Journal
CELLS
Volume 11, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/cells11101643
Keywords
SPLICS; organelle contact sites; split-GFP; stable cell lines; piggyBac system
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Funding
- Ministry of University and Research (Bando SIR 2014) [RBSI14C65Z]
- Universita degli Studi di Padova [CALI_-SID16_01, GRIC128SP0]
- Universita degli Studi di Padova (University of Padova)
- Ministry of University and Research (PRIN2017)
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The study presents a new vector library of Split-GFP-based contact sites (SPLICS) cloned into a piggyBac system for stable and inducible expression of reporters in a cell line of interest. Stable HeLa cell lines expressing SPLICS between different organelles were generated and used to investigate the effect of modulating cholesterol traffic on membrane contact sites. The results highlight the importance of different interfaces for cholesterol sensing and distribution within the cell.
The study of organelle contact sites has received a great impulse due to increased interest in the understanding of their involvement in many disease conditions. Split-GFP-based contact sites (SPLICS) reporters emerged as essential tools to easily detect changes in a wide range of organelle contact sites in cultured cells and in vivo, e.g., in zebrafish larvae. We report here on the generation of a new vector library of SPLICS cloned into a piggyBac system for stable and inducible expression of the reporters in a cell line of interest to overcome any potential weakness due to variable protein expression in transient transfection studies. Stable HeLa cell lines expressing SPLICS between the endoplasmic reticulum (ER) and mitochondria (MT), the ER and plasma membrane (PM), peroxisomes (PO) and ER, and PO and MT, were generated and tested for their ability to express the reporters upon treatment with doxycycline. Moreover, to take advantage of these cellular models, we decided to follow the behavior of different membrane contact sites upon modulating cholesterol traffic. Interestingly, we found that the acute pharmacological inhibition of the intracellular cholesterol transporter 1 (NPC1) differently affects membrane contact sites, highlighting the importance of different interfaces for cholesterol sensing and distribution within the cell.
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