Myc Influences Mitochondrial Dynamics Figure 6. Western analysis of mitochondrial fusion and fission proteins. Western analysis was performed on whole cell lysates from (a) myc+/+, myc2/2 and myc2/2wtMyc cells and from (b) Myc ER cells exposed to 4-HT for the indicated times. b-actin is used as a loading control during each experiment. Densitometry was performed and the values were used to calculate a ratio of the protein expression relative to the level of b-actin. This ratio was normalized and the value for myc2/2 for (a) and untreated MycER cells for (b) was set to 1. Experiments were repeated a minimum of three times and the standard error between the experiments is shown. doi:10.1371/journal.pone.0037699.g006 mitochondria by virtue of being fused to the mitochondrial localization signal for subunit VIII of cytochrome c oxidase (Figure 7a). These cells were then co-plated and either maintained in 4-HT or had the inducing agent removed from the medium 24 or 48 hours prior to inducing plasma membrane fusion with polyethylene glycol as previously described [25,59]. The degree of fusion was measured by quantifying GFP and DsRed co- localization in the resulting heterokaryons (Figure 7b). MycER cells maintained in 4-HT displayed approximately 70% fusion of mitochondria, within two hours of PEG fusion, whereas cells from which 4HT had been removed showed a significantly reduced degree of fusion (Figure 7c). Taken together, these data indicate that mitochondrial biogenesis in response to Myc induction is associated with a coordinated and complex alteration of both fusion and fission proteins, which ultimately favors the former activity. Moreover, and similar to the changes seen in mitochon- drial mass and polarization (Figure 2a), this process is rapidly responsive to changes in Myc levels. Discussion Previous studies have shown that Myc regulates mitochondrial biogenesis and that many enzymes of the OXPHOS and glycolytic pathways are under direct Myc control [12,15,19]. Precisely how Myc coordinates these activities or other aspects of mitochondrial structure and function remains poorly understood. We have shown here that changes in mitochondrial structure and function are temporally regulated in a highly controlled manner in response to changes in Myc levels. These changes are not symmetrical but instead occur more rapidly in response to Myc’s inactivation than to its activation. Most notably, membrane potential and inter- connectivity are lost much more rapidly than mitochondrial mass upon Myc inactivation (Figure 2a). Although we do not understand the basis for this temporal asymmetry, it is tempting to speculate that Myc is primarily responsible for maintaining mitochondrial membrane potential and may do so by increasing membrane fusion. We are currently conducting studies to address this phenomenon. Furthermore, the relatively slow response of mitochondria biogenesis to Myc induction relative to other features of the transformed state suggests that changes in metabolism are not necessary for transformation per se, but rather serve to provide for the more robust proliferative demands of transformed cells [17,18,60]. These results are in keeping with our recent finding that a point mutation in the transactivation domain of Myc (Q131R) retains potent transforming and tumorigenic competence despite being significantly impaired in its ability to up- regulate both glycolysis and OXPHOS [43]. Among the more notable features of myc2/2 cells was the relative paucity of mitochondria in the perinuclear region (Figure 2d). This localization appears to be an on-going, active process as mitochondria at these sites were preferentially lost in MycER cells following 4-HT withdrawal. Close contact between PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37699