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dc.contributor.authorMetz, J
dc.contributor.authorCastro, I
dc.contributor.authorSchrader, M
dc.date.accessioned2017-09-07T09:23:26Z
dc.date.issued2017-09-05
dc.description.abstractOrganelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.en_GB
dc.description.sponsorshipThis work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/K006231/1 and BB/N01541X/1 to M. Schrader). J. Metz and M. Schrader are supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF and WT105618MA). M. Schrader is supported by Marie Curie Initial Training Network action PerFuMe (316723).en_GB
dc.identifier.citationVol. 7, issue 17en_GB
dc.identifier.doi10.21769/BioProtoc.2536
dc.identifier.urihttp://hdl.handle.net/10871/29242
dc.language.isoenen_GB
dc.publisherBio-protocolen_GB
dc.rights© 2017 Bio-protocol LLCen_GB
dc.subjectPeroxisome motilityen_GB
dc.subjectLive-cell imagingen_GB
dc.subjectOrganelle cooperationen_GB
dc.subjectMembrane contacten_GB
dc.subjectGFP-PTS1en_GB
dc.subjectACBD5en_GB
dc.subjectACBD4en_GB
dc.titlePeroxisome Motility Measurement and Quantification Assayen_GB
dc.typeArticleen_GB
dc.identifier.issn2331-8325
dc.descriptionThis is the final version of the article. Available from Bio-protocol via the DOI in this record.en_GB
dc.identifier.journalBio-protocolen_GB
refterms.dateFOA2019-02-19T13:30:33Z


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