High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Millington, M.; Grindlay, Guillermo; Altenbach, Kirsten; Neely, Robert K.; Kölch, Walter; Benčina, Mojca; Read, Nick D.; Jones, Anita C.; Dryden, David T. F.; Magennis, Steven W.

doi:10.1016/j.bpc.2007.01.008

Cited by 54 publications

(72 citation statements)

References 35 publications

(40 reference statements)

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“…-bound YC3.60 sample again indicates a population of YC3.60 molecules, which do not exhibit FRET. Both the heterogeneity of the fluorescence decay of ECFP and the presence of a population of non-interacting donor molecules in the construct severely complicate quantitative analysis of FRET in terms of discrete lifetimes (Millington et al 2007;Borst et al 2008;Wlodarczyk et al 2008).…”

Section: Resultsmentioning

confidence: 99%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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Section: Resultsmentioning

confidence: 99%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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“…ECFP and EYFP are characterized by at least two lifetimes each, and EYFP may be in a nonabsorbing dark state, which does not function as a FRET acceptor. Therefore, three lifetimes were discerned for an ECFP-EYFP fusion protein indicating that different donor-acceptor configurations were present characterized by distinct FRET efficiencies (52). Thus, apparent FRET efficiencies by intensity based FRET (including acceptor bleaching and ratiometric methods) are averages of real FRET efficiencies of the different species.…”

Section: Discussionmentioning

confidence: 99%

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

et al. 2013

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Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. V C 2013 International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; FRET; laser scanning cytometry; high throughput; protein-protein interactions FLUORESCENCE or F€ orster resonance energy transfer (FRET) is a nonradiative process, in which energy is transferred from an excited fluorescent donor dye to a neighboring acceptor within its 2-10 nm vicinity by dipole-dipole coupling (1). In order for FRET to take place, the emission spectrum of the donor should overlap with the absorption spectrum of the acceptor, and the two dyes should have a proper relative orientation (2,3). The process is characterized by the FRET efficiency, E, which is the probability that a donor in the excited state transfers its energy to a nearby acceptor. E depends on the 6th power of the separation distance, R, between the donor and the acceptor:where R 0 is the F€ orster radius, at which E 5 0.5. Because of its sensitive distance dependence, FRET can be used as a molecular ruler to assess intra-or inter-molecular distances (4), molecular conformation or association state. FRET has several effects ...

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“…Finally, the transformation procedure was stopped with the addition of 5 ml STC buffer. This mixture was added to 30 In vitro characterization of RaVC. For in vitro characterization, the protein was expressed in E. coli BL21(DE3) cells.…”

Section: Methodsmentioning

confidence: 99%

“…S1 in the supplemental material). Substantial codon optimization was achieved by replacing some fragments of the pHluorin gene with complementary fragments from Citrine or Venus genes; Venus (39) and Citrine (15) are improved yellow fluorescent proteins that have been shown to be expressed well in A. niger (30). We also introduced a point mutation at amino acid site 206, replacing alanine with lysine (47), to create a monomeric RaVC variant.…”

Section: Codon Optimization Was Used To Improve Ravc Expressionmentioning

confidence: 99%

Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe

et al. 2009

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A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 m of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

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High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Cited by 54 publications

References 35 publications

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe

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