Characterisation of Mitochondrial Dynamics and Mitochondrial DNA Inheritance in Drosophila melanogaster

Mitochondria are dynamic organelles and there is mounting evidence that mitochondrial dysfunction is linked to a variety of diseases. During spermatogenesis in <i>Drosophila melanogaster</i>, the organisation of mitochondrial networks is altered during each stage of development. This thesis is focused on a novel mitochondrial structure, termed the mitoball, which occurs in the premeiotic spermatocytes in the 16 cell stage. The mitoball is a transient aggregation of mitochondria on one side of the cytoplasm, and little is known about the functionality and dynamics of this structure. First, I found that the mitoball is conserved in multiple insect species. Second, live imaging of the mitoball revealed that the mitoball consists of individual mitochondria which move around in the region of the mitoball. Third, mitochondrial DNA (mtDNA) replication was found to occur in the mitoball stage. Fourth, mitoballs were associated with other cellular structures like the endoplasmic reticulum network, Golgi bodies, and the fusome. Next, I used a reverse genetic screen to identify genes linked to mitochondrial trafficking along microtubules that were required for mitoball assembly: <i>milton</i>, <i>miro</i>, and <i>khc</i>. I also generated an X0 male and found that the loss of the Y chromosome did not affect mitoball formation. To discover new genes linked to the mitoball, I performed a whole genome forward genetic screen using EMS on the second and third chromosome. In total, 8027 EMS mutant lines were screened. Several lines with defective mitochondrial morphology were recovered and sequenced. One EMS line had no mitoballs and had an early stop codon in <i>milton</i> and <i>ssp3</i>. Further mapping of this line revealed that the early stop codon in <i>milton</i> was linked to the absence of mitoballs phenotype and the early stop codon in <i>ssp3</i> was linked to defects in later stages. Altogether, the mitoball structure has promising characteristics as a system to study mitochondrial dynamics. Furthermore, premeiotic clustering of mitochondria may also be a hallmark of insect spermatogenesis. As a multi-copy genome, mutant mtDNA molecules often co-exist with wild-type counterparts, and the competition in transmission among these genomes is central to the inheritance and progression of mitochondrial disease. Currently, it is not possible to easily manipulate animal mtDNA for genetic studies. It is thus difficult to map sequences in mtDNA that can confer a transmission advantage to certain mitochondrial genomes. One of my projects is designed to employ irradiation and chemical based methods to generate mutant and recombinant mtDNA in order to identify and map mtDNA sequences linked to a transmission advantage. Through this project, several recombinant mitochondrial genomes were isolated and this system allows for the mapping of the mitochondrial genome through the generation of recombinant genomes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Li, Andy Yu Zhi

Advisor dc:contributor.advisor

• Ma, Hansong

• Confocal
• Drosophila
• Genetics
• Heteroplasmy
• Insect
• Microscopy
• Mitochondria
• MtDNA

Licence

• https://www.repository.cam.ac.uk/bitstreams/e481d5ce-738b-44f6-8654-159f4c5f0510/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.96884

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/350244