I Table of Figures A Novel Biosensor for the Long-Term Optical Integration of Neuronal Activity Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Jonathan A.G. Mackinnon München, den 26.04.2012 II Table of Figures Betreuer: Dr. Oliver Griesbeck Erstgutachter: Dr. Oliver Griesbeck Zweitgutachter: Prof. Dr. Elisabeth Weiss Tag der mündlichen Prüfung: 16.07.2012 I Table of Figures Table of Contents Table of Figures ................................................................................................................................ I Abbreviations ................................................................................................................................. III 1 Abstract ................................................................................................................................ 1 2 Introduction .......................................................................................................................... 3 2.1 2-deoxyglucose (2DG) ...................................................................................................... 4 2.2 Genetically encoded biosensor mechanisms ................................................................... 6 2.2.1 Reversible Biosensors ............................................................................................... 9 2.2.2 Irreversible Biosensors ............................................................................................ 12 2.3 Genetically Encoded Ca2+ Indicators .............................................................................. 16 2.3.1 The canonical EF-hand and Ca2+ binding proteins .................................................. 16 2.3.2 Rational design of GECIs ......................................................................................... 19 2.4 Research Objective ......................................................................................................... 23 2.5 Integrator design approaches ........................................................................................ 24 2.5.1 Approach 1: Ca2+-dependent protease FRET Integrator ......................................... 24 2.5.2 Approach 2: Artificial Ca2+-dependent protease FRET Integrator .......................... 25 2.5.3 Approach 3: Bimolecular Fluorescence Complementation Integrator................... 27 3 Materials and Methods ...................................................................................................... 29 3.1 Molecular Biology ........................................................................................................... 29 3.1.1 Polymerase Chain Reaction .................................................................................... 29 3.1.2 Site-directed mutagenesis ...................................................................................... 30 3.1.3 Restriction of DNA .................................................................................................. 32 3.1.4 Dephosphorylation of Vector DNA ......................................................................... 33 3.1.5 DNA ligation ............................................................................................................ 33 3.1.6 Preparation of chemically-competent E.coli .......................................................... 34 3.1.7 Preparation of electro-competent E.coli ................................................................ 34 3.1.8 Transformation of chemically-competent E.coli .................................................... 35 3.1.9 Transformation of electro-competent E.coli .......................................................... 35 II Table of Figures 3.1.10 Transposon Reaction and Screening ....................................................................... 35 3.2 Protein Biochemistry ...................................................................................................... 38 3.2.1 Protein Expression .................................................................................................. 38 3.2.2 Protein Purification ................................................................................................. 38 3.2.3 Western Blot ........................................................................................................... 39 3.3 Spectroscopy .................................................................................................................. 40 3.3.1 Fluorescent spectra of purified proteins ................................................................ 40 3.3.2 Spectroscopic determination of EC50 ...................................................................... 41 3.4 Cell Culture ..................................................................................................................... 42 3.4.1 Preparation of dissociated hippocampal neurons .................................................. 42 3.4.2 Passaging immortal cell lines .................................................................................. 43 3.4.3 Transient transfection of mammalian cells ............................................................ 43 3.5 Generation of Semliki Forest Virus ................................................................................ 44 3.6 Dissociated Hippocampal neuron assays ....................................................................... 45 3.6.1 12-well plate Electrical Field Stimulation ............................................................... 45 3.6.2 Electrical Field Stimulation for live-cell fluorescence microscopy ......................... 46 3.6.3 Fluo-4 AM Ca2+ dye imaging ................................................................................... 47 3.6.4 LIVE/DEAD Viability/Cytotoxicity Assay .................................................................. 47 3.7 Materials ........................................................................................................................ 48 3.7.1 Instruments ............................................................................................................. 48 3.7.2 Consumables ........................................................................................................... 49 3.7.3 Buffers and Solutions .............................................................................................. 50 3.7.4 Chemicals ................................................................................................................ 53 3.7.5 Plasmids, bacterial strains and cell-lines ................................................................ 54 4 Results ................................................................................................................................ 55 4.1 Approach 1: Ca2+-dependent protease FRET Integrator ................................................ 56 4.1.1 Exploring the feasibility of a µ-Calpain Integrator .................................................. 56 4.2 Approach 2: Artificial Ca2+-dependent protease FRET Integrator ................................. 59 4.2.1 Approach 2a: Conformation-dependent Protease Integrator ................................ 59 III Table of Figures 4.2.2 Approach 2b: Split TEV Integrator .......................................................................... 63 4.2.3 Calibration and application of Electrical Field Stimulation ..................................... 64 4.3 Approach 3: Bimolecular Fluorescence Complementation Integrator .......................... 66 4.3.1 In vitro optimisation of Integrator .......................................................................... 66 4.3.2 Integrator refinement for response to endogenous Ca2+ transients ..................... 76 4.3.3 Semliki Forest Virus delivery of Integrator ............................................................. 89 4.3.4 Application of Bimolecular Fluorescence Complementation developed protein-pair for the development of other split-protein based strategies ............................................ 98 5 Discussion ........................................................................................................................... 99 5.1 Overview of Integrator development ............................................................................ 99 5.2 Approach 1: Ca2+-dependent protease FRET Integrator .............................................. 100 5.3 Approach 2: Artificial Ca2+-dependent protease with FRET reporter .......................... 101 5.4 Approach 3: Bimolecular Fluorescence Complementation Integrator ........................ 103 5.4.1 In vitro optimisation of split-FP Integrator ........................................................... 103 5.4.2 Complementation Integrator refinement for response to endogenous Ca2+ transients .......................................................................................................................... 107 5.4.3 Summary of complementation Integrator development ..................................... 111 5.4.4 Functional validation and virus characterisation .................................................. 112 6 Conclusion ........................................................................................................................ 117 7 Bibliography ...................................................................................................................... 118 Acknowledgements 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