Einblicke in die molekularen Mechanismen zur Regulierung der Aktivität von Multidomain-Proteinen in lebenden Zellen mit Hilfe von FRET-FLIM-Untersuchungen [Elektronische Ressource] = Insights into molecular mechanisms regulating the activity of multidomain proteins in living cells using FRET-FLIM / von Deepak Kumaran Nair

Mjose

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Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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














Dedicated to my family and teachers for their love and
support, which have encouraged and motivated me to
achieve what I have…
ii
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
iii TABLE OF CONTENTS
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
1.6.1 Lck ......................................................................................................................................................... 9
1.7 Discs Large family of proteins and the generation of modular scaffolds ................................... 10
1.7.1 SAP97/hDlg ......................................................................................................................................... 11
1.8 Aims............................................................................................................................................. 12
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 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
2.6.1 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
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
3.1.1 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 .......................................................................................................................... 38
3.1.7 Primers 39
3.1.8 Animals.............................. 39
3.2 Methods ........