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Monitoring the Mitochondrial Dynamics in Mammalian Cells

  • Luca Simula
  • Silvia CampelloEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1782)

Abstract

Mitochondria exist in a dynamic state inside mammalian cells. They undergo processes of fusion and fission to adjust their shape according to the different cell needs. Different proteins tightly regulate these dynamics: Opa-1 and Mitofusin-1 and Mitofusin-2 are the main profusion proteins, while Drp1 and its different receptors (Mff, Fis1, MiD49, MiD51) regulate mitochondrial fission. The dynamic nature of the mitochondrial network has become evident and detectable, thanks to recent advances in live imaging video microscopy and to the availability of mitochondria-tagged fluorescent proteins. High-resolution confocal reconstruction of mitochondria over time allows researchers to visualize mitochondria shape changes in living cells, under different experimental conditions. Moreover, in recent years, different techniques in living cells have been developed to study the process of mitochondria fusion in more details. Among them are fluorescence recovery after photobleaching (FRAP) of mitochondria-tagged GFP (mtGFP), use of photoactivatable mtGFP, polyethylene glycol (PEG)-based fusion of mtGFP and mtRFP cells, and Renilla luciferase assay (for population studies). In addition, in combination with imaging, the analysis of the expression levels of the different mitochondria-shaping proteins, along with that of their activation status, represents a powerful tool to investigate potential modulations of the mitochondrial network. Here, we review this aspect and then mention a number of techniques, with particular attention to their relative protocols.

Key words

Mitochondrial dynamics Fusion Fission Microscopy Live cells 

Notes

Acknowledgments

This work was funded by the Italian Ministry of Health (GR-2011-02351643) and IG-19826 by AIRC to SC and by grants from Fondazione Roma and FISM.

Supplementary material

Supplementary Movie 1.

 Time-lapse confocal video microscopy of a mtYFP-expressing Jurkat cell. Confocal 3D reconstruction of the mitochondrial network in a mtYFP-expressing Jurkat cell over time. Acquisitions have been performed with a 488 nm laser, by 0.4 μm z-stack steps, each 10 s (MP4 269 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of Rome Tor VergataRomeItaly
  2. 2.Department of Pediatric Hematology and OncologyIRCCS Bambino Gesù Children’s HospitalRomeItaly
  3. 3.IRCCS, Fondazione Santa LuciaRomeItaly

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