Project 1:Selective Replication of Functional mtDNA During Oogenesis Restricts the Transmission of a Deleterious Mutation Mutations in mitochondrial DNA (mtDNA) can severely impair cellular energy homeostasis and have been implicated in various pathological processes1. Despite a high mutation rate coupled with the lack of recombination resulting from transmission exclusively through the female germline, crippling mitochondrial mutations are exceedingly rare in populations2. Two recent studies demonstrated strong purifying selection against deleterious mtDNA mutations in the mouse female germline3,4, indicating a selection based on the fitness of individual mitochondria. However, the mechanisms underlying the positive selection of healthy mitochondria have yet to be elucidated. Here we directly visualize mtDNA replication during oogenesis and describe a mechanism for the positive selection of healthy mitochondria in Drosophila. In contrast to the common view that mtDNA replication is either a random process or loosely coupled to nuclear replication, we found that mtDNA replication commenced prior to oocyte determination during the late germarium stage. At this stage, mitochondria are tightly associated with and mtDNA replication is concentrated around the fusome, a cytoplasmic structure mediating transport of mitochondria into the oocyte. We isolated a temperature-sensitive lethal mtDNA mutation, mt:CoIT300I, and found that at the restrictive temperature, mt:CoIT300I displayed reduced mtDNA replication in region 2B of the germarium. Additionally, we produced heteroplasmic flies carrying both wild type and mt:CoIT300I mtDNA genomes, and observed a decrease in the frequency of a lethal mutation both in the female germline and over multiple generations at the restrictive temperature. Furthermore, we determined that selection against mt:CoIT300I occurs at a late germarium stage, which overlaps with the timing of selective replication of wild type genomes. These findings establish a previously uncharacterized developmental mechanism for selective amplification of healthy mtDNA, which may be evolutionarily conserved to prevent transmission of deleterious mutations. This work is currently under revision with Nature. Project 2: Genetic method for selective elimination of damaged mitochondria Mitochondrial turnover has been postulated as a mechanism for mitochondrial quality control. However, it remains a question whether cells are indeed able to eliminate defective mitochondria selectively. Quantitative and live imaging assays are required to measure selective mitochondrial degradation and visualize this process in real time, while a genetic approach is essential to probe mitochondrial turnover in a physiological context. We expressed a toxic bacterial protein, PorB, to damage a subpopulation of total cellular mitochondria in cultured Drosophila cells and tissues. Damaged mitochondria concentrated with PorB were segregated from the mitochondrial network through a fission/fusion process and selectively removed by lysosomes through the autophagy pathway in otherwise healthy cells. We demonstrated for the first time the Parkin-dependent degradation of damaged mitochondria in an animal tissue, the Drosophila flight muscle. Our work proves in principle that defective mitochondria are selectively removed in healthy cells, and also provides a novel genetic approach to monitor mitochondrial turnover and dissect the underlying mechanisms. This work is currently under preparation for re-submission. Project 3: A Drosophila model reveals novel pathogenic mechanism of mtDNA mutation. Animal models carrying inheritable mtDNA mutations are much needed to dissect the pathogenic mechanisms underlying mtDNA mutations and understand their pattern of segregation. In this study, we isolated a conditional lethal mtDNA mutation, mt:CoIT300I that affect Cytochrome C oxidase subunit one locus (CoI) in Drosophila utilizing a selection scheme based on mitochondrially targeted restriction enzyme (Mito-XhoI). Homoplasmic mt:CoIT300I flies were lethal at 29C, and displayed muscle atrophy and neuronal dysfunctions at permissive temperature. mt:CoIT300I mitochondria had reduced COX activity and impaired capacity of Ca2+ uptake that led to cytosolic Ca2+ accumulation consequently. Genetic analysis revealed that Ca2+ mishandling contributed to the pathogenesis of mt:CoIT300I . Furthermore, we generated heteroplasmic flies carrying both wild type and mt:CoIT300I genome. We demonstrated that expression of Mito-XhoI effectively eliminate wild type mtDNA in heteroplasmic background. Applying this genetic scheme, we generated various tissues harboring homoplasmic mt:CoIT300I by tissue specific expression of Mito-XhoI, which allowed us to study the tissue specific and age-related phenotypes caused by mt:CoIT300I at restrictive condition. This manuscript describing this work is under preparation and expected to be submitted soon. Project 4. Division Cycle Contributes to Mitochondrial Deficiencies and Aging of Stem Cells In addition to chronological age, stem cells are also subject to proliferative aging during the adult life span. However, the consequences and contribution of proliferative aging to stem cells activities have not been well investigated. Using Drosophila female germ line stem cells as a model, we found that the replication cycle contributes to the age dependent decline of female fecundity, and is a major factor causing developmental abnormalities in the eggs of old females. The proliferative aging does not cause telomere shortening, but leads to an accumulation of mtDNA mutations that disrupt the mitochondrial respiration chain. Our results suggested that proliferation cycle cause accumulation of damaging mutation on mtDNA, and may represent a conserved mechanism underlying stem cell aging. A manuscript describing this work will be submitted soon. Project 5: A mitochondrial CAMP signaling and mtDNA replication. Prune emerged from a genome wide RNAi screening for genes involved in mtDNA replication. RNAi knock-down of prune led to 70% reduction of mtDNA in Drosophila S2 cells. Prune are predicted to be mitochondrial protein and contains a phosphodiesterase domain. We found that Prune localized to mitochondria matrix and displayed CAMP specific phosphodiesterase activity. Prune mutants flies had reduced mtDNA and mtRNA level and displayed severe neuro-degeneration. Moreover knock down of prune increase CAMP level in mitochondria, suggesting it might be involved in CAMP-PKA pathway in mitochondria. we are now testing a few of possible PKA substrates that might be regulated by prune, and trying to probe how CAMP-PKA signaling regulates mtDNA replication. We also found a mitochondria AKAP ( A-kinase anchor protein), spoonbill was required for mtDNA replication. Spoonbill localizes to mitochondrial outer membrane and binds to PKA and RNAs. Knock-down of spoonbill dramatically disrupted mtDNA replication in cultured cells and tissues. Interestingly, spoonbill was quickly turned over upon mitochondria inhibition by a uncoupler. CCCP, and present in much lower level in flies carrying a deleterious mtDNA mutation. These preliminary observation suggest that spoonbill might be play an essential role in sensing mitochondrial activity and regulating mtDNA replication accordingly. Project 6: Targeted mutagenesis of mammalian mtDNA through direct transformation of engineered mtDNA. We are continuing to develop new techniques to directly transform mammalian mitochondria. We are now trying several approaches: directly transformation with in-vitro engineered mtDNA plasmids;targeting phage recombination system into mitochondria and constructing a DNA element thatcan replicate autonomously inside mitochondria.
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|Zhang, Yi; Chen, Yong; Gucek, Marjan et al. (2016) The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication. EMBO J 35:1045-57|
|Chen, Zhe; Qi, Yun; French, Stephanie et al. (2015) Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function. Mol Biol Cell 26:674-84|
|Zhang, Fan; Qi, Yun; Zhou, Kiet et al. (2015) The cAMP phosphodiesterase Prune localizes to the mitochondrial matrix and promotes mtDNA replication by stabilizing TFAM. EMBO Rep 16:520-7|
|Qi, Y; Liu, H; Daniels, M P et al. (2015) Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration. Cell Death Differ :|
|Ni, Ting; Wei, Gang; Shen, Ting et al. (2015) MitoRCA-seq reveals unbalanced cytocine to thymine transition in Polg mutant mice. Sci Rep 5:12049|
|Ma, Hansong; Xu, Hong; O'Farrell, Patrick H (2014) Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster. Nat Genet 46:393-7|