Characterisation of peroxisome-organelle contacts and cooperation

Abstract

Peroxisomes are organelles which are vital for human health and development. They represent dynamic subcellular compartments which play cooperative roles in essential cellular metabolic processes such as lipid metabolism and redox balance. For example, cooperation between peroxisomes and the endoplasmic reticulum (ER) is essential for the production of myelin lipids which are required for normal neurological function. We recently discovered that peroxisome-ER interaction is mediated by physical linkages in the form of membrane contact sites. These contact sites are mediated by the interaction of peroxisomal ACBD5 and ER-resident VAPB proteins. ACBD5-deficient patients have recently been identified who display retinal dystrophy, white matter disease and accumulation of very-long-chain fatty acids, which can only be degraded in peroxisomes. There is currently a need to develop simple and robust tools to allow efficient visualisation and quantification of these membrane contact sites to further their characterisation and investigate their function. Moreover, these should allow the dynamics of membrane contact sites under physiological conditions to be assessed. This study presents the optimisation of two systems to investigate peroxisome-ER interactions, the proximity ligation assay, Duolink® and a split fluorescent reporter system, split superfolder green fluorescent protein. These allow peroxisome-ER interactions to be visualised and measured in situ with a fluorescence-based readout when the organelles are in close proximity. These systems are powerful and modifiable and will help further characterise peroxisome-ER (or other organelle) membrane contacts and shed light on the interplay between peroxisomes and the ER.