Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system

Yuriko Kakimoto,Yasushi Tamura,Rieko Kojima,Shinya Tashiro,Toshiya Endo,Yuki Morozumi

doi:10.1038/s41598-018-24466-0

Yuriko Kakimoto, Yasushi Tamura + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-24466-0

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Journal: Scientific Reports	Publication Date: Apr 18, 2018
Citations: 85	License type: open-access

Affiliation: Yamagata University, Kyoto Sangyo University

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.

Highlights

One of the functional and structural hallmarks of eukaryotic cells is the presence of complex membrane structures called organelles
Since Ifa[38] and Tom[71] are anchored to the ER membrane and mitochondrial outer membrane (MOM) with their N-terminal transmembrane (TM) segments from the cytosol, respectively, the split-GFP fragments fused to their C-terminus should be exposed to the cytosol
We found that GFP signals generated from Ifa38-GFP1-10 and Tom71-GFP11 showed punctate structures (Fig. 2C and Supplementary Fig. S1), which resemble the foci of the reference contact sites of ERMES (Fig. 2B), these fusion proteins are, like Ifa[38] or Tom[71] alone, expected to be uniformly distributed on each organelle as far as they do not interact with each other

Summary

Introduction

One of the functional and structural hallmarks of eukaryotic cells is the presence of complex membrane structures called organelles. Organelles enclose specific sets of enzymes and maintain spatial individuality to fulfill each organelle’s specialized functions These classic views on organelle functions and structures have been radically challenged by the findings of physical connections between distinct organelles. ERMES form clusters, which can be observed as a limited number of stable dots around the overlapping regions between mitochondria and the ER tubules under a fluorescent microscope by tagging the ERMES core components with a fluorescent protein such as GFP2–6. This indicates that specific regions of the mitochondrial surface form direct contact sites with the ER membrane through ERMES. LDs were reported to interact with several other organelles such as the ER, vacuole, mitochondria, and peroxisomes organelle-tethering factors associated with LDs are largely unknown[29,30,31]

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