Imaging Dynamin-Related Protein 1 (Drp1)-Mediated Mitochondrial Fission in Living Cells
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 c
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Introduction Mitochondria are double membrane-bound organelles present in all eukaryotic cells [1]. Mitochondria are involved in many essential cellular functions, including ATP production, intracellular calcium signaling, and programmed cell death (apoptosis) [2]. These pivotal functions are intimately connected, however, to mitochondrial morphology. Mitochondria form highly dynamic networks that continuously undergo regulated cycles of fission and fusion [3]. Depending on the metabolic state of the cell, mitochondria are mostly either tied in highly branched, filamentous, reticular networks or fragmented into discrete, punctate, granular entities spread throughout the cytoplasm [4]. Most often, mitochondria exist as a mixture of both. A dynamic balance between the opposing processes of
Electronic supplementary material: The online version of this chapter (https://doi.org/10.1007/978-1-07160676-6_16) contains supplementary material, which is available to authorized users. Rajesh Ramachandran (ed.), Dynamin Superfamily GTPases: Methods and Protocols, Methods in Molecular Biology, vol. 2159, https://doi.org/10.1007/978-1-0716-0676-6_16, © Springer Science+Business Media, LLC, part of Springer Nature 2020
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Felipe Montecinos-Franjola and Rajesh Ramachandran
mitochondrial fission and fusion governs the overall shape of mitochondria in any given cell. Dynamin superfamily proteins (DSPs) regulate the mitochondrial fission–fusion balance [5, 6]. Drp1 mediates mitochondrial fission and is recruited from the cytosol to the mitochondrial surface at pre-marked fission sites [7]. Membrane-integrated DSPs mitofusins (Mfn) and optic atrophy 1 (OPA1), anchored in the outer and inner mitochondrial membranes, respectively, conversely mediate mitochondrial fusion [5]. In the context of fission, how Drp1 oligomerization dynamics on the mitochondrial membrane surface is coupled to mitochondrial division stills remains poorly understood. Live-cell confocal microscopy harnessing genetically encoded biomarkers of the mitochondria combined with GFP fusion variants of Drp1 enables correlation of Drp1 membrane dynamics to mitochondrial fission in real time. Here, we detail methods used in the confocal fluorescence imaging of mitochondrial dynamics (Subheading 2) and Drp1-mediated mitochondrial fission (Subheading 3) in living, cultured mammalian cells.
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Section I: Live-Cell Imaging of Mitochondrial Dynamics A popular method to visualize the mitochondrial network in living cells is confocal fluorescence microscopy that allows for real-time monitoring of mitochondrial dynamics at high resolution and low noise. Specific labeling of the mitochondria can be achieved either with the use of synthetic fluorophores or with genetically encoded biomarkers. A common synthetic dye for staining mitochondria is MitoTracker that offers the advantage of covalent attachment to the mitochondria and is insensitive to cell fixation and/or death [8]. However, many synthetic fluorescent dyes can be toxic to cells even when illuminated for short
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