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Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors

Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors
Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

Journal Article
2040-3364
5904-5911
Elsutohy, Mohamed M.
95f0a2f6-1598-4913-9d1c-a12cc93832ec
Chauhan, Veeren M.
f1c327f6-4150-4b58-a133-a2c389f05c8e
Markus, Robert
1fda3cce-58a3-4216-878b-e73781c6d054
Kyyaly, Mohammed Aref
7bd69b33-fec8-405c-9f40-b7157f0242f0
Tendler, Saul J.B.
782c4f23-5107-4dbc-aa5e-b44f7134da8d
Aylott, Jonathan W.
073f0c85-e621-4683-a455-de45b9f9c30c
Elsutohy, Mohamed M.
95f0a2f6-1598-4913-9d1c-a12cc93832ec
Chauhan, Veeren M.
f1c327f6-4150-4b58-a133-a2c389f05c8e
Markus, Robert
1fda3cce-58a3-4216-878b-e73781c6d054
Kyyaly, Mohammed Aref
7bd69b33-fec8-405c-9f40-b7157f0242f0
Tendler, Saul J.B.
782c4f23-5107-4dbc-aa5e-b44f7134da8d
Aylott, Jonathan W.
073f0c85-e621-4683-a455-de45b9f9c30c

Elsutohy, Mohamed M., Chauhan, Veeren M., Markus, Robert, Kyyaly, Mohammed Aref, Tendler, Saul J.B. and Aylott, Jonathan W. (2017) Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors. Nanoscale, 9 (18), 5904-5911. (doi:10.1039/c7nr00906b).

Record type: Article

Abstract

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

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More information

Accepted/In Press date: 8 April 2017
e-pub ahead of print date: 10 April 2017
Published date: 11 May 2017
Keywords: Journal Article

Identifiers

Local EPrints ID: 418753
URI: https://eprints.soton.ac.uk/id/eprint/418753
ISSN: 2040-3364
PURE UUID: 23a3c5e1-41bd-45ed-a1af-eb18ea8a82fb
ORCID for Mohammed Aref Kyyaly: ORCID iD orcid.org/0000-0002-1684-9207

Catalogue record

Date deposited: 21 Mar 2018 17:30
Last modified: 03 Dec 2019 01:33

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