Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom. in Botany Anastasia Eskova born in: Moscow, Russia Oral examination: 07 June 2013 Systematic analysis of integrin α2β1 internalization Referees: Prof. Dr. Roland Eils Dr. Vytaute Starkuviene-Erfle Contents List of publications .............................................................................................................. 5 Acknowledgements .............................................................................................................. 6 Abbreviations ....................................................................................................................... 7 Summary .............................................................................................................................. 8 Zusammenfassung .............................................................................................................. 10 1. Introduction .................................................................................................................... 13 1.1. Integrins .................................................................................................................. 13 1.2. Integrin structure and function ............................................................................... 14 1.3. Intracellular trafficking pathways .......................................................................... 19 1.4. Cytoskeleton in membrane trafficking ................................................................... 25 1.5. Integrin trafficking ................................................................................................. 30 1.5.1. Early steps of integrin endocytosis ................................................................. 34 1.5.2. Integrin recycling............................................................................................ 36 1.5.3. Integrin degradation........................................................................................ 37 1.5.4. Cytoskeleton in integrin trafficking ................................................................ 38 1.6. Integrin α2β1 .......................................................................................................... 40 1.7. Integrin trafficking in disease ................................................................................. 42 2. Objectives ...................................................................................................................... 44 3. Materials and Methods ................................................................................................... 46 3.1 Materials, antibodies, plasmids, reagents ................................................................ 46 3.2 Cell culture .............................................................................................................. 46 3.3 Transfection with siRNA and cDNA ....................................................................... 47 3.4 Western Blotting ...................................................................................................... 48 3.5 RT-PCR ................................................................................................................... 49 3.6. Non-clustered and clustered α2 integrin internalization assay ............................... 49 3.7. Non-clustered integrin α2 internalization on CYTOOchips .................................. 51 3.8. EGF and Transferrin endocytosis assays ................................................................ 51 3.9. Microscopy ............................................................................................................. 52 3.10. Image analysis and statistical data analysis for small-scale experiments ............ 53 3.11. Image analysis and statistical data analysis for RNAi screening on cell arrays ... 54 3.12. Co-localization analysis ....................................................................................... 56 2 3.13. Bioinformatic analysis of screening results ......................................................... 57 4. Results ............................................................................................................................ 58 4.1. Fluorescent-microscopy-based α2 integrin internalization assay .......................... 58 4.1.1. Quantification of microscopy-based integrin internalization assay ............... 61 4.2. Clustered and non-clustered integrin α2β1 pursues different internalization routes ....................................................................................................................................... 64 4.2.1. Non-clustered α2 integrin traffics through Rab5-Rab4-Rab11 positive pathway..................................................................................................................... 64 4.1.2. Endocytosis of non-clustered α2 integrin depends on clathrin and caveolin . 67 4.3. Fluorescent microscopy-based RNAi screen identifies potential regulators of non- clustered α2 integrin endocytic trafficking ................................................................... 68 4.3.1. Bioinformatic analysis of the primary screening results ................................ 71 4.3.2. Validation ........................................................................................................ 75 4.3.3. Expression of α2 integrin is affected by knockdown of number of hits ......... 78 4.3.4. Endocytosis and expression of α2 integrin does not depend on local cell densities .................................................................................................................... 79 4.4. KIF15 is a novel regulator of membrane trafficking .............................................. 81 4.4.1. KIF15 specifically regulates α2 integrin internalization ................................ 81 4.4. Integrin endocytosis on micropatterned substrates ................................................ 91 5. Discussion ...................................................................................................................... 93 5.1. Development of fluorescent-microscopy-based assay for analysis of non-clustered α2 integrin internalization ............................................................................................. 93 5.2. Internalization of non-clustered α2β1 requires clathrin and caveolin .................... 96 5.3. Screening of cytoskeleton-associated proteins for involvement in integrin endocytosis .................................................................................................................... 97 5.4. Validation of the primary screening results .......................................................... 100 5.5. KIF15 is a novel regulator of membrane trafficking ............................................ 104 5.6. Integrin α2 internalization of micropatterned surfaces ........................................ 108 6. Conclusions and outlook .............................................................................................. 110 7. Collaborations .............................................................................................................. 112 8. References .................................................................................................................... 113 Appendix .......................................................................................................................... 132 3 Appendix I. List of genes tested in the primary screening for regulators of integrin α2 internalization .............................................................................................................. 132 Appendix II. Results of the primary screening for regulators of integrin α2 internalization for individual tested siRNAs ............................................................... 156 Appendix III. Expression of kinesins in HeLa cells ................................................... 217 Appendix IV. Results of the validation experiments ................................................... 219 4 List of publications Eskova A., B. Knapp, D. Matelska, T. Lisauskas, R. Pepperkok, R. Russell, R. Eils, L. Kaderali, H. Erfle, V. Starkuviene. KIF15 emerges as a novel regulator of integrin endocytic trafficking (manuscript submitted) Erfle, H., A. Eskova, J. Reymann, V. Starkuviene. Cell arrays and high content screening. 2011. Methods of Molecular Biology, 785, 277-87. List of presentations 2011. "Dynamic Endosomes: Mechanisms Controlling Endocytosis" (24-29 September 2011, Chania, Grece) Poster presentation. Eskova A., B. Knapp, D. Matelska, H. Demirdizen, H. Erfle, Eils, R. Pepperkok, L. Kaderali, R. Russell, R. V. Starkuviene. High- content analysis of kinesins as regulators of integrin traffic. 2010. "International Conference on Systems Biology of Human Disease 2010" (16-18 June 2010, Boston, USA) Poster presentation. Eskova A., B. Knapp, D. Matelska, H. Demirdizen, H. Erfle, Eils, R. Pepperkok, L. Kaderali, R. Russell, R. V. Starkuviene. High- content analysis of kinesins as regulators of integrin traffic. 2010. 8th EMBO-Annaberg workshop “Protein and Lipid function in secretion and endocytosis”, Goldegg am See, Austria. Poster presentation. Eskova A., Knapp B., Erfle H., Kaderali L., Pepperkok R., Starkuviene V. Microscopy-based RNAi assay to identify regulators of