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In situ localization of mitochondrial DNA replication in intact mammalian cells
Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical informat...
Formato: | Texto |
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Lenguaje: | English |
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The Rockefeller University Press
1996
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2133381/ https://www.ncbi.nlm.nih.gov/pubmed/8922374 |
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collection | PubMed |
description | Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network. |
format | Text |
id | pubmed-2133381 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1996 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21333812008-05-01 In situ localization of mitochondrial DNA replication in intact mammalian cells J Cell Biol Articles Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network. The Rockefeller University Press 1996-11-02 /pmc/articles/PMC2133381/ /pubmed/8922374 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Articles In situ localization of mitochondrial DNA replication in intact mammalian cells |
title | In situ localization of mitochondrial DNA replication in intact
mammalian cells |
title_full | In situ localization of mitochondrial DNA replication in intact
mammalian cells |
title_fullStr | In situ localization of mitochondrial DNA replication in intact
mammalian cells |
title_full_unstemmed | In situ localization of mitochondrial DNA replication in intact
mammalian cells |
title_short | In situ localization of mitochondrial DNA replication in intact
mammalian cells |
title_sort | in situ localization of mitochondrial dna replication in intact
mammalian cells |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2133381/ https://www.ncbi.nlm.nih.gov/pubmed/8922374 |