Imaging Dynamin-Related Protein 1 (Drp1)-Mediated Mitochondrial Fission in Living Cells
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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.
Key wordsMitochondrial dynamics Fission Drp1 Confocal microscopy Live-cell imaging
The cell lines used in this study were obtained from the labs of Ting-wei Mu (HeLa) and Xin 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.
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)
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