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mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis
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...
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| Published in: | Nucleic acids research 2023-11, Vol.51 (21), p.e107-e107 |
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| Main Authors: | , , , |
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| Language: | English |
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| cites | cdi_FETCH-LOGICAL-c357t-2e3f8ee8c6557b97ec2ed275d3d3b8f5a598e9a1b7ac2dca0c6f2d211c7d222f3 |
| container_end_page | e107 |
| container_issue | 21 |
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| container_title | Nucleic acids research |
| container_volume | 51 |
| creator | Rey, Timo Tábara, Luis Carlos Prudent, Julien Minczuk, Michal |
| description | 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.
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| doi_str_mv | 10.1093/nar/gkad864 |
| format | article |
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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.
Graphical Abstract
Graphical Abstract</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkad864</identifier><identifier>PMID: 37850644</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>DNA, Mitochondrial - ultrastructure ; Humans ; Microscopy ; Mitochondria - ultrastructure ; Single-Cell Analysis</subject><ispartof>Nucleic acids research, 2023-11, Vol.51 (21), p.e107-e107</ispartof><rights>The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. 2023</rights><rights>The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c357t-2e3f8ee8c6557b97ec2ed275d3d3b8f5a598e9a1b7ac2dca0c6f2d211c7d222f3</citedby><cites>FETCH-LOGICAL-c357t-2e3f8ee8c6557b97ec2ed275d3d3b8f5a598e9a1b7ac2dca0c6f2d211c7d222f3</cites><orcidid>0000-0002-2124-937X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37850644$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rey, Timo</creatorcontrib><creatorcontrib>Tábara, Luis Carlos</creatorcontrib><creatorcontrib>Prudent, Julien</creatorcontrib><creatorcontrib>Minczuk, Michal</creatorcontrib><title>mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>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.
Graphical Abstract
Graphical Abstract</description><subject>DNA, Mitochondrial - ultrastructure</subject><subject>Humans</subject><subject>Microscopy</subject><subject>Mitochondria - ultrastructure</subject><subject>Single-Cell Analysis</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><recordid>eNp9kEtLxDAQgIMouj5O3qUnEaSaR9OkR1lcFQQvei5pMtVomqx5HPz3Vnb16Glg-PiY-RA6JfiK4I5dexWvXz-UkW2zgxaEtbRuupbuogVmmNcEN_IAHab0jjFpCG_20QETkuO2aRbITXkVtF2G4jPEagyxUiWHSWUwVbL-1UGtwblqsjnot-BNtMpVvmgHwZrqsyif7Wi1yjb4SnlTRVjHYIq2g4NZqO28Ve4r2XSM9kblEpxs5xF6Wd0-L-_rx6e7h-XNY60ZF7mmwEYJIHXLuRg6AZqCoYIbZtggR654J6FTZBBKU6MV1u1IDSVEC0MpHdkRuth450M-C6TcTzb9fKE8hJJ6KoUUhDSUzujlBtUxpBRh7NfRTip-9QT3P3n7OW-_zTvTZ1txGSYwf-xvzxk43wChrP81fQM5KYeN</recordid><startdate>20231127</startdate><enddate>20231127</enddate><creator>Rey, Timo</creator><creator>Tábara, Luis Carlos</creator><creator>Prudent, Julien</creator><creator>Minczuk, Michal</creator><general>Oxford University Press</general><scope>TOX</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-2124-937X</orcidid></search><sort><creationdate>20231127</creationdate><title>mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis</title><author>Rey, Timo ; Tábara, Luis Carlos ; Prudent, Julien ; Minczuk, Michal</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c357t-2e3f8ee8c6557b97ec2ed275d3d3b8f5a598e9a1b7ac2dca0c6f2d211c7d222f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>DNA, Mitochondrial - ultrastructure</topic><topic>Humans</topic><topic>Microscopy</topic><topic>Mitochondria - ultrastructure</topic><topic>Single-Cell Analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rey, Timo</creatorcontrib><creatorcontrib>Tábara, Luis Carlos</creatorcontrib><creatorcontrib>Prudent, Julien</creatorcontrib><creatorcontrib>Minczuk, Michal</creatorcontrib><collection>Open Access: Oxford University Press Open Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rey, Timo</au><au>Tábara, Luis Carlos</au><au>Prudent, Julien</au><au>Minczuk, Michal</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2023-11-27</date><risdate>2023</risdate><volume>51</volume><issue>21</issue><spage>e107</spage><epage>e107</epage><pages>e107-e107</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>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.
Graphical Abstract
Graphical Abstract</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>37850644</pmid><doi>10.1093/nar/gkad864</doi><orcidid>https://orcid.org/0000-0002-2124-937X</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Nucleic acids research, 2023-11, Vol.51 (21), p.e107-e107 |
| issn | 0305-1048 1362-4962 |
| language | eng |
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| source | Open Access: Oxford University Press Open Journals; PubMed Central |
| subjects | DNA, Mitochondrial - ultrastructure Humans Microscopy Mitochondria - ultrastructure Single-Cell Analysis |
| title | mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis |
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