No 1.5 glass bottomed dishes

Cells transduced with mito-PAGFP (#23348; Addgene) were plated in No. 1.5 glass-bottomed dishes (MatTek). MEFs were incubated with 0.1 μg/ml MitoTracker Red CMXRos (Invitrogen) for 15 min at 37°C with 5% CO₂, washed, and incubated with complete media for at least 45 min before imaging. MEFs were imaged at 37°C with 5% CO₂. A region that was ∼1 µm² was activated using a 405-nm laser, and the same cell was imaged after 50 min. Images were collected with a Nikon Ti-E widefield microscope with a 63× NA 1.4 oil objective (Nikon), a solid-state light source (Spectra X; Lumencor), and an sCMOS camera (Zyla 5.5 Megapixel).

Cells transduced with mito-paGFP (#23348; Addgene) and either Mfn1-FLAG pBabe-hygro or Mfn2-FLAG-P2A-blasticidin pBabe were plated in No. 1.5 glass-bottomed dishes (MatTek). MEFs were incubated with 0.1 μg/ml MitoTracker Red CMXRos for 15 min at 37°C with 5% CO₂, washed, and incubated with complete media for at least 45 min before imaging. MEFs were imaged at 37°C with 5% CO₂. A region that was ∼1 μm² was activated using a 405-nm laser, and the same cell was imaged every 10 min for 50 min. Images were collected with a Nikon Ti-E widefield microscope with a 63× NA 1.4 oil objective (Nikon), a solid-state light source (SPECTRA X; Lumencor), and an sCMOS camera (Zyla 5.5 Megapixel). Images were cropped and deconvolved using 15 iterations of 3D Landweber deconvolution on Offline Deconvolution software (Nikon). Data were then analyzed using Nikon NIS-Elements Analysis software. Background was removed, and maximum intensity projections were created. Each channel was thresholded separately for each cell, and the number of pixels that were both mCherry positive and GFP positive were recorded. Spread of paGFP was calculated as the number of pixels that were both GFP and mCherry positive divided by total number of mCherry pixels after 50 min divided by the same variable at 0 min (immediately after activation). Graphs were created in Prism (GraphPad).

All cells were plated ingrown on no. 1.5 glass-bottomed dishes (MatTek) for ∼48 h before imaging. Cells were incubated with 0.1 μg/ml MitoTracker Red CMXRos with or without three drops NucBlue (Molecular Probes) for 15 min at 37°C with 5% CO₂. After this, MEF cells were rinsed into complete media for at least 45 min before imaging, and the Flp-In TREx cells were incubated with 0.2 μg/ml tetracycline hydrochloride (Thermo Fisher Scientific) for 4 h. A Z-series with a step size of 0.3 μm was collected with a Nikon Ti-E wide-field microscope with a 60 × NA (numerical aperture) 1.4 oil objective (Nikon), a solid-state light source (Spectra X; Lumencor), and an sCMOS camera (Zyla 5.5 megapixel). Each cell line was imaged on at least three separate occasions by a blinded experimenter (n > 100 cells per experiment).