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Genetically encoded calcium indicators based on troponin C and fluorescent proteins [Elektronische Ressource] / vorgelegt von Nicola Heim

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GENETICALLY ENCODED CALCIUM INDICATORS BASED ON TROPONIN C AND FLUORESCENT PROTEINS Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Nicola Heim München, 2005 Erstgutachter: Prof. Dr. Alexander Borst Zweitgutachter: Prof. Dr. Rainer Uhl Tag der mündlichen Prüfung: 24. Oktober 2005 VTABLE OF CONTENTS TABLE OF CONTENTS..........................................................................................................V 1. ABSTRACT....................................................................................................................1 2. INTRODUCTION ............................................................................................................3 2.1 Fluorescence Techniques....................................................................................3 2.1.1 Fluorescence and FRET................................................................................3 2.1.2 The Green Fluorescent Protein .....................................................................8 2.1.3 Fluorescent Calcium Probes .......................................................................11 2.2 Calcium-Binding Proteins...................................................................................15 2.2.1 EF-Hands..........................

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Published 01 January 2005
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GENETICALLY ENCODED CALCIUM INDICATORS
BASED ON TROPONIN C AND FLUORESCENT
PROTEINS




Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften

an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München


vorgelegt von
Nicola Heim

München, 2005






























Erstgutachter: Prof. Dr. Alexander Borst
Zweitgutachter: Prof. Dr. Rainer Uhl
Tag der mündlichen Prüfung: 24. Oktober 2005

