Author ORCID Identifier
Date of Award
Department or Program
Biochemistry and Cell Biology
Henry N. Higgs
Mitochondria are the energy factories of the cell. The dynamic nature of cells demands routine changes in mitochondrial morphology by fusion and division. The dynamin GTPase Drp1 is a central mitochondrial division protein, driving constriction of the outer mitochondrial membrane via oligomerization. At least four regulatory factors control Drp1 activity on the outer mitochondrial membrane (OMM): 1) receptor proteins (Mff, MiD49, MiD51, and Fis1); 2) actin filaments; 3) the mitochondrial phospholipid cardiolipin (CL); and 4) Drp1 post-translational modifications, of which two phosphorylation sites (S579 and S600) are the most well studied. However, the molecular mechanism of how these factors work together in Drp1 activation is unknown.
In this thesis, I take biochemical and cellular approaches to understand how these regulatory factors work individually and together, showing that:
1) Mff oligomerizes in both solution and cells in a concentration-dependent manner through its C-terminal coiled-coil. The dynamic oligomerization of Mff is crucial for activating Drp1. In the solution, oligomerization-defective Mff fails to activate Drp1 and loses its capacity to recruit Drp1 in U2OS cells. Biochemically, actin filaments work synergistically with Mff to enhance Drp1 activity by reducing the effective concentration of Mff.
2) The activation of MiD49 and MiD51 occurs through long-chain acyl coenzyme A (LCACA), leading to their oligomerization and subsequent activation of DRP1 GTPase activity. A point mutation in the LCACA binding pocket diminishes LCACA binding, resulting in reduced MiD51 oligomerization and impaired Drp1 activation both in solution and HeLa cells. Finally, MiD49 or MiD51 oligomers collaborate with Mff, rather than actin filaments, in DRP1 activation.
3) Phosphorylation at S579 and S600 sites maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. The oligomerization state of both phospho-mimetic mutants is shifted toward smaller oligomers.
Taken together, I propose that mitochondrial division is a multifaceted process involving various factors, and the synergy of these factors may serve distinct purposes for specific mitochondrial division events.
1. Molecular Biology of the Cell 2021, DOI: 10.1091/mbc.E21-04-0224.
2. Molecular Biology of the Cell 2023 DOI: 10.1101/2023.08.20.554022
Liu, Ao, "MITOCHONDRIAL DIVISION: SYNERGIZING IN MITOCHONDRIAL DIVISOME" (2023). Dartmouth College Ph.D Dissertations. 219.