In situ observation of mitochondrial biogenesis as the early event of apoptosis

Abstract

Summary: Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.

Keywords

• Biochemistry methods
• Biomolecular engineering
• Cell biology