RRC ID 53900
Author Kakimoto Y, Tashiro S, Kojima R, Morozumi Y, Endo T, Tamura Y.
Title Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.
Journal Sci Rep
Abstract Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.
Volume 8(1)
Pages 6175
Published 2018-4-18
DOI 10.1038/s41598-018-24466-0
PII 10.1038/s41598-018-24466-0
PMID 29670150
PMC PMC5906596
MeSH Biological Transport Endoplasmic Reticulum / metabolism Gene Expression* Genes, Reporter* Mitochondria / metabolism Models, Biological Models, Molecular Molecular Imaging* / methods Organelles / metabolism* Protein Conformation Protein Transport Recombinant Proteins / chemistry Recombinant Proteins / genetics Signal Transduction Structure-Activity Relationship Vacuoles / metabolism
IF 3.998
Times Cited 19
DNA material Tom71-GFP1-10 (RDB16037) Tom71-GFP11 (RDB16038) Tom71-V5-GFP11 (RDB16039) YNR021W-GFP11 (RDB16040) Tom20N-FLAG-GFP1-10 (RDB16041) Tom70N-FLAG-GFP1-10 (RDB16042) Ifa38-GFP1-10 (RDB16043) Ifa38-GFP11 (RDB16044) Ifa38-FLAG-GFP11 (RDB16045) Ifa38-mCherry-GFP11 (RDB16046) ERj1N-V5-GFP11 (RDB16047) Sec63N-V5-GFP11 (RDB16048) Vph1-GFP1-10 (RDB16049) Dpp1-V5-GFP11 (RDB16050) Pex3N-GFP1-10 (RDB16051) Pex3N-FLAG-GFP11 (RDB16052) Erg6-GFP1-10 (RDB16053) Erg6-FLAG-GFP11 (RDB16054)