Mitochondrial dysfunction underlies many metabolic conditions and results in poor health outcomes. Mitochondrial DNA (mtDNA) reside in the mitochondria and encode essential components of the electron transport chain. The electron transport chain generates ATP via oxidative phosphorylation: a key role that mitochondria play in energy production and metabolism. mtDNA can exist in hundreds to thousands of copies per cell and, unlike the nuclear genome, mtDNA copy number per cell can be modulated in response to energy demand, resulting in variable mtDNA copy number. Importantly, proper mtDNA copy number is crucial for survival as insufficient mtDNA copy number can result in mtDNA depletion syndromes leading to myopathies. Taken together, the variability of mtDNA copy number coupled with the consequences of low copy number suggest a mechanism of tightly controlled mtDNA copy number regulation. Though mtDNA replication has been extensively studied, the mechanism of copy number control remains poorly understood due to a lack of appropriate tools. I have adapted Caenorhabditis elegans as a model to study mtDNA copy number regulation in metazoans. Using quantitative techniques I have developed to measure mtDNA copy number in single worms, I have discovered active regulation of mtDNA copy number in the C. elegans germline. Additionally, my preliminary data suggest that an output generated by the electron transport chain is used by the cell to ?count? mtDNA copies. My data and innovations uniquely position me to identify the mechanisms of copy number regulation in the C. elegans germline.
In aim 1 I propose to identify the electron transport chain components necessary for mtDNA copies to be counted, using a large collection of heteroplasmic strains. Heteroplasmies contain mtDNA with two distinct haplotypes and offer means to identify the minimal requirements for mitochondrial genome counting.
In aim 2 I propose to identify the sensor that acts to regulate copy number using a targeted RNAi screen.
In aim 3 I propose to determine whether mtDNA copy number is regulated at the genome or organelle level using high resolution microscopy. Taken together, these experiments will identify mechanisms of mtDNA copy number regulation. Understanding mtDNA copy number regulation will pave the way for therapeutic options for individuals with mtDNA depletion syndromes.
Mitochondrial DNA copy number is an important regulator of cellular metabolism and sufficient copies are required for development. The experiments outlined here seek to identify mechanisms of mitochondrial DNA copy number control. Understanding copy number regulation is essential for generation of therapeutic medical intervention for patients with mitochondrial DNA depletion syndromes.
