Abstract
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.
Highlights
In the complex, condensed environment of the eukaryotic cell, intricate communication and collaboration between different organelles ensures that biological processes are precisely coordinated
Recent work suggests that most, if not all, organelles communicate in this way and numerous protein complexes contributing to organelle interaction and tethering have been identified, in particular those involved in mediating associations between the endoplasmic reticulum (ER) and mitochondria (Eisenberg-Bord et al, 2016; Gatta and Levine, 2017)
Proximity ligation assays can be used as a measure of peroxisome-ER associations
Summary
In the complex, condensed environment of the eukaryotic cell, intricate communication and collaboration between different organelles ensures that biological processes are precisely coordinated. Oligonucleotide proximity probes fused to secondary antibodies can be ligated, allowing signal amplification and subsequent binding of fluorescently labelled probes Split fluorescence assays, such as split superfolder GFP (spGFP), rely on targeting nonfluorescent portions of a fluorescent protein to different organelle membranes. The two portions do not fluoresce but reconstitution of the fluorescent signal occurs when the split portions re-join, indicating close proximity of the two organelles. These assays have both complemented and extended EM studies, allowing greater characterisation of dynamic ER-mitochondria interactions
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
