Einsatz von Fret-Flim zur Beobachtung von Protein-Protein_Wechselwirkungen in lebenden Zellen: Einblicke in Interaktionen des präsynaptischen Proteins Bassoon [Elektronische Ressource] = Investigating protein-protein interactions by fret-flim in living cells: mediated by the presynaptic proteins bassoon / von Mini Jose

Mjose

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

EINSATZ VON FRET-FLIM ZUR BEOBACHTUNG VON PROTEIN-
PROTEIN WECHSELWIRKUNGEN IN LEBENDEN ZELLEN:
EINBLICKE IN INTERAKTIONEN DES PRÄSYNAPTISCHEN
PROTEINS BASSOON

INVESTIGATING PROTEIN-PROTEIN INTERACTIONS BY FRET-
FLIM IN LIVING CELLS: INSIGHTS INTO INTERACTIONS
MEDIATED BY THE PRESYNAPTIC PROTEIN BASSOON


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 Mini Jose

geb. am 26.12.1979 in Kanjirapally, Kerala, India




Gutachter: Prof. Dr. Eckart D. Gundelfinger
Prof. Dr. Fred S. Wouters




eingereicht am 24. April 2007

verteidigt am 25. October 2007





















Dedicated to my loving father, late Prof. Dr. P.K. Jose, Director, School
of Distance Education, Mahatma Gandhi University, Kerala, India without
whose support, I would never have achieved what I have.
















ACKNOWLEDGEMENTS

First of all, I thank my dear friend, husband and colleague Deepak who has always been there
to support me during the hardest of times and for all his help in preparing this work. I thank
my dearest daughter Naina who has tolerated all the hardships with me during the last months
of work. I sincerely thank Dr. Werner Zuschratter for giving me the opportunity to work in his
lab and for the support he has given during the years. I am grateful to Prof. Eckart D.
Gundelfinger for his encouragement, time and constant support he has rendered during my
work. I thank Dr. Ronald Steffen, who was a sincere friend, for all his support and help. I am
grateful to Dr. Wilko Altrock, Dr. Thomas Dresbach, Prof. David Piston, Prof. Roger Y.
Tsien, Prof. Philip W. Beesley and Prof. Thomas Kuner for providing the different fusion
constructs for my work. I especially thank Dr. Ulrich Thomas for his eminent support for the
cloning works. I also thank Dr. Karl-Heinz Smalla for his encouragement and help with
biochemical techniques. I am grateful to Carsten Reissner for all the computational modelling
works. I thank Dr. Klaus Kemnitz and Dr. Roland Hartig for helpful discussions. One of the
very special persons I should thank is Kathrin, without whose help, I would have found it
difficult to finish all the work. I am grateful to Moni, Heidi, Ilona and Corrina for their
excellent technical help, which has helped me a lot during my work. I acknowledge the
members of the Dept. of Neurochemistry/Molecular Biology especially Roser and Ela whose
help and support I should ever remember. I thank the non-scientific staff of IfN including the
mechanical, electronic workshops and the house technicians for their constant help. I
acknowledge the Deutsche Forschungsgemeinschaft for the support through the grant SPP
1128 ZU59/5-1/2 and Leibniz Institute for Neurobiology for funding my work. I thank all
those whom I cannot name, but would be grateful to, who have helped to make the best of the
situation around me to feel homely. I thank my loving mother, brothers, sisters and all
members of my family who have always been there for me throughout my life. Above all, I
thank God for making everything possible.



th24 April 2007, Magdeburg
Min Jose
TABLE OF CONTENTS
TABLE OF CONTENTS
SUMMARY .......................................................................................................... 1
1 PROLOGUE....................................................................................................... 2
1.1 THE NERVOUS SYSTEM ............................................................................................. 2
1.1.1 Neuronal differentiation and development of neuronal connectivity........................ 2
1.1.2 Developmental shift of intracellular chloride in neurons.......................................... 3
1.2 THE SYNAPSE ............................................................................................................... 4
1.3 MOLECULAR ORGANISATION OF THE CAZ.......................................................... 5
1.3.1 Piccolo and Bassoon.................................................................................................. 6
1.3.2 ELKS/CAST/ERC proteins....................................................................................... 9
1.3.3 CtBP family of proteins........................................................................................... 10
1.4 AIMS OF THE STUDY................................................................................................. 10
1.4.1 Studying intracellular chloride changes during neuronal development .................. 11
1.4.2 Studying interactions of synaptic proteins in vivo.................................................. 11
2 THEORY AND INSTRUMENTATION......................................................... 13
2.1 THEORY........................................................................................................................ 13
2.1.1 Fluorescence............................................................................................................ 13
2.1.2 Fluorescence Lifetime Imaging Microscopy (FLIM) ............................................. 14
2.1.2.1 Time-domain FLIM.......................................................................................... 16
2.1.2.2 Frequency-domain FLIM ................................................................................. 17
2.1.3 Förster’s Resonance Energy transfer (FRET) 17
2.1.3.1 Time-resolved FRET........................................................................................ 17
2.1.3.2 Decay Associated Spectra (DAS) .................................................................... 20
2.1.3.3 Intensity-based FRET....................................................................................... 22
2.1.4 FRET-FLIM: an optimal tool for monitoring interactions in living cells............... 23
2.2 INSTRUMENTATION.................................................................................................. 24
2.2.1 Time correlated single photon counting.................................................................. 24
2.2.2 Experimental set up for FRET-FLIM...................................................................... 25
2.2.2.1 Laser system..................................................................................................... 25
2.2.2.2 Optical system 25
2.2.2.3 Electronics........................................................................................................ 27
2.2.3 Calibration of the set up .......................................................................................... 29
2.2.4 Data analysis ........................................................................................................... 30
3 MATERIALS AND METHODS..................................................................... 32
3.1 MATERIALS................................................................................................................. 32
3.1.1 Media and supplements for different cell types including Bacteria, COS-7, and
hippocampal neurons 32
3.1.2 Buffers..................................................................................................................... 32
3.1.3 Iontophoretic drugs ................................................................................................. 33
3.1.4 Cell strains............................................................................................................... 33
3.1.5 Animals. 33
3.1.6 Antibodies ............................................................................................................... 33
3.1.7 Expression constructs.............................................................................................. 34
3.1.8 Primers.. 34
3.2 METHODS..................................................................................................................... 35
3.2.1 Molecular Biological Techniques ........................................................................... 35
3.2.1.1 Transformation of electrocompetent cells........................................................ 35
3.2.1.2 Transformation of heat-shock competent cells ................................................ 35
3.2.1.3 Preparation of plasmid DNA from E. coli 35 TABLE OF CONTENTS
3.2.1.4 Restriction reactions of plasmid DNA ............................................................. 35
3.2.1.5 Agarose gel electrophoresis ............................................................................. 36
3.2.1.6 Polymerase chain reaction (PCR) .................................................................... 36
3.2.1.7 Site-directed mutagenesis................................................................................. 36
3.2.1.8 Cloning of DNA fragments into vectors ......................