Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy
Summary: Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE d...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723007268 |
| _version_ | 1827584860629237760 |
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| author | Fizza Askari Rupinder Kaur |
| author_facet | Fizza Askari Rupinder Kaur |
| author_sort | Fizza Askari |
| collection | DOAJ |
| description | Summary: Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE domain into the GFP-containing vector backbone, transforming FYVE-GFP into C. glabrata, and preparing slides with FYVE-GFP-expressing C. glabrata cells. We then detail procedures for acquiring and analyzing images and quantifying signal data. This protocol is adaptable to subcellular localization analysis of other low-abundant lipid and protein molecules.For complete details on the use and execution of this protocol, please refer to Askari et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-08T23:37:23Z |
| format | Article |
| id | doaj.art-0aa8ae125c3144689bc297e40688f328 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-08T23:37:23Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-0aa8ae125c3144689bc297e40688f3282023-12-14T05:23:46ZengElsevierSTAR Protocols2666-16672024-03-0151102759Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopyFizza Askari0Rupinder Kaur1Laboratory of Fungal Pathogenesis, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500039, Telangana, IndiaLaboratory of Fungal Pathogenesis, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500039, Telangana, India; Corresponding authorSummary: Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE domain into the GFP-containing vector backbone, transforming FYVE-GFP into C. glabrata, and preparing slides with FYVE-GFP-expressing C. glabrata cells. We then detail procedures for acquiring and analyzing images and quantifying signal data. This protocol is adaptable to subcellular localization analysis of other low-abundant lipid and protein molecules.For complete details on the use and execution of this protocol, please refer to Askari et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723007268Cell BiologyMicroscopy |
| spellingShingle | Fizza Askari Rupinder Kaur Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy STAR Protocols Cell Biology Microscopy |
| title | Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy |
| title_full | Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy |
| title_fullStr | Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy |
| title_full_unstemmed | Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy |
| title_short | Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy |
| title_sort | protocol for determination of phosphatidylinositol 3 phosphate levels and localization in candida glabrata by confocal microscopy |
| topic | Cell Biology Microscopy |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723007268 |
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