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mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.

Published version
Peer-reviewed

Repository DOI


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Authors

Tábara, Luis Carlos 
Prudent, Julien 
Minczuk, Michal 

Abstract

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R = 0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy. mtFociCounter provides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.

Description

Keywords

Humans, DNA, Mitochondrial, Microscopy, Mitochondria, Single-Cell Analysis

Journal Title

Nucleic Acids Res

Conference Name

Journal ISSN

0305-1048
1362-4962

Volume Title

51

Publisher

Oxford University Press (OUP)
Sponsorship
BBSRC (BB/W008467/1)
MRC (MC_UU_00028/3)
MRC (MC_UU_00028/5)
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