The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro

Richard D Byrne, Banafshé Larijani, Dominic L Poccia

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

Original languageEnglish
Title of host publicationThe Nuclear Envelope
EditorsS. Shackleton, P. Collas, E Schirmer
Place of PublicationNew York, U. S. A.
PublisherHumana Press
Pages123-132
Number of pages10
ISBN (Electronic)9781493935307
ISBN (Print)9781493935284
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1411

Keywords

  • Animals
  • Female
  • Fluorescence Resonance Energy Transfer/methods
  • Male
  • Membrane Fusion
  • Membrane Proteins/metabolism
  • Microscopy, Fluorescence/methods
  • Molecular Imaging
  • Nuclear Envelope/metabolism
  • Ovum/metabolism
  • Protein Binding
  • Protein Interaction Mapping
  • Sea Urchins

Cite this

Byrne, R. D., Larijani, B., & Poccia, D. L. (2016). The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro. In S. Shackleton, P. Collas, & E. Schirmer (Eds.), The Nuclear Envelope (pp. 123-132). (Methods in Molecular Biology ; Vol. 1411). New York, U. S. A.: Humana Press. https://doi.org/10.1007/978-1-4939-3530-7_7