The Role of Cortactin in Arp2/3-Dependent Processes and Actin Nucleation Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Julia Margit Oelkers aus Osterholz-Scharmbeck 1. Referent: Prof. Dr. Martin Korte 2. Referent: Prof. Dr. Klemens Rottner eingereicht am: 04.04.2011 mündliche Prüfung (Disputation) am: 23.05.2011 Druckjahr 2011 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Lai FPL, Szczodrak M, Oelkers JM, Ladwein M, Acconcia F, Benesch S, Auinger S, Faix J, Small JV, Polo S, Stradal TEB, Rottner K (2009) Cortactin promotes migration and platelet-derived growth factor-induced actin reorganization by signaling to Rho- GTPases. Molecular Biology of the Cell, 20(14):3209-23. Posterpräsentationen Oelkers JM, Jacob S, Kerkhoff E, Wedlich-Söldner R, Small JV, Stradal TEB, Köstler SA, Rottner K (2010) From test tube to microtub(ul)e: Assaying actin nucleation in vivo. 33rd Annual Meeting of the German Society for Cell Biology and the "Frontiers in melanoma research" Meeting of the German Melanoma Research Network, Regensburg Oelkers JM, Jacob S, Lai FPL, Block J, Szczodrak M, Kerkhoff E, Wedlich-Söldner R, Backert S, Schlüter K, Stradal TEB, Small JV, Koestler SA, Rottner K (2010) A novel in vivo actin polymerization assay: targeting nucleators to microtubules. International Meeting on “Actin Dynamics”, Jena Oelkers JM, Jacob S, Lai FPL, Block J, Szczodrak M, Kerkhoff E, Backert S, Schlüter K, Stradal TEB, Small JV, Koestler SA, Rottner K (2011) A novel in vivo actin polymerization assay: targeting nucleators to microtubules. Jahrestagung des Bonner Forum Biomedizin, Bonn Vorträge The use of microtubules as platforms for assaying actin nucleation in vivo. (2010) Workshop “Cellular Morphogenesis”, Gif-sur-Yvette, Frankreich. Short talk i Table of contents VORVERÖFFENTLICHUNGEN DER DISSERTATION ...................................... I TABLE OF CONTENTS ..................................................................................... II 1 INTRODUCTION ........................................................................................... 1 1.1 The cytoskeleton of mammalian cells ............................................................. 1 1.2 Actin polymerization ........................................................................................ 2 1.3 Actin-dependent structures ............................................................................. 3 1.3.1 Lamellipodia ........................................................................................ 3 1.3.2 Circular dorsal ruffles .......................................................................... 5 1.3.3 Filopodia and microspikes ................................................................... 6 1.3.4 Stress fibers ........................................................................................ 7 1.3.5 Focal adhesions and podosomes ........................................................ 7 1.4 Rho-GTPases ................................................................................................. 8 1.5 Actin nucleation ............................................................................................ 10 1.5.1 The Arp2/3 complex .......................................................................... 11 1.5.1.1 Nucleation promoting factors ........................................................ 12 1.5.1.2 Class I NPFs ................................................................................. 12 1.5.1.3 Class II NPFs ................................................................................ 15 1.5.2 Formins ............................................................................................. 19 1.5.3 WH2-domain containing nucleators .................................................. 20 1.6 Regulators of actin filaments and monomers ................................................ 21 1.6.1 Ena/VASP ......................................................................................... 21 1.6.2 ADF/Cofilin ........................................................................................ 22 1.6.3 Capping protein ................................................................................. 23 1.6.4 Profilin ............................................................................................... 24 1.6.5 Fascin ................................................................................................ 24 1.6.6 α-actinin ............................................................................................ 25 1.7 Manipulation of the actin cytoskeleton by pathogenic bacteria ..................... 26 1.7.1 Listeria monocytogenes .................................................................... 26 1.7.2 Shigella flexneri ................................................................................. 27 1.8 Aims of the thesis.......................................................................................... 28 ii 2 MATERIALS AND METHODS .................................................................... 29 2.1 Chemicals, media and buffers ...................................................................... 29 2.2 Cell culture reagents and plasticware ........................................................... 29 2.3 Enzymes and reagents for molecular biology ............................................... 29 2.4 Vectors .......................................................................................................... 29 2.5 Bacterial cultures .......................................................................................... 29 2.6 Media for bacterial culture ............................................................................. 30 2.7 Conditions for bacterial culture ..................................................................... 30 2.8 Molecular biological standard techniques ..................................................... 31 2.8.1 Plasmids ............................................................................................ 31 2.8.2 Oligonucleotide primers ..................................................................... 32 2.8.3 Generation of DNA constructs ........................................................... 32 2.8.4 DNA sequencing ............................................................................... 32 2.8.5 Restriction digest and dephosphorylation ......................................... 33 2.8.6 DNA extraction from agarose gels .................................................... 33 2.8.7 Ligation .............................................................................................. 33 2.8.8 Generation of CaCl2-competent E. coli ............................................. 33 2.8.9 Transformation of E. coli ................................................................... 33 2.8.10 Preparation of plasmids from E. coli .................................................. 34 2.8.11 Quantification of DNA ........................................................................ 34 2.9 Protein biochemistry ..................................................................................... 34 2.9.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis ............. 34 2.9.2 Coomassie Blue staining ................................................................... 34 2.9.3 Preparation of protein extracts from cultured cells ............................ 35 2.9.4 Measurement of protein concentration .............................................. 35 2.10 Immunobiological methods ........................................................................... 35 2.10.1 Primary antibodies ............................................................................. 35 2.10.2 Secondary reagents .......................................................................... 36 2.10.3 Western blotting ................................................................................ 36 2.11 Tissue culture, transfections and treatments ................................................ 37 2.11.1 Media and solvents ........................................................................... 37 2.11.2 Cell lines ............................................................................................ 38 2.11.3 Cell culture prior to microscopic analysis .......................................... 38 2.11.4 Transfections ..................................................................................... 39 2.11.5 Gentamicin protection assay ............................................................. 39 2.11.6 Cells treatments ................................................................................ 40 2.12 Immunofluorescence microscopy and live-cell imaging ................................ 40 iii 2.12.1 Labeling of the actin cytoskeleton ..................................................... 40 2.12.2 Fixation procedures, stainings and analysis ...................................... 40 2.12.3 Electron microscopy .......................................................................... 41 2.12.4 Live cell imaging and data analysis ................................................... 41 2.12.5 Fluorescence recovery after photobleaching (FRAP)