V
TABLE OF CONTENTS
TABLE OF CONTENTS..........................................................................................................V
1. ABSTRACT....................................................................................................................1
2. INTRODUCTION ............................................................................................................3
2.1 Fluorescence Techniques....................................................................................3
2.1.1 Fluorescence and FRET................................................................................3
2.1.2 The Green Fluorescent Protein .....................................................................8
2.1.3 Fluorescent Calcium Probes .......................................................................11
2.2 Calcium-Binding Proteins...................................................................................15
2.2.1 EF-Hands....................................................................................................15
2.2.2 The Troponin Family..................................................................................16
3. ABBREVIATIONS.........................................................................................................21
4. MATERIALS AND METHODS.......................................................................................23
4.1 Working with DNA ..............................................................................................23
4.1.1 Spectrometric Determination of DNA Concentration.................................23
4.1.2 Gene Amplification by PCR .......................................................................23
4.1.3 Site-directed Mutagenesis by PCR .............................................................24
4.1.4 Restriction of DNA.....................................................................................25
4.1.5 Ligation of DNA Fragments25
4.1.6 Preparation of Competent E. Coli...............................................................25
4.1.7 Quick Transformation of Chemically Competent E. Coli...........................26
4.2 Working with Proteins.........................................................................................26
4.2.1 Recombinant Protein Expression in Bacteria..............................................26
4.2.2 Purification of Recombinantly Expressed Proteins.....................................26
4.3 Cell Culture.........................................................................................................27
4.3.1 Preparation of Dissociated Rat Neurons .....................................................27
4.3.2 Transfection of Dissociated Rat Neurons ...................................................28
4.3.3 Transfection of HEK293 Cells....................................................................28
4.4 Histology and Immunohistochemistry.................................................................29
4.4.1 Cryosections of PFA-fixed Mouse Brains ..................................................29
4.4.2 Acute Slice Preparation of Mouse Brains ...................................................29
4.4.3 Antibody Staining of Cultured Cells...........................................................29
VI TABLE OF CONTENTS
4.4.4 Antibody Staining of Fixed Brain Sections ................................................ 30
4.5 Transgenic Animals............................................................................................30
4.5.1 DNA Preparation for Mouse Oocyte Injections.......................................... 30
4.5.2 Creation and Breeding of Transgenic mice 31
4.5.3 Genotyping of Transgenic Mice ................................................................. 31
4.6 Microscopy and Imaging .................................................................................... 32
4.6.1 FRET-Setup................................................................................................ 32
4.6.2 Determining the Ratio Change of Ratiometric Indicator Proteins .............. 33
4.6.3 Measuring Kd Values................................................................................. 33
4.6.4 Measurig Dissociation Kinetics.................................................................. 34
4.7 Materials.............................................................................................................35
4.7.1 Instruments................................................................................................. 35
4.7.2 Consumables .............................................................................................. 35
4.7.3 Buffers, Solutions, and Media.................................................................... 35
4.7.4 Chemicals and Products ............................................................................. 37
4.7.5 DNA Plasmids and E. coli Strains.............................................................. 39
4.7.6 Primer Sequences.......................................................................................39
5. RESULTS .................................................................................................................... 43
5.1 Construction of New Fluorescent Indicator proteins .......................................... 43
5.1.1 On the Design of Troponin Linker Domains.............................................. 43
5.1.2 Construction of CFP/YFP Constructs with csTnC, humTnC, and TPC1.... 46
5.1.3 Construction of Non-Aequoria Constructs ................................................. 50
5.1.4 Tuning Promising Constructs (I): Truncating and Multiplying the EF-hands
of csTnC 55
5.1.5 Tuning Promising Constructs (II): Mutating EF-hands of TN-L15............ 57
5.1.6 In vitro Kinetics.......................................................................................... 59
5.2 Functionality of TnC-Indicators in Cell Culture: TN-L15, TN-humTC and their
derivatives...................................................................................................................... 61
5.2.1 Cytosolic Expression in HEK293............................................................... 61
5.2.2 Cytosolic Expression in Dissociated Neurons............................................ 63
5.2.3 Subcellular Targeting of TnC-based Indicators and Their Functionality in
HEK293 Cells ............................................................................................................ 64
5.2.4 Subcellular Targeting in Neurons: TN-L15 D107A-Ras and Others.......... 68
5.3 Transgenic Mice Expressing TN-L15 in the Cytosol of Neurons ....................... 71 VII TABLE OF CONTENTS
5.3.1 Choosing an Adequate Promoter and Indicator Construct ..........................71
5.3.2 Expression Patterns and Expression Levels................................................73
5.3.3 Imaging of Live Brain Tissue .....................................................................78
6. DISCUSSION................................................................................................................81
6.1 New Ratiometric Calcium Indicator Probes and Their Physical Properties........81
6.1.1 Generating New Functional Constructs ......................................................81
6.1.2 In Vitro Characterization of Selected Constructs........................................84
6.2 Tests in Live Cells ..............................................................................................86
6.2.1 Cell Culture Experiments............................................................................86
6.2.2 Transgenic Mouse Lines.............................................................................88
6.3 Further Outlook...................................................................................................91
7. REFERENCES ..............................................................................................................93
THANKS AND ACKNOWLEDGEMENTS ..............................................................................105
CURRICULUM VITAE........................................................................................................106 1
1. ABSTRACT
Genetically encoded calcium probes allow the visualization and quantification of
intracellular calcium dynamics with great specificity and sensitivity. Until now, all
genetically encoded calcium indicators have shared a common design that consists of
mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as the
calcium binding moiety, in several configurations. However, most of these calmodulin-
based probe types show deficiencies such as reduced dynamic ranges when expressed
within transgenic organisms and a lack of calcium sensitivity in certain subcellular
targetings. A likely reason for this reduced sensitivity is that calmodulin is an ubiquitous
signal protein in cell metabolism and thus stringently regulated. Thus, we chose to develop
novel types of calcium probes based on the muscle calcium sensor troponin C, a protein
that is not a constituent of non-muscle cells and therefore less likely to interact with
cytosolic activities. By going through a series of cloning optimization steps, a set of new
ratiometric calcium indicators was created using domains of skeletal and cardiac muscle
troponin C variants as calcium binding moieties. These constructs showed in vitro FRET
ratio changes of up to 140 %, had calcium dissociation constants ranging from 470 nM to
29 μM, and were functional in intracellular targetings in which previous indicators had
failed. The new indicators expressed homogenously with no signs of aggregation in
HEK293 cells as well as in rat hippocampal neurons, and large and dynamic ratio changes
could be quantified after drug stimulation in cell culture. Membrane labeling experiments
with the indicator construct TN-L15 were successful in HEK293 cells and hippocampal
neurons. When targeted to the plasma membrane, the indicator readily responded to
agonist-induced increases in cytosolic calcium and kept its full dynamic range. In the last
part of this work, transgenic mouse lines were created expressing one of the new calcium
indicators in the cytosol of neurons. Imaging experiments in live tissue cultures and brain
slices revealed responses to rises in calcium that were superior to previously published
indicator performance in mouse lines expressing other calcium probes.
The novel troponin C-based probes of intracellular calcium developed in this work
have the potential for monitoring calcium dynamics in applications in which previous
calmodulin-containing calcium indicators failed, possibly because they interact less with
the cellular biochemical machinery and are thus more compatible with transgenic
expression in tissue and whole organisms.