Analysis of Phosphatidylinositol Transfer at ER-PM Contact Sites in Receptor-Stimulated Live Cells.

Autor: Chang CL; Department of Cell and Molecular Biology, St. Jude Children's Hospital, Memphis, TN, USA., Lee WR; Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA., Li WT; Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA., Liou J; Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA. Jen.Liou@UTSouthwestern.edu.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2025; Vol. 2888, pp. 23-34.
DOI: 10.1007/978-1-0716-4318-1_3
Abstrakt: 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)P 2 ) in the plasma membrane (PM) important for Ca 2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P 2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM contact sites, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P 2 replenishment following receptor stimulation as a readout of PI transfer using a PI(4,5)P 2 biosensor and total internal reflection microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM contact sites and mediate PI(4,5)P 2 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.
(© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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