Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission.

The c-Myc (Myc) oncoprotein regulates numerous phenotypes pertaining to cell mass, survival and metabolism. Glycolysis, oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis are positively controlled by Myc, with myc-/- rat fibroblasts displaying atrophic mitochondria, structural and functional defects in electron transport chain (ETC) components, compromised OXPHOS and ATP depletion. However, while Myc influences mitochondrial structure and function, it is not clear to what extent the reverse is true. To test this, we induced a state of mitochondrial hyper-fission in rat fibroblasts by de-regulating Drp1, a dynamin-like GTPase that participates in the terminal fission process. The mitochondria from these cells showed reduced mass and interconnectivity, a paucity of cristae, a marked reduction in OXPHOS and structural and functional defects in ETC Complexes I and V. High rates of abortive mitochondrial fusion were observed, likely reflecting ongoing, but ultimately futile, attempts to normalize mitochondrial mass. Cellular consequences included reduction of cell volume, ATP depletion and activation of AMP-dependent protein kinase. In response to Myc deregulation, apoptosis was significantly impaired both in the absence and presence of serum, although this could be reversed by increasing ATP levels by pharmacologic means. The current work demonstrates that enforced mitochondrial fission closely recapitulates a state of Myc deficiency and that mitochondrial integrity and function can affect Myc-regulated cellular behaviors. The low intracellular ATP levels that are frequently seen in some tumors as a result of inadequate vascular perfusion could favor tumor survival by countering the pro-apoptotic tendencies of Myc overexpression.

Three-dimensional analysis of the islet vasculature.

The pancreatic islets of Langerhans are highly vascularized structures scattered throughout the pancreas that contain a capillary network 5-10 times denser than that of the exocrine pancreas. A simple method for three-dimensional (3D) analysis of this intricate intraislet vasculature has been difficult because of the intrinsic opacity of the pancreas. We developed a whole-mount imaging technique that allows relatively easy visualization of the islet vasculature. In combination with confocal microscopy and the use of 3D imaging software, we were able to readily reconstruct the 3D architecture of an islet, allowing delineation of the islet volume, length of the intraislet vessels, and the number of vessel branch-points. This technique allows for straightforward 3D image analysis that may help toward understanding islet function.