The long-term goal of this proposal is to identify the molecular mechanism of mitochondrial fission and its role in apoptosis. Defects in mitochondrial fission have severe consequences and even death. Yet little is known about how fission works and is coordinated with other cellular processes. Genetic and cellular studies, primarily in Saccharomyces cerevisiae, by other investigators have led to the development of a model for mitochondrial fission. In this model, the protein Fis1 regulates fission by mediating the assembly of a dynamin- related GTPase, Dnm1, and an adaptor protein, Mdv1, at the sites of constriction on the mitochondrial outer membrane. Although the data from the qualitative model are suggestive, the model does not explain how this important process is regulated. By integrating cell biological, biochemical, and biophysical data with low- and high-resolution structures of the fission machinery, the current proposal aims to develop a comprehensive model for mitochondrial fission. The first specific aim is to define the protein-protein interactions in solution that are important to fission through experiments that will define domains that support these interactions, the stoichiometries and affinities of these interactions and their consequences on Dnm1 activity. Low-resolution images of the proposed fission machinery will be obtained by electron microscopy. High-resolution structures of Fis1, the binary Fis1/Mdv1 complex, and the ternary Fis1/Mdv1/Dnm1 complex will be pursued by NMR spectroscopy and x-ray crystallography. The second specific aim is to identify yeast Fis1 residues important for homodimerization, Mdv1 binding, and Dnm1 binding. We will identify mutants of Fis1 that affect oligomerization and test these mutants for altered activities to define their importance in assembly of the fission machinery. The third specific aim is to determine whether Fis1 affects the assembly of Dnm1 and Mdv1 on the membrane through experiments with membranes derived from synthetic lipids and isolated mitochondria. These experiments should also allow determination of the order of assembly. The resulting data from all three approaches will be integrated into a complete picture of how mitochondrial fission is accomplished. The analyses also promise considerable general insight into the basis of dynamin-based membrane dynamics, as well as protein-protein and protein-lipid interactions. Human homologues of the mitochondrial fission machinery exist and are reported to be important in regulating apoptosis, which is linked to many important diseases. Therefore, detailed information on mitochondrial fission might be helpful in designing strategies to inhibit or induce apoptosis.

Public Health Relevance

Mitochondria perform many essential functions that are thought to require frequent mitochondrial fission and fusion events, which are accomplished by distinct protein machineries. A point mutant in the mitochondrial fission protein, Dnm1, caused infant death. Additionally, mitochondrial fission increases during apoptosis, a process whose misregulation contributes to many human diseases. The work proposed will illuminate mechanistic details of these processes and represents an important step towards the discovery of new therapeutic strategies for human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Medical College of Wisconsin
Schools of Medicine
United States
Zip Code
Tan, Frederick J; Zuckerman, Jonathan E; Wells, Robert C et al. (2011) The C. elegans B-cell lymphoma 2 (Bcl-2) homolog cell death abnormal 9 (CED-9) associates with and remodels LIPID membranes. Protein Sci 20:62-74
Wells, Robert C; Hill, R Blake (2011) The cytosolic domain of Fis1 binds and reversibly clusters lipid vesicles. PLoS One 6:e21384
Jeyaraju, D V; McBride, H M; Hill, R B et al. (2011) Structural and mechanistic basis of Parl activity and regulation. Cell Death Differ 18:1531-9
Tooley, James E; Khangulov, Victor; Lees, Jonathan P B et al. (2011) The 1.75 A resolution structure of fission protein Fis1 from Saccharomyces cerevisiae reveals elusive interactions of the autoinhibitory domain. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:1310-5
Chang, Chuang-Rung; Manlandro, Cara Marie; Arnoult, Damien et al. (2010) A lethal de novo mutation in the middle domain of the dynamin-related GTPase Drp1 impairs higher order assembly and mitochondrial division. J Biol Chem 285:32494-503
Hill, R Blake; Pellegrini, Luca (2010) The PARL family of mitochondrial rhomboid proteases. Semin Cell Dev Biol 21:582-92
Picton, Lora K; Casares, Salvador; Monahan, Ann C et al. (2009) Evidence for conformational heterogeneity of fission protein Fis1 from Saccharomyces cerevisiae. Biochemistry 48:6598-609
Siskind, Leah J; Feinstein, Laurence; Yu, Tingxi et al. (2008) Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels. J Biol Chem 283:6622-30
Tan, Frederick J; Husain, Michelle; Manlandro, Cara Marie et al. (2008) CED-9 and mitochondrial homeostasis in C. elegans muscle. J Cell Sci 121:3373-82
Wells, Robert C; Picton, Lora K; Williams, Sarah C P et al. (2007) Direct binding of the dynamin-like GTPase, Dnm1, to mitochondrial dynamics protein Fis1 is negatively regulated by the Fis1 N-terminal arm. J Biol Chem 282:33769-75

Showing the most recent 10 out of 14 publications