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

2021 Fiscal Year Research-status Report

• Project/Area Number: 21K15627
• Research Institution: Osaka University
• Principal Investigator: 鐘 其静 大阪大学, 医学系研究科, 特任研究員(常勤) (50796076)
• Project Period (FY): 2021-02-01 – 2024-03-31
• 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

3: Progress in research has been slightly delayed.

Reason

Currently, we are trying to establish the protocol for CRIPSR library screening. However, there is a delay in getting 96 well culture insert plate due to broken supply chain of laboratory materials from abroad.

Strategy for Future Research Activity

For the establishment of screening protocol, we will seed HeLa cells labelled with mitotracker Red into 96 well culture inserts and after subjecting the cells with treatments for specified duration, the inserts will be removed and the extracellular mitochondria will be left to sediment to well bottom. Automated imaging and quantification by In Cell Analyzer will help determine the number of released mitochondria. We will further work on calculating the statistical parameters, the Z’ factor, signal to noise (S/N) ratio, coefficient of variation at the maximum signal (positive control), and coefficient of variation at the minimum signal (negative control) to optimize the quality of sceening assay.

Causes of Carryover

We plan to purchase HeLa Cas9 stably overexpressing cells and CRIPSR library.