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Cdc42 and Beta1 Integrin in Cell Migration

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Cdc42 and Beta1 Integrin in Cell Migration Cdc42 and β1 integrin in cell migration Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Aleksandra Czuchra München 2005 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. Aleksandra Czuchra München, den 19.09.2005 2 Die vorliegende Arbeit wurde zwischen August 2002 und August 2005 unter Anleitung von Dr. Cord Brakebusch am Max-Planck-Institut für Biochemie in Martinsried durchgeführt. Wesentliche Teile dieser Arbeit sind in folgender Publikation veröffentlicht: Czuchra, A., Wu, X., Meyer, H., van Hengel, J., Schroeder, T., Geffers, R., Rottner, K. and Brakebusch, C. (2005) Cdc42 is not essential for filopodium formation, directed migration, cell polarization and mitosis in fibroblastoid cells. Mol Biol Cell 16, 4473-4484. Promotionsgesuch eingereicht am 19.09.2005 Tag der mündlichen Prüfung 12.12.2005 Erster Gutachter: Prof. Dr. Charles N. David Zweiter Gutachter: Prof. Dr. Michael Schleicher Sondergutachter: Dr. Cord Brakebusch 3 Table of contents TABLE OF CONTENTS……………………………………………………………………………...4 ABBREVIATIONS…………………………………………………………………………………...10 1 SUMMARY ................................................................................................................................... 13 2 INTRODUCTION......................................................................................................................... 14 2.1 INTEGRINS................................................................................................................................... 14 2.1.1 INTEGRIN CYTOPLASMIC DOMAINS ........................................................................................... 15 2.1.2 INTEGRIN ACTIVATION .............................................................................................................. 16 2.1.3 INTEGRIN SIGNALLING .............................................................................................................. 19 2.1.4 INTEGRINS IN SKIN .................................................................................................................... 24 2.2 THE RHO GTPASES FAMILY ...................................................................................................... 29 2.2.1 REGULATION OF THE RHO GTPASES FAMILY ........................................................................... 31 2.2.2 THE CDC42 FAMILY .................................................................................................................. 32 2.2.3 TOOLS OF TRADE: USE OF DOMINANT-NEGATIVE INHIBITORY MUTANTS OF THE RHO-FAMILY GTPASES ............................................................................................................................................... 33 2.3 CELL MIGRATION ....................................................................................................................... 34 2.3.1 CDC42 IN CELL MIGRATION....................................................................................................... 35 2.3.1.1 Cdc42 as regulator of protrusion formation........................................................................... 36 2.3.1.2 Cdc42 as master regulator of cell polarity ............................................................................. 38 2.3.2 INTEGRINS IN CELL MIGRATION ................................................................................................ 41 2.4 SIGNALLING NETWORKS LINKING INTEGRINS AND RHO GTPASES TO PROMOTE MIGRATION ..................................................................................................................................................... 43 2.5 AIM OF THIS STUDY .................................................................................................................... 46 3 MATERIALS AND METHODS ................................................................................................. 48 3.1 MATERIALS ................................................................................................................................. 48 3.2 ANIMALS...................................................................................................................................... 48 3.3 GENERATION OF GENETICALLY MODIFIED MICE..................................................................... 48 3.3.1 PREPARATION OF FEEDER CELL LINES ...................................................................................... 48 3.3.2 PREPARATION OF DNA FOR ELECTROPORATION INTO EMBRYONIC STEM CELLS..................... 49 3.3.3 ELECTROPORATION OF ES CELLS.............................................................................................. 49 3.3.4 SELECTION, PICKING AND FREEZING OF POSITIVE ES CELL CLONES......................................... 50 3.3.5 PREPARATION OF ES CELLS FOR BLASTOCYST INJECTION........................................................ 50 3.3.6 PRODUCTION OF CHIMERIC ANIMALS........................................................................................ 50 3.3.7 BREEDING SCHEMES.................................................................................................................. 51 4 Table of contents 3.4 WOUNDING AND PREPARATION OF WOUND TISSUE.................................................................. 51 3.5 HISTOLOGICAL METHODS.......................................................................................................... 52 3.5.1 ISOLATION AND EMBEDDING OF MOUSE SKIN ........................................................................... 52 3.5.1.1 Embedding and cutting skin for cryosections ........................................................................ 52 3.5.1.2 Embedding and cutting skin for paraffin sections.................................................................. 52 3.5.2 HAEMATOXYLIN AND EOSIN STAINING..................................................................................... 53 3.5.3 LACZ STAINING......................................................................................................................... 53 3.6 IMMUNOLOGICAL METHODS...................................................................................................... 54 3.6.1 IMMUNOFLUORESCENCE (IF) .................................................................................................... 54 3.6.1.1 Immunofluorescence on skin sections ................................................................................... 54 3.6.1.2 Immunofluorescence on adherent cells.................................................................................. 55 3.6.1.3 Immuonfluorescence on suspension cells .............................................................................. 55 3.6.1.4 Primary antibodies ................................................................................................................. 55 3.6.1.5 Secondary antibodies ............................................................................................................. 56 3.6.2 IMMUNOHISTOCHEMISTRY........................................................................................................ 56 3.6.2.1 BrdU staining ......................................................................................................................... 56 3.6.2.2 Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling (TUNEL) .......... 57 3.6.3 FLUORESCENCE ACTIVATED CELL SORTER (FACS) ................................................................ 57 3.7 CELLS AND CELL CULTURE METHODS ...................................................................................... 58 3.7.1 CELL LINES................................................................................................................................58 3.7.2 CULTURE CONDITIONS AND MEDIA........................................................................................... 58 3.7.2.1 Culturing of fibroblastoid cell lines and Phoenix cells .......................................................... 58 3.7.2.2 Culturing of ES cells and endodermal cells ........................................................................... 59 3.7.2.3 Culturing of primary keratinocytes ........................................................................................ 59 3.7.2.4 Culturing of cells on coverslips ............................................................................................. 60 3.7.3 PASSAGING AND TRYPSINIZING OF CELLS................................................................................. 60 3.7.4 FREEZING OF CELLS................................................................................................................... 60 3.7.5 THAWING OF CELLS................................................................................................................... 61 3.8 BIOCHEMICAL METHODS ........................................................................................................... 61 3.8.1 EXTRACTION OF PROTEINS FROM CELLS ................................................................................... 61 3.8.1.1 Serum induced signalling....................................................................................................... 61 3.8.1.2 Wounding induced signalling ...............................................................................................
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