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Regulation of Keratin Filament Network Dynamics

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Regulation of Keratin Filament Network Dynamics Regulation of keratin filament network dynamics Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom Biologe Marcin Maciej Moch aus Dzierżoniów (früher Reichenbach, NS), Polen Berichter: Universitätsprofessor Dr. med. Rudolf E. Leube Universitätsprofessor Dr. phil. nat. Gabriele Pradel Tag der mündlichen Prüfung: 19. Juni 2015 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. This work was performed at the Institute for Molecular and Cellular Anatomy at University Hospital RWTH Aachen by the mentorship of Prof. Dr. med. Rudolf E. Leube. It was exclusively performed by myself, unless otherwise stated in the text. 1. Reviewer: Univ.-Prof. Dr. med. Rudolf E. Leube 2. Reviewer: Univ.-Prof. Dr. phil. nat. Gabriele Pradel Ulm, 15.02.2015 2 Publications Publications Measuring the regulation of keratin filament network dynamics. Moch M, and Herberich G, Aach T, Leube RE, Windoffer R. 2013. Proc Natl Acad Sci U S A. 110:10664-10669. Intermediate filaments and the regulation of focal adhesion. Leube RE, Moch M, Windoffer R. 2015. Current Opinion in Cell Biology. 32:13–20. "Panta rhei": Perpetual cycling of the keratin cytoskeleton. Leube RE, Moch M, Kölsch A, Windoffer R. 2011. Bioarchitecture. 1:39-44. Intracellular motility of intermediate filaments. Leube RE, Moch M, Windoffer R. Under review in: The Cytoskeleton. Editors: Pollard T., Dutcher S., Goldman R. Cold Springer Harbor Laboratory Press, Cold Spring Harbor. Multidimensional monitoring of keratin filaments in cultured cells and in tissues. Schwarz N, and Moch M, Windoffer R, Leube RE. In preparation for: Methods in Enzymology. Conference presentations Quantification of keratin network dynamics from confocal microscopy images (20 minutes presentation). Moch M, Herberich G, Aach T, Windoffer R, Leube RE. 2013. Focus on Microscopy. Maastricht, Netherlands. Regulation of keratin network dynamics by epidermal growth factor and focal adhesion kinase (15 minutes presentation). Moch M, Herberich G, Aach T, Windoffer R, Leube RE. 2012. 35th Annual Meeting of the German Society for Cell Biology. Dresden, Germany. Computational analysis of keratin network dynamics in response to epidermal growth factor stimulation and plectin downregulation (15 minutes presentation). Moch M, Herberich G, Aach T, Windoffer R, Leube RE. 2013. 55th Symposium of the Society for Histochemistry. Prague, Czech Republic. Quantification of live keratin network dynamics (15 minutes presentation). Moch M, Herberich G, Aach T, Windoffer R, Leube RE. 2012. Physics of Cancer. Leipzig, Germany. 3 Publications Posters Computational analysis of keratin network dynamics. Moch M, Herberich G, Schwarz N, Windoffer R, and Leube RE. Seeing is believing Imaging the Processes of Life. 2013. EMBL Heidelberg. Germany. Downregulation of plectin does not affect keratin network organization, inward-directed motion of keratin filaments and keratin network turnover in A431 cells. Moch M, Herberich G, Schwarz N, Windoffer R, and Leube RE. 8th European Conference on Intermediate Filaments in Health and Disease. 2013. Amsterdam. The Netherlands. Computational analysis of keratin network dynamics. Moch M, Herberich G, Aach T, Windoffer R, and Leube RE. 2012 Annual Meeting of the American Society for Cell Biology. San Francisco. USA. Imaging of EGF- and adhesion-dependent keratin filament dynamics. Moch M, Windoffer R, and Leube RE. Seeing is believing Imaging the Processes of Life. 2011. EMBL Heidelberg. Germany. Regulation of keratin network dynamics by extra cellular matrix contacts. Moch M, Schwarz N, Windoffer R, and Leube RE. 34th Annual Meeting of the German Society for Cell Biology. 2011. Bonn, Germany. Focal adhesion kinase-dependent keratin filament dynamics. Moch M, Kölsch A, Windoffer R, and Leube RE. 11th DGZ Young Scientist Meeting Imaging Cell Migration. 2009. Martiensried. Germany. Grants The company of biologists travel grant for 8th European Conference on Intermediate filaments in Health and Disease. 2013. Amsterdam, The Netherlands. COST Nanonet travel grant for 55th Symposium of the Society for Histochemistry. 2013. Prague, Czech Republic. Travel grant of the German Academic Exchange Service for Annual Meeting of the American Society for Cell Biology. 2012. San Francisco, CA, USA. 4 Table of contents Table of contents Abstract ....................................................................................................................................................... 10 1. Introduction ............................................................................................................................................. 12 Cytoskeleton ............................................................................................................................................ 12 1.1. Actin ................................................................................................................................................. 13 1.2. Microtubules..................................................................................................................................... 15 1.3. Intermediate filaments ...................................................................................................................... 16 1.3.1. Structure .................................................................................................................................... 16 1.3.2. Localisation ............................................................................................................................... 17 1.3.3. Transcription and translation ..................................................................................................... 17 1.3.4. Dynamics ................................................................................................................................... 18 1.3.5. Function and interaction with other proteins ............................................................................. 20 1.4. Extracellular matrix adhesion complexes ......................................................................................... 22 1.4.1. Focal adhesions ......................................................................................................................... 22 1.4.2. Hemidesmosomes ...................................................................................................................... 23 1.5. Epidermal growth factor signalling .................................................................................................. 27 1.5.1. Epidermal growth factor (EGF) ................................................................................................ 27 1.5.2. Epidermal growth factor receptor (EGFR) ................................................................................ 28 1.6. Src and FAK signalling .................................................................................................................... 30 1.6.1. Focal adhesion kinase (FAK) mediated signalling .................................................................... 30 1.6.2. Src.............................................................................................................................................. 31 1.7. Aim of the thesis .............................................................................................................................. 32 2. Materials and methods ............................................................................................................................ 33 2.1. Materials ........................................................................................................................................... 33 2.1.1. Chemicals .................................................................................................................................. 33 2.1.2. Molecular ladders ...................................................................................................................... 34 2.1.3. Kits ............................................................................................................................................ 35 2.1.4. Enzymes .................................................................................................................................... 35 2.1.5. Inhibitors and growth factors .................................................................................................... 36 2.1.6. Cell culture media, matrix proteins, and antibiotics .................................................................. 36 2.1.7. Antibodies ................................................................................................................................. 37 2.1.8. Labware ..................................................................................................................................... 39 2.1.9. Laboratory equipment ............................................................................................................... 40 2.1.10. Microscopes ............................................................................................................................ 42 5 Table of contents 2.1.11. Bacteria strains ........................................................................................................................ 44 2.1.12. Primers ...................................................................................................................................
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