Characterization of PICK1 overexpression in fibroblast NIH3T3 cells

• Main Authors: Chia-Yin Hua, 花珈吟
• Other Authors: Wey-Jinq Lin
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/53620799229407218170

碩士 === 國立陽明大學 === 生物藥學研究所 === 101 === PICK1 (Protein interacting with C-kinase 1) is a scaffold protein that predominantly localized to mitochondria in NIH3T3 cells and can function as a selective anchoring protein for PKCα. Recruitment of PKCα to mitochondria stabilizes mitochondrial membrane potential and confers a higher resistance to drug-induced apoptosis. Mitochondria are dynamic organelles. Disruption of fusion causes severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Our previous study showed that down-regulation of PICK1 by shRNA in NIH3T3 cells decreased ATP production, reduced oxygen consumption, increased lactate production, increased mitochondrial fragmentation, and increased cell death. These studies indicated a novel and important role of PICK1 in maintaining mitochondria functions. Furthermore, our previous studies showed that knockdown PICK1 promoted glucose starvation-induced autophagy. In this study, I showed that overexpression of PICK1 in NIH3T3 cell also increased cell death under low glucose conditions. The LC3-II protein level was increased, but activation of caspasee-3 was not detected. These results suggested the glucose starvation induced autophagy. Furthermore, overexpression of PICK1 promoted mitochondrial fragmentation under low glucose conditions. I further examiend changes of mitochondrial membrane potential using JC-1, which can form aggregates inside mitochondria in a membrane potential-dependent manner and can be detected by fluorescent microscopy. PICK1-overexpressing stable clones showed altered mitochondrial membrane potential under low glucose conditions. These results suggested overexpression of PICK1 decreased cell viability and increased mitochondrial fragmentation.