Analysis of phosphatidylinositol transfer at ER-PM junctions in receptor-stimulated live cells

Chi Lun Chang, Jen Liou

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages1-11
Number of pages11
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1949
ISSN (Print)1064-3745

Keywords

  • ER-PM junctions
  • PI
  • PI transfer protein
  • PI(4,5)P biosensor
  • PI(4,5)P replenishment

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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