Abstract
Mitochondria form highly dynamic networks that continuously undergo fission and fusion. Dynamin-related protein 1 (Drp1), a key regulator of mitochondrial division, self-assembles into a helical polymer around pre-marked scission sites and generates the constriction force necessary to sever the organelle. Live-cell fluorescence imaging of Drp1 oligomerization dynamics and mitochondrial fission can provide unprecedented insights into the spatiotemporal relationship between these coupled processes. The high-resolution images provided by the laser scanning confocal microscope facilitate the observation of the finer details of mitochondrial structure as well as Drp1 polymer dynamics in real time. We provide a detailed description of the confocal imaging methods used to characterize mitochondrial dynamics in living cells with an emphasis on Drp1-mediated mitochondrial fission.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
Change history
22 September 2020
The chapter was inadvertently published with the incorrect unit mm (millimeters) as “Step size, mm (z-stack)” instead of μm (micrometers) as “Step size, μm (z-stack)”.
References
Friedman JR, Nunnari J (2014) Mitochondrial form and function. Nature 505:335–343
Youle RJ, van der Bliek AM (2012) Mitochondrial fission, fusion, and stress. Science 337:1062–1065
Labbe K, Murley A, Nunnari J (2014) Determinants and functions of mitochondrial behavior. Annu Rev Cell Dev Biol 30:357–391
Mishra P, Chan DC (2016) Metabolic regulation of mitochondrial dynamics. J Cell Biol 212:379–387
Ramachandran R (2018) Mitochondrial dynamics: the dynamin superfamily and execution by collusion. Semin Cell Dev Biol 76:201–212
Ramachandran R, Schmid SL (2018) The dynamin superfamily. Curr Biol 28:R411–R416
Friedman JR, Lackner LL, West M, DiBenedetto JR, Nunnari J, Voeltz GK (2011) ER tubules mark sites of mitochondrial division. Science 334:358–362
Chazotte B (2009) Labeling mitochondria with fluorescent dyes for imaging. Cold Spring Harb Protoc 2009:pdb prot4948
Minamikawa T, Sriratana A, Williams DA, Bowser DN, Hill JS, Nagley P (1999) Chloromethyl-X-rosamine (MitoTracker Red) photosensitises mitochondria and induces apoptosis in intact human cells. J Cell Sci 112(Pt 14):2419–2430
Olenych SG, Claxton NS, Ottenberg GK, Davidson MW (2007) The fluorescent protein color palette. Curr Protoc Cell Biol 21:25
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Simula L, Campello S (2018) Monitoring the mitochondrial dynamics in mammalian cells. Methods Mol Biol 1782:267–285
Wakabayashi J, Zhang Z, Wakabayashi N, Tamura Y, Fukaya M, Kensler TW, Iijima M, Sesaki H (2009) The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice. J Cell Biol 186:805–816
Macdonald PJ, Stepanyants N, Mehrotra N, Mears JA, Qi X, Sesaki H, Ramachandran R (2014) A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission. Mol Biol Cell 25:1905–1915
Mitra K, Lippincott-Schwartz J (2010) Analysis of mitochondrial dynamics and functions using imaging approaches. Curr Protoc Cell Biol Chapter 4:Unit 4 25 21–Unit 4 25 21
Smirnova E, Griparic L, Shurland DL, van der Bliek AM (2001) Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell 12:2245–2256
Strack S, Cribbs JT (2012) Allosteric modulation of Drp1 mechanoenzyme assembly and mitochondrial fission by the variable domain. J Biol Chem 287:10990–11001
Acknowledgments
The cell lines used in this study were obtained from the labs of Ting-wei Mu (HeLa) and **n Qi (Drp1−/− MEFs), both of Case Western Reserve University School of Medicine. FM-F is grateful to Yanlin Fu and Di Hu (CWRU) for advice on the handling of cell cultures and co-transfection. This work was supported by National Institutes of Health grant R01GM121583 awarded to R.R.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Live-cell time-lapse video microscopy of mitochondrial dynamics. HeLa cells expressing mCherry-Mito-7 (red) were excited using the 543 nm laser line, and 512 × 512 images were acquired every 4 s. In this video, two events are shown: the fission of a mitochondrial fragment followed by the separation and subsequent fusion of the resulting daughter fragment with another mitochondrial filament. The video was assembled from 100 frames, and a subset of these images is shown in Fig. 2. Scale bar 4 μm (AVI 45,147 kb)
Animated movie of the mitochondrial network in Drp1−/− MEFs transiently expressing mCherry-Mito-7 (red). The animation shows slices of the high-resolution z-stack series used for 3D reconstruction (from the top to the bottom of the cell). See Fig. 3 for experimental details. Scale bar 10 μm (AVI 26,373 kb)
Animated movie of the 3D-reconstructed mitochondrial network in Drp1−/− MEFs transiently expressing mCherry-Mito7 (red). The mitochondrial network is represented as volume and prepared using default settings of the “3D viewer” plugin in ImageJ. Scale bar 10 μm (AVI 40,821 kb)
Time-lapse video microscopy of Drp1-mediated mitochondrial fission. Live HeLa cells expressing mCherry-Mito-7 (red) and mEGFP-Drp1 (green) were excited using the 488 and 543 nm laser lines, respectively, and images were acquired every 5 s. The original lariat-shaped mitochondrial filament undergoes fission catalyzed by Drp1 assembled over the filament as a “collar” (green puncta). The video was assembled from 50 frames, and a subset of these images are shown in Fig. 4. Scale bar 5 μm (AVI 17,905 kb)
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Montecinos-Franjola, F., Ramachandran, R. (2020). Imaging Dynamin-Related Protein 1 (Drp1)-Mediated Mitochondrial Fission in Living Cells. In: Ramachandran, R. (eds) Dynamin Superfamily GTPases. Methods in Molecular Biology, vol 2159. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0676-6_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0676-6_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0675-9
Online ISBN: 978-1-0716-0676-6
eBook Packages: Springer Protocols