Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic
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...
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| Language: | English |
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MDPI AG
2022-05-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/11/10/1643 |
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| author | Flavia Giamogante Lucia Barazzuol Elena Poggio Marta Tromboni Marisa Brini Tito Calì |
| author_facet | Flavia Giamogante Lucia Barazzuol Elena Poggio Marta Tromboni Marisa Brini Tito Calì |
| author_sort | Flavia Giamogante |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T03:10:37Z |
| format | Article |
| id | doaj.art-658382a25dbf48b5a3cbbdb029c60dcd |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T03:10:37Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-658382a25dbf48b5a3cbbdb029c60dcd2023-11-23T10:27:36ZengMDPI AGCells2073-44092022-05-011110164310.3390/cells11101643Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol TrafficFlavia Giamogante0Lucia Barazzuol1Elena Poggio2Marta Tromboni3Marisa Brini4Tito Calì5Department of Biomedical Sciences, University of Padova, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyDepartment of Biology, University of Padova, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyDepartment of Biology, University of Padova, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, 35131 Padova, ItalyThe 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.https://www.mdpi.com/2073-4409/11/10/1643SPLICSorganelle contact sitessplit-GFPstable cell linespiggyBac system |
| spellingShingle | Flavia Giamogante Lucia Barazzuol Elena Poggio Marta Tromboni Marisa Brini Tito Calì Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic Cells SPLICS organelle contact sites split-GFP stable cell lines piggyBac system |
| title | Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic |
| title_full | Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic |
| title_fullStr | Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic |
| title_full_unstemmed | Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic |
| title_short | Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic |
| title_sort | stable integration of inducible splics reporters enables spatio temporal analysis of multiple organelle contact sites upon modulation of cholesterol traffic |
| topic | SPLICS organelle contact sites split-GFP stable cell lines piggyBac system |
| url | https://www.mdpi.com/2073-4409/11/10/1643 |
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