Article
In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag.
Department of Chemistry, Columbia University, New York 10027, USA.
Nature Methods (impact factor:
19.28).
05/2005;
2(4):255-7.
DOI:10.1038/nmeth749
pp.255-7
Source: PubMed
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Citations (0)
- Cited In (6)
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Article: Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag.
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ABSTRACT: Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF') that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF' (5'-fluorescein-SLF') allowed the labeling of proteins genetically fused to FKBP-F36V in living cells. Here we describe the identification of novel fluorescent SLF'dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF'-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF' labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells.Cytometry Part A 11/2008; 75(3):207-24. · 3.73 Impact Factor -
Article: Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.
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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. · 9.68 Impact Factor -
Article: Fluorescent probes for super-resolution imaging in living cells.
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ABSTRACT: In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.Nature Reviews Molecular Cell Biology 01/2009; 9(12):929-43. · 39.12 Impact Factor
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Keywords
chemical methods
complex cellular environment
eDHFR
fast kinetics
functionalities
green fluorescent protein
mechanistic questions
protein function
selectively tag Escherichia coli dihydrofolate reductase
wider variety
