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Merck

Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.

Contact (Thousand Oaks (Ventura County, Calif.)) (2019-06-15)
Alexa Bishop, Maki Kamoshita, Josiah B Passmore, Christian Hacker, Tina A Schrader, Hans R Waterham, Joseph L Costello, Michael Schrader
RESUMEN

Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.

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Sigma-Aldrich
Anti-PMP70 antibody, Mouse monoclonal, clone 70-18, purified from hybridoma cell culture
Sigma-Aldrich
Anti-ACBD5 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution