Parkinson’s Disease (PD) is a common neurodegenerative disorder with its pathologic mechanisms including mitochondrial dysfunction. Parkin is a key protein involved in the pathway of the clearance of damaged mitochondria. Our previous study has identified a mitochondrial protein p32 as a novel parkin interactor. But p32 is mainly found in the matrix of mitochondria by electron microscopy images and subcellular immunoblotting results, however immunocytochemistry studies have suggested that parkin is distributed in cytosol. In this study, we decided to investigate the distribution pattern of p32 with the help of mitochondrial markers and determine whether p32 may play a role in the parkin-mediated clearance of damaged mitochondria. We established a cellular model of mitophagy with carbony...[ Read more ]

Parkinson’s Disease (PD) is a common neurodegenerative disorder with its pathologic mechanisms including mitochondrial dysfunction. Parkin is a key protein involved in the pathway of the clearance of damaged mitochondria. Our previous study has identified a mitochondrial protein p32 as a novel parkin interactor. But p32 is mainly found in the matrix of mitochondria by electron microscopy images and subcellular immunoblotting results, however immunocytochemistry studies have suggested that parkin is distributed in cytosol. In this study, we decided to investigate the distribution pattern of p32 with the help of mitochondrial markers and determine whether p32 may play a role in the parkin-mediated clearance of damaged mitochondria. We established a cellular model of mitophagy with carbonyl cyanide m-chlorophenyl hydrazine (CCCP), an uncoupler causing mitochondrial depolarization and damage and found that overexpression and knockdown of p32 had effects on the CCCP-induced mitophagy. To further delineate the distribution of p32, we applied the Stochastic Optical Reconstruction Microscopy (STORM). By using photo-switchable fluorescent probes, STORM temporally separates the otherwise spatially overlapping images of individual biomolecules, improving the resolution to 20~50 nm. We took series of STORM pictures by immunostaining mitochondrial proteins, Tom20, COX-IV and p32 in cultured HEK293 cell lines and in primary cultured mouse neurons, with treatment of different toxics. The photos indicate that mitochondrial outer-membrane protein Tom20 formed nanoscale clusters that are quite resistant to diverse treatment, while p32 did not. In the future, we will focus on the distributions of mitochondrial proteins by means of diverse imaging methods.