From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 144

CHAPTER FIVE

From FRET Imaging to Practical
Methodology for Kinase Activity
Sensing in Living Cells

, } , ‡ and Pauline Vandame } , ‡ , Corentin Spriet * , ‡ ,

François Sipieter *

, ‡ ,
Jean-François Bodart } , ‡ , Franck B. Riquet } , ‡ ,1 and Laurent Héliot *

, ‡ , Pierre Vincent ‡ ,} , Dave Trinel *

Aymeric Leray *

, ‡ ,1
* BiophotonicTeam(BCF), IRICNRS,UniversityLille1-UniversityLille2USR3078,Villeneuved'Ascq, France
‡ Groupement deRechercheMicroscopie Fonctionnelle du vivant, GDR2588-CNRS, Villeneuve d'Ascq, France
} Neurobiologie des processus adaptatifs—UMR 7102, UPMC, Paris, France
} Laboratoire de R´gulation des Signaux de division EA4479 University Lille1, Villeneuve d'Ascq, France
1 Corresponding authors: Emails: [e-mail]; [e-mail]

Contents
1.

Introduction 147

2. Fluorescence Generalities 151

2.1 Introduction 151

2.2 The absorption process 151

2.3 The emission process 153

3. FRET Measurement 156

3.1 Introduction 156

3.2 FRET basis 157

3.3 Overlap integral J( l ) 158

2

3.4 Orientation factor k 159

3.5 Energy transfer efficiency 160

3.6 FRET measurements of molecular populations 161

3.7 Conclusion on FRET principles and applications 162

3.8 Few considerations on fluorescent proteins 163

4. FRET Measurements: Methods and Instrumentation 164

4.1 Intensity-based approaches 167

5. Data Analysis 173

5.1 Ratiometric imaging 173

5.2 Fluorescence lifetime imaging 176

6. Design and Optimization of Genetically Encoded KARs 186

6.1 Substrate peptide identification 187

6.2 Phosphoamino acid-binding domain 189

6.3 Optimal linker combinations in FRET-based biosensors 190

6.4 Choosing a FRET pair 191