Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

John N Koberstein; Melissa L Stewart; Chadwick B Smith; Andrei I Tarasov; Frances M Ashcroft; Philip J S Stork; Richard H Goodman

doi:10.1073/pnas.2204407119

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

John N Koberstein
, Melissa L Stewart
, Chadwick B Smith
, Andrei I Tarasov
, Frances M Ashcroft
, Philip J S Stork
, Richard H Goodman

Research output: Contribution to journal › Article › peer-review

39 Citations (Scopus)

Abstract

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of β-cell glycolytic heterogeneity and dynamics.

Original language	English
Article number	e2204407119
Pages (from-to)	e2204407119
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	119
Issue number	31
Early online date	26 Jul 2022
DOIs	https://doi.org/10.1073/pnas.2204407119
Publication status	Published (in print/issue) - 2 Aug 2022

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. We thank Markus Grompe and Sunghee Chai for the β-cell–specific adeno-associated virus vector; Xander Viray and James Frank for help with calcium measurements; Matthew Schleisman for assistance with FACS; Yibing Jia for help with DNA sequencing; Stephanie Kaech for her expertise in microscopy; Erkan Karakas and Carsten Shultz for helpful discussions; and Paul Klevit and Melissa Kirigiti from the Oregon National Primate Research Center for technical assistance in preparing islets. Portions of this work were carried out in the Oregon Health and Science University Imaging Core, Flow Cytometry Core, Molecular Technologies Core, and by R01 AG055431 (to R.H.G.).

Publisher Copyright:
Copyright © 2022 the Author(s).

Funding

Funding Information: ACKNOWLEDGMENTS. We thank Markus Grompe and Sunghee Chai for the β-cell–specific adeno-associated virus vector; Xander Viray and James Frank for help with calcium measurements; Matthew Schleisman for assistance with FACS; Yibing Jia for help with DNA sequencing; Stephanie Kaech for her expertise in microscopy; Erkan Karakas and Carsten Shultz for helpful discussions; and Paul Klevit and Melissa Kirigiti from the Oregon National Primate Research Center for technical assistance in preparing islets. Portions of this work were carried out in the Oregon Health and Science University Imaging Core, Flow Cytometry Core, Molecular Technologies Core, and by R01 AG055431 (to R.H.G.). Publisher Copyright: Copyright © 2022 the Author(s).

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Biosensing Techniques
Fructose
Fructosediphosphates
Glycolysis
Repressor Proteins - metabolism
fructose 1,6-bisphosphate
glycolysis
β-cells

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10.1073/pnas.2204407119

Cite this

Koberstein, J. N., Stewart, M. L., Smith, C. B., Tarasov, A. I., Ashcroft, F. M., Stork, P. J. S., & Goodman, R. H. (2022). Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate. Proceedings of the National Academy of Sciences of the United States of America, 119(31), e2204407119. Article e2204407119. https://doi.org/10.1073/pnas.2204407119

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abstract = "Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of β-cell glycolytic heterogeneity and dynamics.",

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note = "Funding Information: ACKNOWLEDGMENTS. We thank Markus Grompe and Sunghee Chai for the β-cell–specific adeno-associated virus vector; Xander Viray and James Frank for help with calcium measurements; Matthew Schleisman for assistance with FACS; Yibing Jia for help with DNA sequencing; Stephanie Kaech for her expertise in microscopy; Erkan Karakas and Carsten Shultz for helpful discussions; and Paul Klevit and Melissa Kirigiti from the Oregon National Primate Research Center for technical assistance in preparing islets. Portions of this work were carried out in the Oregon Health and Science University Imaging Core, Flow Cytometry Core, Molecular Technologies Core, and by R01 AG055431 (to R.H.G.). Publisher Copyright: Copyright {\textcopyright} 2022 the Author(s).",

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Koberstein, JN, Stewart, ML, Smith, CB, Tarasov, AI, Ashcroft, FM, Stork, PJS & Goodman, RH 2022, 'Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate', Proceedings of the National Academy of Sciences of the United States of America, vol. 119, no. 31, e2204407119, pp. e2204407119. https://doi.org/10.1073/pnas.2204407119

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate. / Koberstein, John N; Stewart, Melissa L; Smith, Chadwick B et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 119, No. 31, e2204407119, 02.08.2022, p. e2204407119.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

AU - Koberstein, John N

AU - Stewart, Melissa L

AU - Smith, Chadwick B

AU - Tarasov, Andrei I

AU - Ashcroft, Frances M

AU - Stork, Philip J S

AU - Goodman, Richard H

N1 - Funding Information: ACKNOWLEDGMENTS. We thank Markus Grompe and Sunghee Chai for the β-cell–specific adeno-associated virus vector; Xander Viray and James Frank for help with calcium measurements; Matthew Schleisman for assistance with FACS; Yibing Jia for help with DNA sequencing; Stephanie Kaech for her expertise in microscopy; Erkan Karakas and Carsten Shultz for helpful discussions; and Paul Klevit and Melissa Kirigiti from the Oregon National Primate Research Center for technical assistance in preparing islets. Portions of this work were carried out in the Oregon Health and Science University Imaging Core, Flow Cytometry Core, Molecular Technologies Core, and by R01 AG055431 (to R.H.G.). Publisher Copyright: Copyright © 2022 the Author(s).

PY - 2022/8/2

Y1 - 2022/8/2

N2 - Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of β-cell glycolytic heterogeneity and dynamics.

AB - Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of β-cell glycolytic heterogeneity and dynamics.

KW - Biosensing Techniques

KW - Fructose

KW - Fructosediphosphates

KW - Glycolysis

KW - Repressor Proteins - metabolism

KW - fructose 1,6-bisphosphate

KW - glycolysis

KW - β-cells

UR - https://www.scopus.com/pages/publications/85135126624

U2 - 10.1073/pnas.2204407119

DO - 10.1073/pnas.2204407119

M3 - Article

C2 - 35881794

SN - 0027-8424

VL - 119

SP - e2204407119

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 31

M1 - e2204407119

ER -

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

Abstract

Bibliographical note

Funding

UN SDGs

Keywords

Access to Document

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