Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy.

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. Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Citation

Fizza Askari, Rupinder Kaur. Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy. STAR protocols. 2024 Mar 15;5(1):102759

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PMID: 38088931

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