Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells
- 19 July 2010
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 107 (31), 13582-13587
- https://doi.org/10.1073/pnas.1002025107
Abstract
Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein–protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein–protein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 μs) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P < 10-6), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.Keywords
This publication has 49 references indexed in Scilit:
- The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D ActivityPublished by Elsevier BV ,2009
- Luminescent Terbium Protein Labels for Time‐Resolved Microscopy and ScreeningAngewandte Chemie-International Edition, 2009
- An In Vivo Covalent TMP-Tag Based on Proximity-Induced ReactivityACS Chemical Biology, 2009
- Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexesProceedings of the National Academy of Sciences of the United States of America, 2008
- Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerizationNature Methods, 2008
- Visualization of molecular interactions by fluorescence complementationNature Reviews Molecular Cell Biology, 2006
- Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAATNature Genetics, 2003
- THE GREEN FLUORESCENT PROTEINAnnual Review of Biochemistry, 1998
- A novel genetic system to detect protein–protein interactionsNature, 1989
- Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine SecretionJournal of Neurochemistry, 1989