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  • EINBLICKE IN DIE MOLEKULAREN MECHANISMEN ZUR REGULIERUNG DER

    AKTIVITÄT VON MULTIDOMAIN-PROTEINEN IN LEBENDEN ZELLEN MIT HILFE VON FRET-FLIM UNTERSUCHUNGEN

    INSIGHTS INTO MOLECULAR MECHANISMS REGULATING THE ACTIVITY OF MULTIDOMAIN PROTEINS IN LIVING CELLS USING FRET-FLIM

    Dissertation

    zur Erlangung des akademischen Grades

    doctor rerum naturalium

    (Dr. rer. nat.)

    genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg

    Von Master of Science in Physics Deepak Kumaran Nair

    geb. am 10.03.1980 in Mararikulam, Kerala, India

    Gutachter: Prof. Dr. Stephan Diekmann Privatdozent Dr. Reinhard König

    eingereicht am 01. Oktober 2007

    verteidigt 19. März 2008

  • ii

    Dedicated to my family and teachers for their love and support, which have encouraged and motivated me to achieve what I have…

  • iii

    ACKNOWLEDGEMENTS

    I thank my wife Mini, whose love and support has inspired me in research and life. I thank my daughter Naina, who along with my wife has silently suffered all the hardships in the last few months.

    I sincerely thank Prof. Eckart Gundelfinger for motivating me to learn and appreciate cell biology and I will always be grateful for his help and consideration shown towards the successful completion of my work. I thank Dr. Werner Zuschratter and Dr. Roland Hartig for giving me the opportunity to work in their laboratories. I thank them for their belief in me, by giving me the opportunity to work in both biology and physics. I thank Prof. Burkhart Schraven and Dr. Reinhard König for their encouragement and opinions.

    One of the special people I would like to thank is Kathrin; without whose timely and sincere effort I would not have completed many experiments in time.

    I am grateful to Prof. Thomas Kuner, Prof. Athar Chishti, Prof. Hannes Stockinger, Prof. Philip Beesley Dr. Michael Kreutz, Dr. Toshihiko Hanada and Dr. Karl-Heinz Smalla for providing the constructs used in my work.

    I am very indebted to Dr. Ronald Steffen, a friend, a co-worker and a very patient scientist who taught me to appreciate the complexities of photophysical processes. I am extremely thankful to Dr. Ulrich Thomas who spent a lot of his time to make me understand various aspects of molecular biology and protein biochemistry.

    I am very thankful to Moni, Heidi, and Ilona who always found time to help me in my need. I also thank Ela, Roser and Falco who had spent their valuable time to make me a better cell biologist. I am grateful to the assistance from mechanical and electrical workshops to make the work more comfortable.

    I would like to thank the members of the Neurochemistry Department at IFN and Institute of Immunology, Magdeburg for their valuable suggestions and help in improving my work.

    This list will not be complete without mentioning many people whom I cannot name but only thank for their love, support, and concern.

    I would also like to thank Deutsche Forshungsgemeinshaft for funding me through the project FOR-521-HA 3498/1.

    I would like to thank my teachers who have moulded me into what I am and I hope that I have held their esteem with this humble effort. I thank my parents and my family who taught me to work hard in whatever I did. I thank them for their love, prayers, and the encouragement they gave whenever I expressed my interest for higher studies. Finally, I thank god for giving me good teachers and a loving family.

    01-10-2007 Deepak Nair

  • TABLE OF CONTENTS

    iv

    TABLE OF CONTENTS SUMMARY .......................................................................................................... 1 1 INTRODUCTION.............................................................................................. 2

    1.1 Immune system.............................................................................................................................. 2 1.2 Adaptive immune response............................................................................................................ 2 1.3 T cells and B cells.......................................................................................................................... 3 1.4 Antigen presenting cells................................................................................................................. 3 1.5 Immunological synapse:-formation and molecular organisation................................................... 4 1.6 Src kinases: structure and function ................................................................................................ 7

    1.7 Discs Large family of proteins and the generation of modular scaffolds ................................... 10

    1.8 Aims............................................................................................................................................. 12

    2 THEORETICAL FOUNDATIONS AND INSTRUMENTATION................ 17 2.1 Fluorescence of organic molecules.............................................................................................. 17 2.2 Theory of spectral separation....................................................................................................... 19 2.3 Fast excited state reactions .......................................................................................................... 22 2.4 FRET............................................................................................................................................ 24 2.5 Fluorescence Lifetime Imaging Microscopy to probe FRET ...................................................... 26 2.6 FLIM-FLMS................................................................................................................................ 27

    2.7 Fluorescence tags to image macromolecular dynamics............................................................... 35 2.8 Photophysics of GFP based FRET............................................................................................... 36

    3 MATERIALS AND METHODS..................................................................... 37 3.1 Materials ...................................................................................................................................... 37

    3.2 Methods ....................................................................................................................................... 39

    4 RESULTS......................................................................................................... 45 4.1 Photophysics of FRET between CFP and YFP in living cells..................................................... 45

    4.2 Activity-dependent conformational changes of Lck in living cells ............................................. 53

    1.6.1 Lck ......................................................................................................................................................... 9

    1.7.1 SAP97/hDlg ......................................................................................................................................... 11

    1.8.1 Real-time conformational changes of Lck ........................................................................................... 13 1.8.2 Calicum-dependent conformational changes of SAP97 and PSD95.................................................... 14 1.8.3 Role of Lck-SAP97 association in synaptic stabilisation..................................................................... 16

    2.6.1 Time and Space Correlated Single Photon Counting (TSCSPC) ......................................................... 27 2.6.2 Detectors .............................................................................................................................................. 27 2.6.3 Instrumentation .................................................................................................................................... 28 2.6.4 Steady state imaging............................................................................................................................. 30 2.6.5 Calibration of the setup ........................................................................................................................ 30 2.6.6 Data analysis ........................................................................................................................................ 32

    3.1.1 Chemicals............................................................................................................................................. 37 3.1.2 Bacteria and mammalian cell culture media and antibiotics ................................................................ 37 3.1.3 Buffers.................................................................................................................................................. 37 3.1.4 Cell strains............................................................................................................................................ 37 3.1.5 Antibodies ............................................................................................................................................ 38 3.1.6 GFP fusion constructs .........