Abstract
Peroxisomes are ubiquitous cell organelles involved in many metabolic and signaling functions. Their assembly requires peroxins, encoded by PEX genes. Mutations in PEX genes are the cause of Zellweger Syndrome spectrum (ZSS), a heterogeneous group of peroxisomal biogenesis disorders (PBD). The size and morphological features of peroxisomes are below the diffraction limit of light, which makes them attractive for super-resolution imaging. We applied Stimulated Emission Depletion (STED) microscopy to study the morphology of human peroxisomes and peroxisomal protein localization in human controls and ZSS patients. We defined the peroxisome morphology in healthy skin fibroblasts and the sub-diffraction phenotype of residual peroxisomal structures (‘ghosts’) in ZSS patients that revealed a relation between mutation severity and clinical phenotype. Further, we investigated the 70 kDa peroxisomal membrane protein (PMP70) abundance in relationship to the ZSS sub-diffraction phenotype. This work improves the morphological definition of peroxisomes. It expands current knowledge about peroxisome biogenesis and ZSS pathoethiology to the sub-diffraction phenotype including key peroxins and the characteristics of ghost peroxisomes.
Highlights
Background was measured in rawStimulated Emission Depletion (STED) data images in non-stained areas and the maximum background intensity was subtracted in ImageJ using the math tool
We stained the peroxisomal membrane of human skin fibroblasts with anti-PEX14 antibodies and coupled secondary antibodies for analysis by a 775 nm STED microscope
We quantified the diameter of 90 peroxisomes and found a mean peroxisomal diameter of 98.1 ± 17.1 (±SD) (Fig. 1F), which is smaller than the peroxisome size previously measured by super-resolution microscopy[43]
Summary
We used STED microscopy and automated imaging analysis to analyze the size and morphology of peroxisomal ghosts in ZSS patient fibroblasts from different complementation groups. In PEX13−/− patient fibroblasts we found ghost structures with luminal ACAA1 content (Supplementary Figure S4A) and complex structures accumulating ACAA1 (Supplementary Figure S4B), which are reminiscent of the membrane systems that have been reported to be biogenetic intermediates[47] This quantitative analysis revealed that the ghosts in ZSS cells appear more circular (less elongated) when compared to control cells (Fig. 5D). To investigate whether there is a correlation between the integral membrane proteins abundance and ghost size, we quantitated PMP70 by Western blotting in whole-cell lysates from ZSS patient fibroblasts with mutations in PEX1−/− (p.G843D, p.I700fsX42), PEX13−/− (p.W313G), PEX10−/− (p.L272fs), PEX2−/− (p.F278LfsX3), and PEX12−/+ (p.L123del) as well as from human control fibroblasts. Our results confirm that different degrees of severity of ZSS, which correlate with residual import function, are associated with distinctive morphological ghost phenotypes (Table 1)
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