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.
Highlights
Organelles are critical membrane-bound structures that developed in eukaryotic cells to allow them to efficiently perform multiple chemical reactions within single cells by isolating and concentrating specific enzymes and metabolites
The split-GFP proteins expressed on the endoplasmic reticulum (ER) and the mitochondrial outer membrane (MOM) mostly showed dot-like signals like the ERMES dots (Figure 1B, a), indicating that the split-GFP proteins worked as a fluorescent probe for the ER-mitochondria-contact sites (Figure 1C)
We noticed that the reconstituted split-GFP proteins resulted in some large foci or elongated tubular signals, which were never observed for the ERMES dots, albeit only a minor portion of the total (∼10% of the total signals) (Figure 1B, b)
Summary
Organelles are critical membrane-bound structures that developed in eukaryotic cells to allow them to efficiently perform multiple chemical reactions within single cells by isolating and concentrating specific enzymes and metabolites. In addition to the ER-mitochondria contacts, multiple organelle-contact sites as well as tethering factors have been identified in yeast. Extensive studies have identified a number of factors that tether distinct organelle membranes, it is still unknown how the contact sites are regulated in terms of number and size. We previously developed split-GFP probes that could visualize inter-organelle-contact sites as research tools to tackle these problems (Kakimoto et al, 2018). Similar studies using split-GFP were performed by other research groups and demonstrated its usability for visualization and quantification of inter-organelle contacts (Cieri et al, 2018; Shai et al, 2018; Yang et al, 2018). A previous study reported that the split-GFP probes expressed on peroxisome and the ER could act as an artificial tether and increase their organelle contacts (Bishop et al, 2019)
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