@inbook{4232858188d940dc98d975a9ca25a6c8,
title = "Analysis of phosphatidylinositol transfer at ER-PM junctions in receptor-stimulated live cells",
abstract = "Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P2 replenishment following receptor stimulation, as a readout of PI transfer, using a PI(4,5)P2 biosensor and total internal reflection fluorescence microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM junctions and mediate PI(4,5)P2 replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.",
keywords = "ER-PM junctions, PI, PI transfer protein, PI(4,5)P biosensor, PI(4,5)P replenishment",
author = "Chang, {Chi Lun} and Jen Liou",
note = "Publisher Copyright: {\textcopyright} Springer Science+Business Media, LLC, part of Springer Nature 2019.",
year = "2019",
doi = "10.1007/978-1-4939-9136-5_1",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "1--11",
booktitle = "Methods in Molecular Biology",
}