Characterization of peroxisomes and peroxisome deficient cell lines by super-resolution microscopy and biochemical methods

Abstract

Stimulated Transmission Emission Depletion (STED) microscopy was used to characterize the nanoscopic morphology of wild-type peroxisomes under normal and proliferative conditions in mammalian cells and in yeast Saccharomyces cerevisiae (in this text referred to as yeast). In yeast, the new peroxisomal protein Pex35 was characterized by analyzing the subdiffraction size and morphology of peroxisomes in wildtype, and pex35 mutant strains. Only STED microscopy could reveal the Pex35 overexpression phenotype, which resembled hyper-vesiculation of clumped peroxisomes within a subdiffraction volume. This remarkable subdiffraction phenotype supports the hypothesis that PEX35 regulates peroxisome abundance by an Arf1 dependent vesiculation mechanism. In mammalian HeLa cells, the colocalization of the translocon complex and peroxisomal fission factors on peroxisome membranes were analyzed at the nanoscale. RING (PEX2-PEX10-PEX12) and docking (PEX13-PEX14) subcomplexes of the translocon machinery were found to localize on distinct membrane substructures. We also used STED microscopy, and developed an automated imaging analysis pipeline in CellProfiler (www.cellprofiler.org) to analyze the sub-diffraction morphology of the cellular peroxisomal ghost phenotype in Peroxisome biogenesis disorder patient cells (Zellweger Syndrome Spectrum phenotype). The size of the peroxisomal ghosts was found to correlate with import deficiency, integral peroxisomal membrane (PMP70) protein abundance, and the clinical severity of the patients. This is the first time that the membrane ghost phenotype associated with Zellweger Syndrome Spectrum disorder could be shown to correlate with the clinical severity and import deficiency in patients’ fibroblasts. Overall, this work forwards the characterization of the peroxisome organelle in wildtype conditions and human peroxisomal disorders.