Myc Influences Mitochondrial Dynamics Figure 5. Electron Transport Chain complexes. (a) Blue native gel electrophoresis. A representative blue native gel is depicted. (b) Quantitation of complex formation. The histogram is representative of the optical density of Coomassie blue staining for at least three separate experiments. In comparison to the myc+/+ cells, p values were generated and the strains with values ,0.05 are indicated with an asterisk. (c) Complex I in situ gel activity. Enzymatic activity staining was performed for Complex I. Depicted is a representative experiment. Note that Complex I activity could be detected in the previously identified Complex I band as well as the bands that represent higher order supercomplexes (SC). Total activity of Complex I+SC was calculated for myc+/+ cells and arbitrarily set to 100%. (d) Complex V in situ gel activity. Enzymatic activity staining was performed for Complex V. Shown is a representative experiment. Total activity of Complex V (represented as the sum of individual Vm and Vd activities) was calculated for myc+/+ cells and arbitrarily set to 100%. doi:10.1371/journal.pone.0037699.g005 Similar in situ measurements of Complex V ATP synthase activity showed an even more profound reduction in myc2/2 cells than indicated by the total protein levels depicted in Figure 5b. In this assay, total Complex V activity averaged only 15% of that measured in myc+/+ cells, with Vm and Vd activities being reduced comparably (Figure 5d). As had been seen with Complex I, the re-expression of Myc in myc2/2wtMyc cells only partially restored Complex V activity, with virtually the entire correction occurring at the level of Vm. Overall, these results clearly show that Myc overexpression does not restore either normal respiratory chain function or structure despite normalizations of mitochon- drial size, mass and ATP content. Regulation of mitochondrial fusion and fission proteins by Myc Mitochondria are dynamic and interconnected organelles whose overall structural and functional integrity are maintained by a balance between fusion and fission [24,25,32]. Because the smaller and poorly communicating mitochondria of myc2/2 cells could reflect alterations in either or both of these pathways, we examined several key fusion and fission proteins. Those in the former category included the inner membrane-embedded GTPase, Opa1, which plays important roles in the maintenance of mitochondrial shape and cristae formation [32] and the mitofusins Mfn1 and Mfn2 [53], which interact with each other as well as Opa1, and positively regulate membrane potential, metabolism and survival [22,24,25,32,54,55,56]. Proteins in the latter category included the dynamin-like protein Dlp1 and Fis1, which are believed to trigger fission as a result of their interaction on the outer mitochondrial surface [57,58]. Western analysis of the above cell lines showed both fusion and fission proteins to be expressed at somewhat higher levels in myc+/ + cells when compared to myc2/2 cells (Figure 6a). This ranged from a minimal increase in levels of Mfn1 to larger increases in the levels of Fis1 and Opa1. Although our findings were mostly similar in myc2/2wtMyc cells, Opa1 and Dlp1 levels generally exceeded those of myc+/+ cells. These results were largely confirmed in MycER cells where long-term activation of MycER by 4-HT was again associated with variable increases in most of the proteins, with the largest and most significant increase observed for Mfn1, whose level increased more than two-fold when compared to untreated myc2/2wtMyc cells (Figure 6b). Surprisingly, there was no decline in fission or fusion protein levels upon 4-HT removal, despite the previously observed loss of mitochondrial mass (Figure 2a). Given the foregoing results, as well as our evidence indicating that loss of Myc decreased the connections between mitochondria (Figure 2d), we next quantified the amount of fusion activity in MycER cells (Figure 7). Cells that were maintained in 4-HT for over four weeks were stably transduced with lentiviral vectors expressing GFP and DsRed proteins that were targeted to PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37699