2023 Fiscal Year Research-status Report
細胞外ミトコンドリア放出による生体恒常性維持と加齢・疾患の関連について
| Project/Area Number |
21K15627
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
鐘 其静 大阪大学, 大学院医学系研究科, 特任研究員(常勤) (50796076)
|
|---|
| Project Period (FY) |
2021-02-01 – 2025-03-31
|
| Keywords | Mitochondria / Extracellular release / Mitophagy |
|---|
| Outline of Annual Research Achievements |
Mitochondrial quality control, which is crucial for maintaining mitochondrial function and cell fate determination, is mainly achieved through mitophagy. We recently discovered an alternative quality control pathway mediated by extracellular mitochondria release. Using mitochondrial toxin treatment and parkin gene overexpression and knockdown approaches, we found that mitochondrial stress and perturbation of mitophagy pathway induce greater extracellular mitochondria release, suggesting that impairment of conventional mitophagy prompts alternative pathway.
However, the detailed mechanisms and the resulting biological consequences remain indefinite. Here I propose to study the mechanisms of mitochondria secretion and its cell-autonomous and non-cell autonomous biological consequences. Briefly, the study of mechanisms involves CRISPR library screening assay to identify intrinsic factors that regulate mitovesicle release. Elucidation of the biological significance of mitochondria release includes determination on how mitochondria release affects the donor and recipient cells. A better understanding of the machinery at molecular level and physiological relevance can pave the way for therapeutic implication.
|
| Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
The project progression is delayed due to the maternity and childcare leave of the principle investigator. However, we have established Hela Cas9 stably overexpressing cells with DsRed2-labelled mitochondria and purchased LentiArray Human Membrane Trafficking CRISPR library for screening. This library targets 141 genes with up to 4 gRNA per gene target.
|
| Strategy for Future Research Activity |
Future plan involves the optimization for lentivirus transduction condition, MOI determination, incubation period and the requirement of polybrene using the positive control HPRT1-targeting lentivirus with EmGFP before performing screening. Gene editing efficiency has to be verified using genomic cleavage detection kit. Detection of extracellular mitochondria will be performed using CQ1 confocal quantitative image cytometer,
|
| Causes of Carryover |
The grant will be used to purchase reagents, assay plates and kits.
|
