DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT DER BIOLOGIE DER LUDWIG-MAXIMILIANS- UNIVERSITÄT MÜNCHEN ANALYSIS OF RHOH FUNCTION IN VIVO TATJANA DORN MÜNCHEN 2007 Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe angefertigt habe. Ich habe weder anderweitig versucht, eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile derselben einer anderen Prüfungskommission vorgelegt. Tatjana Dorn München, 13.06.2007 Die vorliegende Arbeit wurde zwischen Januar 2004 und Mai 2007 unter Anleitung von Prof. Dr. Cord Brakebusch am Max-Planck-Institut für Biochemie in Martinsried und an der Universität von Kopenhagen durchgeführt. Wesentliche Teile dieser Arbeit sind in folgender Publikation veröffentlicht: Dorn, T., Kuhn, U., Bungartz, G., Stiller, S., Bauer, M., Ellwart, J., Peters, T., Scharffetter-Kochanek, K., Semmrich, M., Laschinger, M., Holzmann, B., Klinkert, W.E.F., Straten, P.T., Køllgaard, T., Sixt, M. and Brakebusch, C. (2007). RhoH is important for positive thymocyte selection and T cell receptor signaling. Blood 109: 2346- 2355. Promotionsgesuch eingereicht am: 13.06.2007 Tag der mündlichen Prüfung am: 01.08.2007 Erster Gutachter: Prof. Dr. Charles N. David Zweiter Gutachter: Prof. Dr. Michael Schleicher Sondergutachter: Prof. Dr. Cord Brakebusch Table of contents Table of contents Abbreviations............................................................................................................................ 8 1 Summary........................................................................................................................... 10 2 Introduction ...................................................................................................................... 11 2.1 Family of Rho GTPases........................................................................................................ 11 2.1.1 Overview of Rho GTPase family ..................................................................................... 11 2.1.2 Regulation of Rho GTPases ............................................................................................. 13 2.1.2.1 Regulation by GEFs, GAPs and GDIs.................................................................... 13 2.1.2.2 Regulation by lipid modification ............................................................................ 15 2.1.2.3 Regulation by gene expression ............................................................................... 15 2.1.2.4 Regulation by phosphorylation and ubiquitinylation.............................................. 16 2.1.2.5 Crosstalk between Rho GTPases ............................................................................ 16 2.1.3 Biological function of Rho GTPases................................................................................ 16 2.2 Hematopoiesis........................................................................................................................ 18 2.2.1 Development of myeloid and erythroid cells.................................................................... 18 2.2.2 Development of lymphoid cells........................................................................................ 19 2.2.2.1 B cells ..................................................................................................................... 19 2.2.2.2 NK cells .................................................................................................................. 20 2.2.2.3 T cells...................................................................................................................... 20 2.2.2.4 Natural killer T cells ............................................................................................... 24 2.2.2.5 γδ T cells ................................................................................................................. 25 2.3 TCR signaling........................................................................................................................ 25 2.4 Role of Rho GTPases in hematopoiesis............................................................................... 29 2.4.1 Migration and adhesion .................................................................................................... 29 2.4.2 Differentiation, proliferation and survival........................................................................ 29 2.4.3 Phagocytosis and production of reactive oxygen species................................................. 31 2.5 RhoH function ....................................................................................................................... 31 2.6 Aim of the project ................................................................................................................. 35 3 Materials and methods..................................................................................................... 36 3.1 Materials ................................................................................................................................ 36 3.2 Generation of the RhoH-deficient mice .............................................................................. 36 3.3 Mice........................................................................................................................................ 36 3.4 Generation of the RhoH antibody ....................................................................................... 37 3.4.1 Immunization of a rabbit with the RhoH peptide ............................................................. 37 3.4.2 ELISA of the rabbit anti-serum ........................................................................................ 38 3.5 Bacteria culture..................................................................................................................... 38 3.5.1 Bacteria strains ................................................................................................................. 38 3.5.2 Media and conditions for bacteria culture ........................................................................ 38 3.6 Molecular biology methods .................................................................................................. 39 3.6.1 Primers.............................................................................................................................. 39 3.6.2 Determination of DNA concentration .............................................................................. 39 3.6.3 Agarose gel electrophoresis.............................................................................................. 39 3.6.4 DNA digestion.................................................................................................................. 40 3.6.5 DNA ligation .................................................................................................................... 40 4 Table of contents 3.6.6 Dephosphorylation of the vector ...................................................................................... 40 3.6.7 Extraction of DNA fragments from agarose gel............................................................... 41 3.6.8 Cloning of RhoH constructs ............................................................................................. 41 3.6.9 Mini-preparation of plasmid DNA ................................................................................... 42 3.6.10 Maxi-preparation of plasmid DNA................................................................................... 42 3.6.11 Polymerase chain reaction................................................................................................ 42 3.6.12 DNA sequencing .............................................................................................................. 43 3.6.13 Preparation of competent bacteria .................................................................................... 43 3.6.13.1 Preparation of heat shock competent DH5α........................................................... 43 3.6.13.2 Preparation of electrocompetent BL21(DE3) ......................................................... 44 3.6.14 E.coli transformation ........................................................................................................ 44 3.6.14.1 Heat shock............................................................................................................... 44 3.6.14.2 Electroporation........................................................................................................ 44 3.6.15 Genomic DNA extraction................................................................................................. 45 3.6.15.1 DNA extraction from tail pieces ............................................................................. 45 3.6.15.2 DNA isolation from tail pieces for Southern blot................................................... 45 3.6.16 Southern blotting .............................................................................................................. 45 3.6.16.1 Genomic digestion and DNA gel electrophoresis................................................... 46 3.6.16.2 Labeling of the DNA probe .................................................................................... 46 3.6.16.3 Hybridization, washing, autoradiography............................................................... 47 3.6.17 RNA.................................................................................................................................