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

Jevtic, V., Kindle, P., & Avilov, S. (2020). Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structures Illumination Microscopy. Journal of Visualized Experiments, 160, e60003. doi:10.3791/60003.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0006-8FAE-6 Version Permalink: http://hdl.handle.net/21.11116/0000-0006-8FAF-5
Genre: Journal Article

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video version: https://www.jove.com/video/60003

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 Creators:
Jevtic, Visnja1, Author
Kindle, Petra1, Author
Avilov, Sergiy1, Author              
Affiliations:
1Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society, ou_2243641              

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 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 networkthat undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward techniqueto characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their proteincomponents, 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 intoaccount the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling ofmitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantificationof mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

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Language(s): eng - English
 Dates: 2020-06-04
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.3791/60003
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Title: Journal of Visualized Experiments
  Other : Journal of Visualized Experiments: JoVE
  Abbreviation : J. Vis. Exp.
Source Genre: Journal
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Publ. Info: Rockville Pike, Bethesda MD : JoVE
Pages: - Volume / Issue: 160 Sequence Number: - Start / End Page: e60003 Identifier: ISSN: 1940-087X
CoNE: https://pure.mpg.de/cone/journals/resource/1940087X