Protein activation dynamics in cells and tumor micro arrays assessed by time resolved Förster resonance energy transfer

Véronique Calleja; Pierre Leboucher; Banafshé Larijani

doi:10.1016/B978-0-12-391856-7.00036-6

Protein activation dynamics in cells and tumor micro arrays assessed by time resolved Förster resonance energy transfer

Véronique Calleja, Pierre Leboucher, Banafshé Larijani

Research output: Chapter or section in a book/report/conference proceeding › Book chapter

3 Citations (SciVal)

Abstract

Analytical time resolved Förster resonance energy transfer (FRET) can be exploited for assessing, in cells and tumor micro arrays, the activation status and dynamics of oncoproteins such as epidermal growth factor receptor (EGFR1) and their downstream effectors such as protein kinase B (PKB) and 3-phosphoinositide-dependent protein kinase 1 (PDK1). The outcome of our research involving the application of quantitative imaging for investigating molecular mechanisms of phosphoinositide-dependant enzymes, such as PKB and PDK1, has resulted in a refined model describing the dynamics and regulation of these two oncoproteins in live cells. Our translational research exploits a quantitative FRET method for establishing the activation status of predictive biomarkers in tumor micro arrays. We developed a two-site FRET assay monitored by automated frequency domain Fluorescence lifetime imaging microscopy (FLIM). As a proof of principle, we tested our methodology by assessing EGFR1 activation status in tumor micro arrays from head and neck patients. Our two-site FRET assay, by high-throughput frequency domain FLIM, has great potential to provide prognostic and perhaps predictive biomarkers.

Original language	English
Title of host publication	Methods in Enzymology
Publisher	Elsevier Academic Press Inc
Pages	225-246
Number of pages	22
DOIs	https://doi.org/10.1016/B978-0-12-391856-7.00036-6
Publication status	Published - 16 Feb 2012

Publication series

Name	Methods in Enzymology
Volume	506
ISSN (Print)	0076-6879
ISSN (Electronic)	1557-7988

Bibliographical note

Funding Information:
We are grateful to Peter J. Parker for his critical insights and scientific discussions in these projects. We would like to thank Paul Barber from the Gray Institute for Radiation, Oncology & Biology and Medical Sciences Division (University of Oxford) for the analysis software (TRI2) of the two-photon FLIM. We thank Christopher Applebee for the setup of live imaging on the two-photon FLIM. We also thank Nirmal Jethwa and Christopher Applebee for commenting on the manuscript. This work was supported by Cancer Research UK core funding to London Research Institute and by EU grant-QLK3-CT-2000.

Funding

We are grateful to Peter J. Parker for his critical insights and scientific discussions in these projects. We would like to thank Paul Barber from the Gray Institute for Radiation, Oncology & Biology and Medical Sciences Division (University of Oxford) for the analysis software (TRI2) of the two-photon FLIM. We thank Christopher Applebee for the setup of live imaging on the two-photon FLIM. We also thank Nirmal Jethwa and Christopher Applebee for commenting on the manuscript. This work was supported by Cancer Research UK core funding to London Research Institute and by EU grant-QLK3-CT-2000.

Keywords

FLIM
FRET
Keywords
PDK1
PKB/Akt
Tumor micro arrays

ASJC Scopus subject areas

Biochemistry
Molecular Biology

Access to Document

10.1016/B978-0-12-391856-7.00036-6

Cite this

Protein activation dynamics in cells and tumor micro arrays assessed by time resolved Förster resonance energy transfer. / Calleja, Véronique; Leboucher, Pierre; Larijani, Banafshé.
Methods in Enzymology. Elsevier Academic Press Inc, 2012. p. 225-246 (Methods in Enzymology; Vol. 506).

Research output: Chapter or section in a book/report/conference proceeding › Book chapter

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Protein activation dynamics in cells and tumor micro arrays assessed by time resolved Förster resonance energy transfer

Abstract

Publication series

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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Cite this