(OXPHOS), the depletion of ATP pools, and the loss of inner mitochondrial membrane permeability [13,21]. Fusion might thus complement Myc-mediated de novo biosynthesis by minimizing the number of irreversibly damaged mitochondria and thereby prolonging their life spans [22,23,24,25]. This protective function would be particularly valuable given the fact that mitochondria are the major source of the reactive oxygen species (ROS) that are elevated by Myc overexpression [26,27]. The larger and more efficient mitochondria might also be better able to serve the metabolic needs of the more actively proliferating transformed cell, perhaps in a capacity analogous to that described in hypertrophic cardiac muscle [19,28]. Fusion, however, is only partially effective at preserving mitochondrial integrity. In the face of overwhelming damage, mitochondria fission produces small, dysfunctional organelles that can be selectively eliminated by the autophagosomal machinery [29]. Fission is also used to reduce mitochondrial mass in the face of rapid reductions in metabolic demands [30]. Thus, both mitochondrial mass and function appear to be highly responsive to the metabolic environment and are coordinately orchestrated by a well-balanced combination of de novo synthesis and remodeling via the fission/fusion processes [31,32]. In the current work, we have investigated the kinetics of mitochondrial assembly and disassembly, along with the mecha- nisms underlying these processes, by inducing or inactivating Myc in several cell types. We find that conditional Myc depletion is associated with a rapid decline in mitochondrial structural integrity and function, as well as abnormalities in the electron transport chain (ETC) supercomplexes. In contrast, Myc re- expression leads to relatively slower and asymmetric normalization of mitochondria mass and high rates of OXPHOS, despite only partial reversal of ETC complex abnormalities. The coordination of these structural and functional alterations seems to occur in tandem with changes in the levels of several key proteins involved in mitochondrial fission and fusion. These findings shed light on the mechanisms underlying the increases in mitochondrial mass and metabolic reprogramming that accompany Myc deregulation in cancer. Results Control of mitochondrial structure by Myc Initially, we employed three rat fibroblast lines: one with endogenous levels of Myc (myc+/+) [33], an isogenic line bearing a homozygous deletion of myc (myc2/2) [34], and myc2/2 cells stably transduced with a lentiviral vector encoding wild-type human Myc (myc2/2wtMyc cells). Immunoblotting showed that Myc was overexpressed in myc2/2wtMyc cells relative to myc+/ +cells (Figure S1). Differential Myc expression resulted in significant ultrastructural differences as observed by transmission electron microscopy (Figure 1a). Of particular note was that the mitochondria of myc2/2 cells were not only less abundant and smaller (average length approx. 500 nm) than those of myc+/+ cells (average length approx. 800 nm), but lacked their more elaborate cristae patterns. Additionally, the mean mitochondrial length in myc+/+wtMyc cells was over 50% greater than that of myc+/+ cells (approx. 1300 nm, Figure 1b). From these studies, we conclude that endogenous Myc plays a role in mitochondrial size and structural integrity. In order to study the Myc-induced changes in mitochondria as a function of time, we next utilized myc2/2 cells transduced with a retrovirally-encoded, 4-hydroxytamoxifen (4-HT)-responsive Myc- estrogen receptor (MycER) fusion protein [35], which we hereafter refer to as MycER cells. In the absence of 4-HT, the MycER Myc Influences Mitochondrial Dynamics fusion protein is sequestered in the cytoplasm [35,36]. After binding 4-HT, MycER undergoes a conformational change, is shuttled to the nucleus, and becomes transcriptionally active. We used 10-n-nonyl-acridinium-orange-chloride (NAO) to quantify mitochondrial mass in MycER cells in the logarithmic growth phase. Following the addition of 4-HT, an increase in NAO staining was noted over the ensuing three to four weeks. Upon reaching this stage of ‘‘chronic’’ exposure (i.e. .28 days), 4- HT was removed from the medium and cells were again stained with NAO, which revealed that the previously observed increase in mitochondrial mass was reversible, with NAO staining patterns gradually decreasing, but remaining at elevated levels for as long as 10 days (Figure 2a). The same cells were stained with MitoTrackerH Deep Red which also measures mitochondrial mass, but does so in a manner that is more dependent on the maintenance of mitochondrial membrane potential ([25,37,38]; Figure 2a). We noted a somewhat more gradual increase in this signal following the addition of 4- HT, but a much more rapid decline upon its removal. A similar pattern was seen in the corresponding epifluorescence images of the cells (Figure 2b), suggesting that the maintenance of mitochondrial membrane potential is more dependent on ongoing Myc activity than is mitochondrial mass. In the absence of 4-HT, MycER cells were similar in appearance to myc2/2 cells with regard to mitochondrial morphology (Figure 2c). Upon activation of Myc activity, the normalization of mitochondrial length and cristae patterns appeared to occur somewhat more rapidly than did the normalization of total mitochondrial mass. Similarly, these properties were also lost more rapidly than mass upon removal of 4-HT. Mitochondria are highly interconnected in a manner that often correlates with their mass and function [39]. To determine whether this was the case with the rat fibroblasts being studied here, we generated space filling overlays over three-dimensional reconstructions from serial confocal images obtained on Mito- TrackerH-stained MycER cells exposed to 4-HT for differing periods of time. Both untreated cells and those from which 4-HT had been removed, showed the reduced mitochondrial mass and loss of connectivity previously noted for myc2/2 cells. (Figures 2d and Video S1). In contrast, chronic 4-HT treatment was associated with a dense and highly interconnected mitochondrial network. We also noted a tendency for the mitochondria of these latter cells to be highly concentrated in a peri-nuclear pattern. The removal of 4-HT was associated with a selective migration of mitochondria away from nuclei and a more dispersed distribution. From the above studies, we conclude that mitochondrial mass responds gradually to Myc induction and withdrawal, whereas changes in membrane potential and interconnectivity are more rapid, particularly following 4-HT removal. Finally, a preferential re-localization of mitochondria to sites immediately adjacent to and/or connected with the nuclear membrane occurs following Myc induction. Control of mitochondrial function by Myc The previous studies suggested that Myc-associated changes in mitochondrial mass and structure might also be accompanied by altered mitochondrial function, particularly in light of the rapid decline in MitoTracker Deep Red staining (Figure 2a), which is known to be partially dependent on membrane polarization [40]. To investigate this further, we first compared mitochondrial membrane polarization in living myc2/2, myc+/+, and myc2/ 2wtMyc cells using the JC-1 dye [41]. We measured the ratio of red-shifted JC-1 aggregates, whose formation is favored under conditions of high membrane potential, and green-shifted PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37699