Mitochondria are dynamic organelles whose shape and function are controlled by continual cycles of fusion and fission. In particular, mitochondrial fission has important roles in cell physiology. In many forms of apoptosis, the mitochondria of cells undergo increased mitochondrial fission prior to the execution of cell death. Inhibition of this regulated mitochondrial fission can reduce the level of cell death, indicating that fission is an important component. We propose structural studies that will greatly advance our mechanistic understanding of mitochondrial fission, and lead to methods to manipulate this process. Mitochondrial fission depends on recruitment of the dynamin-related protein, Dnm1, to the mitochondrial surface, where it assembles into a multimeric complex that mediates mitochondrial constriction. Dnm1 recruitment requires the mitochondrial membrane protein Fis1, with the adaptor proteins Mdv1 and Caf4 acting as molecular bridges between Fis1 and Dnm1. To understand how Fis1 recruits the fission machinery, we have developed a co- expression system to produce complexes of Fis1 bound to fragments of Mdv1 or Caf4. Using X-ray crystallography, we will solve the atomic structure of these complexes. In addition, we will use a similar approach, coupled with cryo-electron microscopy, to understand how binding of Mdv1 or Caf4 to Dnm1 activates mitochondrial fission. These structural studies will provide a wealth of insight into how Fis1 physically interacts with Mdv1 and Caf4, and how assembly of the fission complex occurs. We will evaluate the physiological relevance of these structural findings using extensive structure/function analysis of mutants of Fis1, Mdv1, and Caf4. Our structural work has also identified a dimeric form of Fis1, and we will use biochemical and cell biological approaches to understand the oligomerization of Fis1 during mitochondrial fission. Taken together, these studies will provide a structural and mechanistic basis for understanding mitochondrial fission, a process with links to apoptosis, neuronal function, and senescence. The proposed work will lead to a structural understanding of mitochondrial fission, a fundamental cellular process that is important in cell physiology. Mitochondrial fission is an important component of apoptosis, a form of programmed cell death that plays a role in development, tissue morphogenesis, and prevention of cancer. In addition, mitochondrial fission is important for the function of nerve cells and has been linked to aging. These studies will lead to structural insights that will enable investigators to manipulate mitochondrial fission, and thereby potentially modulate processes such as apoptosis, cancer, and nerve function. ? ? ?
