RRC ID 63994
Author Tashiro S, Kakimoto Y, Shinmyo M, Fujimoto S, Tamura Y.
Title Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells.
Journal Front Cell Dev Biol
Abstract Inter-organelle contact sites have attracted a lot of attention as functionally specialized regions that mediate the exchange of metabolites, including lipids and ions, between distinct organelles. However, studies on inter-organelle contact sites are at an early stage and it remains enigmatic what directly mediates the organelle-organelle interactions and how the number and degree of the contacts are regulated. As a first step to answer these questions, we previously developed split-GFP probes that could visualize and quantify multiple inter-organelle contact sites in the yeast and human cultured cells. However, the split-GFP probes possessed a disadvantage of inducing artificial connections between two different organelle membranes, especially when overexpressed. In the present study, we developed a way to express the split-GFP probes whose expressions remained at low levels, with minimal variations between different yeast cells. Besides, we constructed a HeLa cell line in which the expression of the split-GFP probes could be induced by the addition of doxycycline to minimize the artificial effects. The improved split-GFP systems may be faithful tools to quantify organelle contact sites and screen new factors involved in organelle-organelle tethering in yeast and mammalian cells.
Volume 8
Pages 571388
Published 2020-1-1
DOI 10.3389/fcell.2020.571388
PMID 33330450
PMC PMC7714769
IF 5.186
DNA material pMM86 pcDNA3.1-ERj1(1-200)-V5-GFP(1-10) (RDB18663) pMM83 pcDNA3.1-TOMM70(1-70)-3xFLAG-GFP(11) (RDB18662) pMM189 pIRESNeo3-TOMM70(1-70)-3xFLAG-GFP11 (RDB18665) pYC137 pBS-GPDp-DPP1-V5-GFP(11)-CyC1ter-natNT2 (RDB18658) pYC136 pBS-GPDp-Erg6-V5-GFP(11)-CyC1ter-natNT2 (RDB18657) pYC135 pBS-GPDp-Ifa38-V5-GFP(11)-CyC1ter-natNT2 (RDB18656) pYC141 pBS-GPDp-Ifa38-GFP(1-10)-CyC1ter-hphMX (RDB18659) pYC144 pBS-GPDp-Pex3N-GFP(1-10)-CyC1ter-hphMX (RDB18661) pYC143 pBS-GPDp-Tom71-GFP(1-10)-CyC1ter-hphMX (RDB18660) pYU99 pRS316-ADH1p-mCherry-MCS-CYC1ter (RDB19395)