Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

Abstract

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

People
Mandrou, Elena
Author

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

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Thomason, Peter A.
Author

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

See full publications list
Paschke, Peggy I.
Author

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

See full publications list
Paul, Nikki R.
Author

Nikolaou, Savvas and Juin, Amelie and Whitelaw, Jamie A. and Paul, Nikki R. and Fort, Loic and Nixon, Colin and Spence, Heather J. and Bryson, Sheila and Machesky, Laura M. (2024) CYRI-B-mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake. eLife, 13: e83712. ISSN 2050-084X

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

Burgess, Selena G. and Paul, Nikki R. and Richards, Mark W. and Ault, James R. and Askenatzis, Laurie and Claydon, Sophie G. and Corbyn, Ryan and Machesky, Laura M. and Bayliss, Richard (2024) A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. Open Biology, 14 (3): 230376. ISSN 2046-2441

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Tweedy, Luke
Author

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

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Insall, Robert
Author

Farndale, Lucas and Insall, Robert and Yuan, Ke (2025) TriDeNT: Triple deep network training for privileged knowledge distillation in histopathology. Medical Image Analysis, 102: 103479. ISSN 1361-8415

Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert (2024) A reliable system for quantitative g-protein activation imaging in cancer cells. Cells, 13 (13): 1114. ISSN 2073-4409

Kapitany, Valentin and Fatima, Areeba and Zickus, Vytautas and Whitelaw, Jamie and McGhee, Ewan and Insall, Robert and Machesky, Laura and Faccio, Daniele (2024) Single-sample image-fusion upsampling of fluorescence lifetime images. Science Advances, 10 (21): eadn0139. ISSN 2375-2548

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Information
Title:
A reliable system for quantitative g-protein activation imaging in cancer cells
Creators:
Mandrou, Elena and Thomason, Peter A. and Paschke, Peggy I. and Paul, Nikki R. and Tweedy, Luke and Insall, Robert
Identification Number:
10.3390/cells13131114
Date:
27 June 2024
Divisions:
College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Publisher:
MDPI
Additional Information:
Funding was provided by the UKRI Physics of Life programme (grant EP/T002123/1, PIs Prof. Laura Machesky & Prof. Daniele Faccio), the Wellcome Trust (Investigator grant 221786/Z/20/Z, to RHI), the Medical Research Council (programme grant MR/X000702/1, to RHI), and a studentship from the Glasgow Cancer Centre. Core Services and Beatson Advanced Imaging Resource at the CRUK Scotland Institute are core funded by Cancer Research UK, grant A31287.
Library
URI:
https://pub.demo35.eprints-hosting.org/id/eprint/223
Item Type:
Article
Depositing User:
Unnamed user with email ejo1f20@soton.ac.uk
Date Deposited:
11 Jun 2025 16:36
Revision:
24
Last Modified:
12 Jun 2025 10:34
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