期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 31, 页码 13582-13587出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1002025107
关键词
cellular imaging; dihydrofolate reductase; Forster resonance energy transfer; lanthanide luminescence; protein labeling
资金
- National Institutes of Health (National Institute of General Medical Sciences) [R01GM081030]
- National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases) [R01DK061931]
- Chicago Biomedical Consortium with Chicago Community Trust [C-008]
Forster 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 transgeni-cally 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 auto-fluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 mu s) 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.
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