Ratiometric Imaging of pH Probes

Grillo-Hill, Bree K.; Webb, Bradley A.; Barber, Diane L.

doi:10.1016/b978-0-12-420138-5.00023-9

Cited by 61 publications

(95 citation statements)

References 50 publications

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“…We modulated the cellular pHi by treating cells with proton transport inhibitors or ion exchange inhibitors (69,70) and then quantified metabolite levels in cells using gas chromatography coupled to mass spectrometry (GC/MS). These experiments were admittedly limiting since several metabolic pathways affect isocitrate and αKG levels.…”

Section: Resultsmentioning

confidence: 99%

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Luna

Lesecq

White

et al. 2020

Preprint

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§This paper is dedicated to the memory of our dear colleague and friend, Michelle Evon Scott (1990Scott ( -2020. ABSTRACTIsocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (α-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for α-KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ~12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

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Section: Resultsmentioning

confidence: 99%

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Luna

Lesecq

White

et al. 2020

Preprint

Self Cite

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“…Lastly, the general principles described here can be applied to the use of mCherry::pHluorin to measure pHi in other tissues as well. Indeed, our protocol was adapted from a protocol for using mCherry::pHluorin to measure pHi in the Drosophila eye, 7 and we have used similar methodology to image mCherry::pHluorin in the larval brain. Overall, the tools and methods described here should provide new opportunities to investigate the many diverse functions of pHi in vivo within living, intact tissue.…”

Section: Discussionmentioning

confidence: 99%

“…7 First, we perform live imaging experiments to measure fluorescence intensities of pHluorin and mCherry in germaria dissected into a buffer containing NaHCO 3 , which mimics physiological conditions. Next, we generate standard curves by measuring pHluorin and mCherry fluorescence intensities in a Na + free, K + buffer containing the ionophore nigericin adjusted to two different pH values, 6.5 and 7.5.…”

Section: Protocolmentioning

confidence: 99%

“…To circumvent the problem of poor dye penetration, we and others have used a genetically encoded probe, mCherry::pHluorin, 4–7 that can be expressed specifically in the cell types of interest and imaged in live tissue. pHluorin is a variant of GFP with a higher pKa (~7.0 vs ~4.0) that folds more readily at higher pH so the total fluorescence intensity emitted from a population of pHluorin molecules in the cell increases with increasing pHi.…”

Section: Introductionmentioning

confidence: 99%

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Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live <em>Drosophila</em> Ovarian Tissue

Tatapudy

Benitez

Nystul

2017

JoVE

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SHORT ABSTRACT We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values. LONG ABSTRACT Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating of pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin.1 Here, we describe a protocol we routinely use for culturing and imaging Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage from mCherry::pHluorin transgenic wild type and mutant lines; however the methods we describe can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, culturing ovarian tissue, and acquiring and analyzing images to obtain pHi values.

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“…However, detection of multiple colors is an important requirement for biological applications where multiple structures are labeled with different fluorophores. For example, dual color imaging is an important tool for many studies in which it is important to know whether two proteins are co-localized or not in a given structure [162], or to detect intracellular environmental changes by measuring the ratio between intensities of different emission wavelengths of a given fluorescent biosensor [163]. Simultaneous acquisition of two colors can be performed by using a dichroic mirror to separate the signal into two synchronized detectors [88], or by using commercial solutions for simultaneous acquisition in the same camera.…”

Section: 1a Simultaneous Multicolor Detectionmentioning

confidence: 99%

Light-sheet microscopy: a tutorial

et al. 2018

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This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

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Ratiometric Imaging of pH Probes

Cited by 61 publications

References 50 publications

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live <em>Drosophila</em> Ovarian Tissue

Light-sheet microscopy: a tutorial

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