Mechanisms of mitochondrial inheritance - Pamula Admin Childcare Supplement - Project Summary
The survival of eukaryotic species depends on the faithful transmission of both nuclear and mitochondrial
genomes. Mutations in mitochondrial DNA (mtDNA) cause neurodegenerative and neuromuscular diseases in
humans. Strikingly, though mitochondria are inherited exclusively through the maternal lineage, rapid changes
in mtDNA allele frequency can occur, resulting in severe mitochondrial disease in a subset of offspring due to
an increased mutational load. The long-term goal of this project is to decipher the molecular mechanisms
regulating mitochondrial segregation in the germline. To achieve this goal, I will take a multidisciplinary approach
combining genetics, proteomics, biochemistry, and high-resolution quantitative microscopy using the model
organism, Drosophila melanogaster. The following aims will be pursued: (1) Analyze mtDNA allele frequency in
gamete precursor cells termed primordial germ cells (PGCs). During embryogenesis, a small subset of
mitochondria is permanently separated from the rest of the oocyte into PGCs, resulting in an ~1000-fold reduction
in mtDNA content. To examine the consequence of this mitochondrial population bottleneck on the segregation
of mtDNA alleles, I will use a heteroplasmic fly strain harboring both wild-type and mutant mitochondrial
genomes. I will determine mtDNA allele frequency in individual PGCs using high-resolution imaging of single
mtDNA molecules and quantitative PCR and will examine how these ratios change when the size of the
bottleneck is genetically constricted. (2) Determine the network of Long Oskar interacting proteins. Long Oskar
is the master regulator of mitochondrial inheritance. To recruit mitochondria to the site of PGC formation, Long
Oskar stimulates F-actin reorganization, but it does not contact mitochondria directly. To identify proteins
downstream of Long Oskar, I will use proximity labelling and tandem mass spectrometry. I will then map Long
Oskar-binding regions on direct binding partners. (3) Identify nuclear-encoded mitochondrial proteins required
for mitochondrial inheritance. Currently, our understanding of how mitochondria are targeted to sites of PGC
formation is limited by an incomplete parts list of the mitochondrial segregation machinery. I will perform a
comprehensive RNAi screen of mitochondrial membrane-associated proteins to identify those required for
mitochondrial localization. Together, these aims will reveal how the mitochondrial bottleneck impacts the
segregation of mtDNA alleles and will likely inform on the population risk of mitochondrial associated diseases.
In addition, these experiments will identify molecular components of the mtDNA segregation machinery that is
used to transmit mitochondria to germline cells during early Drosophila embryogenesis. Together, these results
have the potential to shed light on how similar events may occur in pre-implantation human embryos.
