GFP-based FRET analysis in live cells

Takanishi, Christina L.; Bykova, Ekaterina A.; Cheng, Wei; Zheng, Jie

doi:10.1016/j.brainres.2006.01.119

Cited by 101 publications

(116 citation statements)

References 18 publications

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“…(15)], which itself depends linearly on e 2 , the ratio of the extinction coefficients of ECFP and EYFP at the donor excitation wavelength, 458 nm [Eqs. (12) and (13) (47)), e 2 cannot be determined unequivocally. The extinction coefficients of ECFP and EYFP at 458 nm (Supporting Information Table S1) were estimated as the product of the peak extinction coefficient of the corresponding dye and the extinction at 458 nm relative to the peak.…”

Section: Ratiometric and Acceptor Photobleaching Fret Results Obtainementioning

confidence: 95%

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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Section: Ratiometric and Acceptor Photobleaching Fret Results Obtainementioning

confidence: 95%

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

et al. 2013

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“…From these two images, the total emission spectrum and the TMRM emission spectrum, respectively, were constructed. Standard emission spectra also were collected from cells labeled with only TMRM or FM and used to separate cross-contamination between FM and TMRM due to spectra overlaps (49).…”

Section: Methodsmentioning

confidence: 99%

Thermosensitive TRP channel pore turret is part of the temperature activation pathway

Yang

Cui

Wang

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperaturesensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand-and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand-and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity.conformational change | fluorescence resonance energy transfer | temperature sensing | thermodynamics T emperature-sensitive transient receptor potential channels, or thermoTRPs, include four heat-activated channels (TRPV1-4) and two cold-activated channels (TRPM8 and TRPA1) that exhibit nicely spaced activation temperatures covering the physiological temperature range (1-4). These are expressed in dorsal root ganglion sensory neurons, keratinocytes, and other cells (1). Temperature changes cause rapid, reversible activation of thermoTRP channels in both native cells (3) and expression systems ( Fig. 1 A and B). Depolarizing currents through these nonselective, cationpermeable channels lead to the generation of action potentials (5) or the release of messenger molecules (6, 7) that encodes temperature information. Structurally, thermoTRPs resemble voltagedependent potassium channels, with four subunits surrounding a central ion permeation pore (4). Each subunit contains six transmembrane segments (S1-S6) and long intracellular N and C termini. The channel pore is formed by S6 and a P-loop that in most thermoTRPs is noticeably longer than those of KcsA and voltagegated potassium channels (8, 9). Unique TRP channel structural elements, such as TRP Box immediately after S6 and N-terminal ankyrin repeats, are found in most thermoTRPs.Despite extensive research, the channel structure bestowing high temperature sensitivity on thermoTRPs remains elusive. ThermoTRP channels are polymodal sensors responsive to a wide range of physical and chemical stimuli, such as transmembrane voltage, ligands, and pH. It has been proposed that heat might control thermoTRP activation by shifting the channel's response to these stimuli (10, 11). Alternatively, synergistic activation by mul...

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“…in which D and A are molar extinction coefficients for the donor and acceptor, respectively, at the donor excitation wavelength (Takanishi et al, 2006;Gao et al, 2007). For the control groups, Kv2.1⌬C1a-Yϩ C-syx and Kv2.1⌬331-Yϩ C-syx, data presented were extracted from eCFP/eYFP molar ratio Ͼ3 at which saturation of FRET occurred.…”

Section: Fret Analysis and Confocal Microscopymentioning

confidence: 99%

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

Etzioni¹,

Siloni²,

Chikvashvilli³

et al. 2011

J. Neurosci.

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Whereas neuronal M-type Kϩ channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.

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GFP-based FRET analysis in live cells

Cited by 101 publications

References 18 publications

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

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

Thermosensitive TRP channel pore turret is part of the temperature activation pathway

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

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