Dynamic imaging of protein-protein interactions by MP-FLIM

被引：7

作者：

Ameer-Beg, SM ^{[1
]}

Peter, M ^{[1
]}

Keppler, MD ^{[1
]}

Prag, S ^{[1
]}

Barber, PR ^{[1
]}

Ng, T ^{[1
]}

Vojnovic, B ^{[1
]}

机构：

[1] Mt Vernon Hosp, Gray Canc Inst, Northwood HA6 2JR, Middx, England

来源：

MULTIPHOTON MICROSCOPY IN THE BIOMEDICAL SCIENCES V | 2005年 / 5700卷

关键词：

multiphoton; TCSPC; FRET; FLIM; fluorescent proteins; time-resolved anisotropy; nuclear factor kappa B; cell signaling;

D O I：

10.1117/12.589202

中图分类号：

TH742 [显微镜];

学科分类号：

摘要：

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live and fixed cell experiments.

引用

页码：152 / 161

页数：10

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