Dual function of LIMK2 in endothelial cells [Elektronische Ressource] / vorgelegt von Pankaj Goyal

Pankaj Goyal

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten
der Ludwig-Maximilians-Universität München
Vorstand: Prof. Dr. med. P. C. Weber






Dual function of LIMK2 in endothelial cells




Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München



vorgelegt von
Pankaj Goyal


aus
Agra, Indien

2005

Mit Genehmigung der Medizinischen Fakultät
der Universität München






1. Berichterstatter: Prof. Dr. med. Wolfgang Siess
2. BerichtM. Schleicher

Mitberichterstatter: Priv. Doz. Dr. R. Wienecke
Prof. Dr. W. M. Franz

Dekan: Prof. Dr. med. D. Reinhardt

Tag der mündlichen Prüfung: 13.07.2005







Table of contents i
Table of contents
Table of contents ...............................................................................................................................i
Abbreviations and units..................................................................................................................vii
1. Introduction ..................................................................................................................................1
1.1. Physiological properties of the endothelium.........................................................................1
1.2. Pathophysiological activation of endothelium ......................................................................2
1.3. Endothelial dysfunction and cytoskeleton.............................................................................3
1.3.1. Actin cytoskeleton..........................................................................................................4
1.3.2. Actin filament-turnover..................................................................................................4
1.3.3. Cellular organization of the actin cytoskeleton..............................................................5
1.3.4. Regulation of actin dynamics by actin binding proteins ................................................7
1.3.5. ADF/Cofilin Family.8
1.3.6. Rho GTPases and actin dynamics ................................................................................11
1.3.7. Rho-GTPases activated protein Kinases ......................................................................12
1.4. LIM-kinase ..........................................................................................................................14
1.4.1. Structure of LIM-Kinases.............................................................................................14
1.4.1.1. LIM domain...........................................................................................................15
1.4.1.2. PDZ domain ..........................................................................................................17
1.4.1.3. Kinase domain.......................................................................................................19
1.4.2. Gene expression and subcellular localization of LIM-kinases.....................................21
1.4.3. Regulation of LIMK activation ....................................................................................21
1.5. Regulation of protein transport between nucleus and cytoplasm........................................22
1.6. Cellular functions of LIMK-kinases24
2. Aim of the study.........................................................................................................................26
3. Materials and methods................................................................................................................27
3.1. General equipments.............................................................................................................27
3.2. Materials..............................................................................................................................28
3.2.1. Chemicals .....................................................................................................................28
3.2.2. Enzymes and reagents for molecular biology ..............................................................29
ii Table of contents
3.2.3. Antibodies.................................................................................................................... 29
3.2.3.1. Primary antibodies ................................................................................................ 29
3.2.3.2. Secondary antibodies ............................................................................................ 30
3.2.3.3. IgG isotype controls and sera................................................................................ 30
3.2.4. Inhibitors........ 30
3.2.5. Ligands......................................................................................................................... 30
3.2.6. Commercial Kits and other materials .......................................................................... 30
3.2.7. Primers........ 31
3.2.7.1. For preparation of cDNA inserts........................................................................... 31
3.2.7.2. For deletion mutation............................................................................................ 31
3.2.7.3. For site directed mutagenesis................................................................................ 32
3.2.7.4. Primers for gene expression analysis.................................................................... 33
3.2.8. Plasmids ....................................................................................................................... 33
3.2.8.1. Original plasmids.................................................................................................. 33
3.2.8.2. Plasmid with insert................................................................................................ 33
3.2.8.3. Plasmid with mutated insert.................................................................................. 34
3.3. Work with E.coli................................................................................................................. 36
3.3.1. Bacterial strains............................................................................................................ 36
3.3.2. Media for bacterial culture........................................................................................... 36
3.3.3. General........ 36
3.3.4. Culturing bacteria......................................................................................................... 37
3.3.4.1. Growth on solid media.......................................................................................... 37
3.3.4.2. Growth of liquid cultures...................................................................................... 37
3.3.4.3. Monitoring the bacterial growth ........................................................................... 37
3.3.5. Transformation of plasmid in E.coli ............................................................................ 37
3.3.5.1. Preparation of competent cells by CaCl method ................................................. 37 2
3.3.5.2. Heat shock transformation of the plasmid ............................................................ 37
3.4. Recombinant DNA processing and manipulation............................................................... 38
3.4.1. DNA amplification of LIMK1 and LIMK2 cDNA...................................................... 38
Table of contents iii
3.4.2. Buffers and solutions....................................................................................................39
3.4.3. Restriction endonuclease digestion of DNA ................................................................39
3.4.4. Purification of the digested DNA.................................................................................40
3.4.5. Ethanol precipitation of DNA ......................................................................................40
3.4.6. Dephosphorylation of linearized plasmid DNA by CIP...............................................40
3.4.7. Ligation of DNA fragments .........................................................................................41
3.4.8. Miniprep: small-scale preparation of plasmid DNA ....................................................41
3.4.9. Endofree Maxiprep: Large scale preparation of plasmid DNA ...................................41
3.4.10. Quantification of DNA and RNA solutions ...............................................................42
3.4.11. Agarose gel electrophoresis........................................................................................42
3.4.12. DNA recovery from agarose gel ................................................................................43
3.4.13. DNA sequencing ........................................................................................................43
3.5. Mutagenesis of the LIMK2 gene.........................................................................................43
3.5.1. PCR based site-directed mutagenesis....................