Description:

Communication of mitochondria through networking supports fuel metabolism and calcium transport, and impacts the cellular response to apoptotic stimuli. A mitochondrial network shares a continuous matrix lumen that enables the diffusion of molecules and allows for electrical coupling and synchronization. However, identifying a network and distinguishing it from closely packed but disconnected mitochondria remains a challenge, and thus it is unclear how common and how dynamic functional networks are. We used the resources at the BioCurrent Research Center- tissue culture, confocal microscopy, chemical databases and fluorescent probes to set up a methodology to functionally define the network in time and space and study its electrical and diffusion properties over time.

Progress:

We used confocal microscopy on cell lines (INS-1 and COS-7 cells) transfected with matrix-targeted photoactivatable GFP. The setup of the multi-photon confocal microscope (LSM 510) at the BRC was unique for its precise resolution that allowed a selective labeling of an individual mitochondrion with a precision of ~0.5mm. After tagging, the morphology (lumen continuity, fusion and fission events) and mitochondrial membrane potential were simultaneously imaged.

We found that each mitochondrial web is made up of multiple disconnected networks. Due to the complexity of mitochondrial web morphology, the network boundaries are poorly predicted by conventional visual inspection. Dissecting the web into individual networks using this approach may explain previously described observations of membrane potential heterogeneity along a mitochondrion. We found that all segments within a single network are invariably equipotential and such heterogeneity may represent the existence of multiple networks. Interestingly, fission events frequently occur without any gross morphological changes. These events, which are invisible under standard confocal microscopy, redefine the mitochondrial network boundaries while electrically dissociating the resultant sections.