Photoactivatable Green Fluorescent Protein-Based Visualization and Quantification of Mitochondrial Fusion and Mitochondrial Network Complexity in Living Cells

Abstract

Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis and stress induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assay rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

Mitochondrial Morphofunction in Mammalian Cells

“…Given the tight connection between mitochondrial and cellular function, physiological analysis of mitochondrial morphofunction is ideally carried out within the context of a living cell (147,148,188,196). If required, this can also entail analysis of mitochondrial fusion dynamics and/or motility using photoactivatable proteins (173,231,248). Micropatterned coverslips have been used to ''standardize'' cell size and shape and allow analysis of mitochondrial structure under controlled conditions (60).…”

Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery

“…After photoactivation of a small region of interest (ROI), diffusion and dilution of the photoactivated GFP fluorescence within the mitochondrial network were tracked in a time course experiment. The decrease in GFP fluorescence intensity within the photoactivated ROI at different time points was used for measuring the rates of mitochondrial fusion (Karbowski et al , ). This assay revealed that overexpression of hFis1 in cells significantly reduced diffusion of the mito‐PAGFP signal away from the photoactivated ROI, compared to empty vector control (Fig C and D), indicating that hFis1 overexpression reduces the rate of mitochondrial fusion.…”

“…The mito‐PAGFP‐based assay was performed as previously described (Karbowski et al , , ). For hFis1 overexpression, WT and Drp1 −/− 293T cells were co‐transfected with mito‐PAGFP (0.5 μg), mito‐DsRed (0.2 μg), and either empty vector (0.5 μg) or Myc‐hFis1 plasmid (0.5 μg).…”

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

“…Probes such as mito-YFP have facilitated the observation of mitochondrial fission and fusion events, as well as the connectivity of the mitochondrial network through FRAP analysis [37–40]. Photoactivatable probes, such as mito-PAGFP, have been used extensively to quantify mitochondrial fusion rates and assess the mitochondrial network [72–76]. Other fluorescent proteins boast unique properties that allow them to act as biosensors for various mitochondrial functions (see [77,78] for a recent reviews).…”