Monitoring the Mitochondrial Dynamics in Mammalian Cells.

Mitochondria exist in a dynamic state inside mammalian cells. They undergo processes of fusion and fission to adjust their shape according to the different cell needs. Different proteins tightly regulate these dynamics: Opa-1 and Mitofusin-1 and Mitofusin-2 are the main profusion proteins, while Drp1 and its different receptors (Mff, Fis1, MiD49, MiD51) regulate mitochondrial fission. The dynamic nature of the mitochondrial network has become evident and detectable, thanks to recent advances in live imaging video microscopy and to the availability of mitochondria-tagged fluorescent proteins. High-resolution confocal reconstruction of mitochondria over time allows researchers to visualize mitochondria shape changes in living cells, under different experimental conditions. Moreover, in recent years, different techniques in living cells have been developed to study the process of mitochondria fusion in more details. Among them are fluorescence recovery after photobleaching (FRAP) of mitochondria-tagged GFP (mtGFP), use of photoactivatable mtGFP, polyethylene glycol (PEG)-based fusion of mtGFP and mtRFP cells, and Renilla luciferase assay (for population studies). In addition, in combination with imaging, the analysis of the expression levels of the different mitochondria-shaping proteins, along with that of their activation status, represents a powerful tool to investigate potential modulations of the mitochondrial network. Here, we review this aspect and then mention a number of techniques, with particular attention to their relative protocols.