Dissertation Aus dem Physiologischen Institut Lehrstuhl: Physiologie – Zelluläre Physiologie (Biomedizinisches Zentrum München) der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. Claudia Veigel Calbindin-D28k and its role in apoptosis: Inhibition of Caspase-3 activity and interaction with Pro-Caspase-3 investigated by in-situ FRET microscopy. Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Johannes Lohmeier aus Bong Town, Liberia 2018 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. Michael Meyer Prof. Dr. Alexander Faussner Mitberichterstatter: Prof. Dr. Nikolaus Plesnila Prof. Dr. Dr. Bernd Sutor Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 14.06.2018 Eidesstattliche Versicherung Lohmeier, Johannes Name, Vorname Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema Calbindin-D28k and its role in apoptosis: Inhibition of Caspase-3 activity and interaction with Pro-Caspase-3 investigated by in-situ FRET microscopy. selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. München, den 23.12.2016 Table of contents 1. Introduction ....................................................................................................................... 1 1.1. Calbindin-D28k (CBD28k) .....................................................................................................1 1.1.1. EF-hand motif ................................................................................................................. 3 1.1.2. Cytoprotection ................................................................................................................ 3 1.1.3. Intermolecular interaction ............................................................................................ 4 1.2. Apoptosis ................................................................................................................................ 7 1.3. Förster resonance energy transfer (FRET) .................................................................... 10 1.3.1. Theoretical background of FRET ............................................................................... 10 1.3.2. FRET microscopy .......................................................................................................... 13 1.3.2.1. Acceptor-Photobleaching (PB) .......................................................................... 14 1.3.2.2. Sensitized Emission (SE) ..................................................................................... 15 1.3.3. Fluorescent proteins (FPs) ......................................................................................... 17 1.3.4. FRET Caspase-3 biosensor ........................................................................................ 19 1.4. Research objectives ........................................................................................................... 20 1.4.1. Pilot experiment: In-situ assay of Caspase-3 activity upon expression of Calbindin-D28k ....................................................................................................................... 20 1.4.2. Investigation of intermolecular interaction between Calbindin-D28k and Pro- Caspase-3 by Acceptor-Photobleaching and Sensitized Emission ............................. 21 2. Materials and methods ............................................................................................... 22 2.1. Molecular biology ............................................................................................................... 26 2.1.1. pCMV5 vector plasmid ................................................................................................ 26 2.1.2. Primer design and Polymerase Chain Reaction (PCR) ........................................ 27 2.1.2.1. mAmetrine primers .............................................................................................. 27 2.1.2.2. Calbindin-D28k primers ...................................................................................... 28 2.1.2.3. tdTomato primers ................................................................................................ 28 2.1.2.4. Caspase-3 primers ............................................................................................... 29 2.1.2.5. Partial IMPase primers ........................................................................................ 29 2.1.3. Restriction enzyme (RE) digestion .......................................................................... 30 2.1.4. Gel electrophoresis (GE) ............................................................................................ 30 2.1.5. Ligation of DNA ............................................................................................................ 31 2.1.6. Purification of DNA ..................................................................................................... 31 2.1.7. DNA sequencing .......................................................................................................... 31 2.1.8. SDS-Page and Western Blot ..................................................................................... 31 2.2. Cellular biology .................................................................................................................... 34 2.2.1. Transformation ............................................................................................................. 34 2.2.2. Isolation and expansion of clones ........................................................................... 34 2.2.3. Plasmid preparation .................................................................................................... 34 2.2.4. Preparation of eukaryotic cell lines ........................................................................ 35 2.2.5. Transient transfection ................................................................................................ 36 2.2.6. Application of cytotoxic agents ............................................................................... 37 2.2.7. Preparation of slides for confocal microscopy ..................................................... 38 2.3. Confocal-Imaging................................................................................................................ 39 2.3.1. Imaging-Configuration ............................................................................................... 40 2.3.2. Data acquisition and analysis ................................................................................... 41 2.4. Miscellaneous ..................................................................................................................... 42 3. Results ............................................................................................................................ 43 3.1. Fusion proteins .................................................................................................................... 43 3.1.1. FRET donor: mAmetrine-CBD28k ............................................................................. 43 3.1.2. FRET acceptor: tdTomato-CASP3 ............................................................................ 44 3.1.3. FRET acceptor: tdTomato-pIMPase ......................................................................... 45 3.2. Pilot experiments ............................................................................................................... 46 3.2.1. Signal increase upon photobleaching ................................................................ 46 3.2.2. mTurquoise2 spectral bleed-through (SBT) ..................................................... 48 3.2.3. In-situ assay of Caspase-3 activity upon expression of Calbindin-D28k .. 49 3.3. Investigation of intermolecular interaction between Calbindin-D28k and Pro- Caspase-3 by Acceptor-Photobleaching and Sensitized Emission ................................. 54 4. Discussion ...................................................................................................................... 59 5. Summary ........................................................................................................................ 70 References .......................................................................................................................... 71 Appendix ............................................................................................................................ 79 Suppl.: pCMV5 plasmid Suppl.: mAmetrine forward and reverse primer Suppl.: CBD28k forward and reverse primer Suppl.: tdTomato forward and reverse primer Suppl.: CASP3 forward and reverse primer Suppl.: IMPase secondary structure Suppl.: pIMPase design and construction Suppl.: mAmetrine (w/ linker and w/o TAA stop codon) + EcoR1 + CBD28k (w/ stop codon) inserted in pCMV5 MCS Suppl.: TdTomato (w/o TAA stop codon) + Kpn1 + GTT-Linker + CASP3 (w/ stop codon) inserted in pCMV5 MCS Suppl.: tdTomato (w/o TAA stop codon) +