific M22426 Mitochondria tracking dye 
Multidrop Combi Reagent Dispenser Thermo Fisher Scientific
Opti-MEM Thermo Fisher Scientific 51985-042
Orca-R2 (C10600) CCD Camera Hamamatsu
Penicillin-Streptomycin  Sigma-Aldrich P0781-100ML
Phosphate buffered saline (PBS) Sigma-Aldrich P4417-100TAB
PowerUp SYBR Green Master Mix Thermo Fisher Scientific A25742
qPCR primer Fw B2M (reference) CAGGTACTCCAAAGATTCAGG 
qPCR primer Fw GPI (reference gene) GACCTTTACTACCCAGGAGA
qPCR primer Fw MT-ND1  TAGCAGAGACCAACCGAACC 
qPCR primer Fw POLG TGGAAGGCAGGCATGGTCAAACC
qPCR primer Fw TFAM GATGAGTTCTGCCTGCTTTAT
qPCR primer Fw TWNK GCCATGTGACACTGGTCATT
qPCR primer Rev B2M (reference) GTCAACTTCAATGTCGGATGG 
qPCR primer Rev GPI (reference gene) AGTAGACAGGGCAACAAAGT
qPCR primer Rev MT-ND1  ATGAAGAATAGGGCGAAGGG 
qPCR primer Rev POLG GGAGTCAGAACACCTGGCTTTGG
qPCR primer Rev TFAM GGACTTCTGCCAGCATAATA
qPCR primer Rev TWNK AACATTGTCTGCTTCCTGGC
ScanR microscope Olympus
siRNA Ctrl Dharmacon D-001810-10-5
siRNA POLG Invitrogen POLGHSS108223
siRNA TFAM Invitrogen TFAMHSS144252
siRNA TWNK Invitrogen C10orf2HSS125597
Suction device NeoLab 2-9335 Suction device for cell culture
Triton X-100 Sigma-Aldrich T9284-500ML
Trypsin Biowest L0931-500
UPlanSApo 20x 0.75 NA objective Olympus

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