Myc Influences Mitochondrial Dynamics Figure 7. PEG Fusion Assay. (a) MycER cells exposed to 4-HT chronically were stably transduced with lentiviruses encoding mitochondrially targeted DsRed or GFP. Photos show representative cells with mitochondrially-targeted fluorescent proteins. (b). Cells were co-plated in the presence or absence of 4-HT and were treated with PEG following incubation with CHX. Cells were fixed stained with DAPI and heterokaryons were identified and imaged by confocal fluorescent microscopy. (c) The degree of mitochondrial fusion was calculated by determining the percentage of co-localized fluorescent signals with the use of the CoLocalizer Pro software package. Cells from which 4-HT was removed from the media for one or two days were compared to cells constantly maintained in 4-HT. doi:10.1371/journal.pone.0037699.g007 mitochondria and the nuclear membrane has been proposed to facilitate the transfer of ATP or metabolic precursors to sites of particularly intense local utilization [61]. Similar perinuclear clustering of mitochondria has previously been described in cells that overexpress Mfn1 [62]. Such selective spatial organization is also seen in striated muscle where mitochondria are in close proximity to the sarcoplasmic reticulum and likely play a role in Ca2+ buffering [63,64]. Whether the intimate and apparently fluid association between mitochondria and nuclei described here also has distinct functional consequences remains to be determined. Regardless of the differences of these mitochondrial subsets, the rapidity with which their spatial reorganization occurs in response to abrupt changes in Myc levels shows a close correlation with the structural and metabolic fluctuations that occur in parallel. Overexpression of Myc in the myc2/2 background restored mitochondrial mass and volume to wild-type levels, while simultaneously giving rise to mitochondria that were longer than those observed in myc+/+ cells (Figure 1). We speculate that these longer mitochondria may be a direct result of increased fusion activity. ETC complexes also remained quite abnormal in myc2/ 2wtMyc cells (Figure 5); thus, the excessive OXPHOS potential of myc2/2wtMyc cells (Figure 3b) cannot be explained by propor- tionate changes in ETC complexes. These cells do possess a higher glycolytic rate than the other cells (Figure S2), which is consistent with our previous findings as well as with reports that Myc is a major regulator of the Warburg effect [16,43,65]; therefore, much of the energy requirement of these cells appears to be met by ATP generated via aerobic glycolysis (Figure 3). That certain ETC complexes and supercomplexes, and their activities, remained abnormal in myc2/2wtMyc cells may therefore be one of the factors leading to the up-regulation of glycolysis. This may also underlie the well-known tendency of Myc over-expression to induce high levels of reactive oxygen species [27] that might be facilitated by structural and/or functional ETC abnormalities leading to an increase leak of electrons, particularly at Complex I [66]. Finally, the ETC abnormalities we have observed may represent a metabolic adaptation that reflects a shift from the production of ATP to the production of the lipid, protein and nucleic acid intermediates needed to meet the higher proliferative demands imposed as a result of Myc deregulation. The mechanism underlying the changes reported here appears to involve alterations in the levels and the activities of proteins that coordinate mitochondrial fission and fusion. That both types of proteins respond positively to Myc was unexpected and does not allow for a simple model of how, collectively, they preside over increases in mitochondrial mass, polarity and fusion. The degree to which deviations in the normal ratios of these proteins in response to Myc deregulation contribute to mitochondrial structural and functional abnormalities is also a question for future work. In addition to regulating mitochondrial dynamics, fusion is necessary to maintain structural and functional homogeneity, to preserve mitochondrial genome integrity, and to ensure the proper balance between energy generation and cellular mass PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37699