Harsha E Rajapakse

University of Illinois at Chicago, Chicago, IL, United States

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Publications (3)25.38 Total impact

  • Harsha E Rajapakse, Lawrence W Miller
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    ABSTRACT: Lanthanide-based or luminescence resonance energy transfer (LRET) microscopy can be used to sensitively image interactions between reporter-labeled proteins in living mammalian cells. With LRET, luminescent lanthanide complexes are used as donors, conventional fluorophores are used as acceptors, and donor-sensitized acceptor emission occurs at time scales that reflect the long (~ms) lanthanide emission lifetime. These long-lived signals can be separated from short-lifetime (~ns) sample autofluorescence and directly excited acceptor fluorescence by using pulsed light to excite the specimen and by implementing a short delay (>100 ns) before detection, thereby increasing measurement sensitivity. As practical implementation of time-resolved LRET microscopy requires several potentially unfamiliar experimental techniques, we explicitly describe herein methods to label proteins in living mammalian cells with luminescent terbium complexes, image interactions between terbium-labeled proteins and green fluorescent protein fusions, and quantitatively analyze LRET images.
    Methods in enzymology 01/2012; 505:329-45. · 1.90 Impact Factor
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    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 micros) 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.
    Proceedings of the National Academy of Sciences 08/2010; 107(31):13582-7. · 9.74 Impact Factor
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    ABSTRACT: Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time-resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.
    Angewandte Chemie International Edition 07/2009; 48(27):4990-2. · 13.73 Impact Factor