Monitoring Phosphatidic Acid Signaling in Breast Cancer Cells Using Genetically Encoded Biosensors
Phospholipids are important signaling molecules that regulate cell proliferation, death, migration, and metabolism. Many phospholipid signaling cascades are altered in breast cancer. To understand the functions of phospholipid signaling molecules, genetically encoded phospholipid biosensors have been developed to monitor their spatiotemporal dynamics. Compared to other phospholipids, much less is known about the subcellular production and cellular functions of phosphatidic acid (PA), partially due to the lack of a specific and sensitive PA biosensor in the past. This chapter describes the use of a newly developed PA biosensor, PASS, in two applications: regular fluorescent microscopy and fluorescence lifetime imaging microscopy-Förster/fluorescence resonance energy transfer (FLIM-FRET). These protocols can be also used with other phospholipid biosensors.
Key wordsPhospholipid Phosphatidic acid PASS Cancer Immunofluorescence Fluorescence lifetime imaging microscopy FLIM Fluorescence resonance energy transfer FRET
This work was supported by a research grant RP130425 from the Cancer Prevention and Research Institute of Texas (CPRIT) and a research grant R01HL119478 from the National Heart, Lung, and Blood Institute of the National Institutes of Health to GD, and a UTHealth Innovation for Cancer Prevention Research Training Program Predoctoral Fellowship grant RP140103 from the CPRIT to ML. The content is solely the responsibility of the authors and does not necessarily represent the official views of the CPRIT and National Institutes of Health.
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