Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells

Chi Lun Chang, Jen Liou

Research output: Contribution to journalArticle

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)
Pages (from-to)1-11
Number of pages11
JournalMethods in molecular biology (Clifton, N.J.)
Volume1949
DOIs
StatePublished - Jan 1 2019

Fingerprint

Phosphatidylinositols
Endoplasmic Reticulum
Cell Membrane
Phospholipid Transfer Proteins
Spatio-Temporal Analysis
Biosensing Techniques
Homeostasis

Keywords

  • ER-PM junctions
  • PI
  • PI transfer protein
  • PI(4,5)P2 biosensor
  • PI(4,5)P2 replenishment

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells. / Chang, Chi Lun; Liou, Jen.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1949, 01.01.2019, p. 1-11.

Research output: Contribution to journalArticle

@article{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)P2 biosensor, PI(4,5)P2 replenishment",
author = "Chang, {Chi Lun} and Jen Liou",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-9136-5_1",
language = "English (US)",
volume = "1949",
pages = "1--11",
journal = "Methods in molecular biology (Clifton, N.J.)",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells

AU - Chang, Chi Lun

AU - Liou, Jen

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - ER-PM junctions

KW - PI

KW - PI transfer protein

KW - PI(4,5)P2 biosensor

KW - PI(4,5)P2 replenishment

UR - http://www.scopus.com/inward/record.url?scp=85061967644&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85061967644&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-9136-5_1

DO - 10.1007/978-1-4939-9136-5_1

M3 - Article

C2 - 30790244

AN - SCOPUS:85061967644

VL - 1949

SP - 1

EP - 11

JO - Methods in molecular biology (Clifton, N.J.)

JF - Methods in molecular biology (Clifton, N.J.)

SN - 1064-3745

ER -