The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging

2013 ◽  
Vol 405 (12) ◽  
pp. 3983-3987 ◽  
Author(s):  
Sandrine Poëa-Guyon ◽  
Hélène Pasquier ◽  
Fabienne Mérola ◽  
Nicolas Morel ◽  
Marie Erard
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Protein A ◽  
Ph Sensor ◽  
Cyan Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Enhanced Cyan Fluorescent Protein

scholarly journals Fluorescence lifetime imaging of pH along the secretory pathway

2021 ◽  
Author(s):  
Peter Linders ◽  
Martin ter Beest ◽  
Geert van den Bogaart
Keyword(s):  
Temporal Resolution ◽  
Fluorescence Lifetime ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Excitation Wavelength ◽  
Fluorescence Lifetime Imaging ◽  
Ph Homeostasis ◽  
Ph Changes ◽  
Lifetime Imaging ◽  
Wide Range

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway. Then, we analyze the dynamic pH changes within cells treated with Brefeldin A, a COPI coat inhibitor. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)-α while it was in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

scholarly journals Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

2012 ◽  
Vol 48 (42) ◽  
pp. 5127 ◽  
Author(s):  
Antonella Battisti ◽  
Michelle A. Digman ◽  
Enrico Gratton ◽  
Barbara Storti ◽  
Fabio Beltram ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

scholarly journals Unravelling the mechanisms controlling heme supply and demand

2021 ◽  
Vol 118 (22) ◽  
pp. e2104008118
Author(s):  
Galvin C.-H. Leung ◽  
Simon S.-P. Fung ◽  
Andrea E. Gallio ◽  
Robert Blore ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Supply And Demand ◽  
Fluorescence Lifetime Imaging ◽  
Prosthetic Group ◽  
Lifetime Imaging ◽  
Free Heme ◽  
Green Fluorescent ◽  
Cellular Compartments

In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

scholarly journals Interaction of Poxvirus Intracellular Mature Virion Proteins with the TPR Domain of Kinesin Light Chain in Live Infected Cells Revealed by Two-Photon-Induced Fluorescence Resonance Energy Transfer Fluorescence Lifetime Imaging Microscopy

2010 ◽  
Vol 84 (24) ◽  
pp. 12886-12894 ◽  
Author(s):  
Ananya Jeshtadi ◽  
Pierre Burgos ◽  
Christopher D. Stubbs ◽  
Anthony W. Parker ◽  
Linda A. King ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Surface Protein ◽  
Fluorescence Lifetime Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Infected Cells ◽  
Tpr Domain

ABSTRACT Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

scholarly journals Photoswitching FRET to monitor protein–protein interactions

2018 ◽  
Vol 116 (3) ◽  
pp. 864-873 ◽  
Author(s):  
Kristin H. Rainey ◽  
George H. Patterson
Keyword(s):  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Protein Protein Interactions ◽  
Lifetime Imaging ◽  
Powerful Approach ◽  
Fluorescent Molecules ◽  
In Cells

FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule’s photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched “off” rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched “on” again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein–protein interactions and fluorescent protein-based biosensors.

scholarly journals Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy

2003 ◽  
Vol 372 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Véronique CALLEJA ◽  
Simon M. AMEER-BEG ◽  
Borivoj VOJNOVIC ◽  
Rudiger WOSCHOLSKI ◽  
Julian DOWNWARD ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Fusion Partner ◽  
Intact Cells ◽  
Lifetime Imaging ◽  
Novel Approach

To be able to detect in situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction. We have developed a novel approach to monitoring conformational changes of proteins in intact cells. A double-labelled fluorescent green fluorescent protein–yellow fluorescent protein (GFP–YFP) fusion protein has been constructed, allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET). Additionally, a novel fusion partner, YFPdark, has been designed to act as a sterically hindered control for EAF-FRET. Any conformational changes will cause a variation in FRET, which, in turn, is detected by fluorescence lifetime imaging microscopy (‘FLIM’). Protein kinase B (PKB)/Akt, a key component of phosphoinositide 3-kinase-mediated signalling, was selected for this purpose. Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable. We report here that platelet-derived-growth-factor (‘PDGF’) stimulation of NIH3T3 cells expressing the GFP–Akt–YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation. We also show that the GFP–Akt–YFP construct conserves fully its functional integrity. This novel approach of monitoring the in situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.

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