La lecture à portée de main
156
pages
English
Documents
2010
Écrit par
Phoopma
Publié par
georg-august-universitat_gottingen
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156
pages
English
Ebook
2010
Le téléchargement nécessite un accès à la bibliothèque YouScribe Tout savoir sur nos offres
Publié par
Publié le
01 janvier 2010
Nombre de lectures
34
Langue
English
Poids de l'ouvrage
13 Mo
Publié par
Publié le
01 janvier 2010
Nombre de lectures
34
Langue
English
Poids de l'ouvrage
13 Mo
INVESTIGATION OF ENDOSOMAL RECYCLING
OF SYNAPTIC VESICLES
Dissertation
in partial fulfilmlent of the requirements for the de gree
“Doctor of Natural Sciences (Dr. rer. nat. )”
in the Molecular Biology Program a t the
Georg August University Göttingen,l tFya cou f Biolo gy
submitted by
Peer Hoopmann
born i n
Thedinghausen, German y
September 2010
I hereby declare that I prepareddi stsehret ation enti t“lIednvestigation of Endosomal
Recycling of Synaptic Vesicl eos”n my own and with no other sources and aids than
quoted.
Peer Hoopman n
LIST OF PUBLICATIONS
Hoopmann P, Punge A, Barysch SV, Westphal V, Bückers OJp,a zo F, Bethani I,
Lauterbach MA, Hell SW, Rizzo.l iE SnOdosomal sorting roefa dliy releasable synaptic
vesicles. Proc Natl Acad Sci U S Ain. press
Hoopmann P, Rizzoli SO, Betz. I WmJaging synaptic vesicle dynamics with FM ydes.
Optical Imaging in Neuroiescnce (Book chapter)i.n press
Opazo F, Punge A,ck Büers J,H oopmann P, Kastrup L, Hell SW, Rizzoli SO (2010)
Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling. Traffic
11:800‐812.
Geumann U, Barysch SV,Ho opmann P, Jahn R, Rizzoli SO (2008) SNARE function is not
involved in early ednosome docking. Mol Biol Cell 19:53‐25337.7
I
TABLE OF CONTENTS
List of Publications________________________________ ___________________________ I
Table of Contents II
Acknowledgements ________________________________ _________________________ IV
Abstract ________________________________ ________________________________ __ V
List of Figures ______________________________ VI
List of Tables________________________________ VI I
List of Abbreviations ________________________________ ______________________ VII I
1. Introduction __________________________ 10
1.1 Pre‐Synaptic Functio________________________________n ________________________________ _10
1.2 Endosomal Trafficking _20
1.3 Endosomes and Synaptic Vesicles ____________________ 26
1.4 Aim of the Project ______ 31
2. Materials and Methods _________________ 32
2.1 Antibodie________________________________s ________________________________ ______________ 32
2.2 Chemicals and Reagents _______________________________ 34
2.3 Microscopy _____________ 35
2.4 Neuronal Cell Culture __38
2.5 Preparation of Lumenally Labeled Synaptic Vesicles 38
2.6 PHluorin Expression and Imagi________________________________ng ____________________ 40
2.7 Phot‐oxidation Electron Microscopy ________________ 43
2.8 Quantum Dot Imaging and Analysis __________________ 45
2.9 Rab5‐GFP Expression and Imaging ___________________ 46
2.10 Blocking Endosome Function with Soluble Syntaxin 13 Frag_______________ment 49
2.11 SynaptiVc esicle Marker Surface Immunostainin________________________________g _51
2.12 Isolated Synaptic Vesicle Immunoassay ____________ 52
2.13 Extraction of Cholesterol from Endosomes _________ 54
3. Results________________________________ _______________________________ 55
3.1 Endosomal SNAREs Participate in Synaptic Vesicle Recyc_____________________ling 55
3.2 Morphological Evidence for Endosomal Fusion of RRP Ve___________________sicles 59
II 3.3 Endocytosed Vesicle Protein ReacBhoinag F ide Endosomes_____________________ 65
3.4 Effects of Inhibiting Endosomal Func________________________________tion ____________ 70
3.5 The Recycling‐Step Necessitating Endosomal Sor______________________________ting 75
3.6 How Can Endosomes Sort Synaptic Vesicle Comnpeonts?__________________________ 83
4. Discussion ________________________________ ____________________________ 84
4. 1 Exchange of Molecules during Vesicle Recycling ____84
4. 2 Mechanisms of Endosomal Sorting of Synaptic Ve____________________________sicles 87
4. 3 Synaptic Vesicle Pools and Endosomal Recycl________________________________ing ___89
4. 4 Perspectives for Synaptic Vesicle Recycling _________ 90
5. Summary and Conclusions________________________________ _______________ 93
5.1 Do Endosomal Proteins CycBle tween Internal Organelles and the Plasma
Membrane upon Stimulation? _______________________________ 94
5.2 Do Synaptic Vesicles Fuse with Endosomes During Recyclin____________________g? 95
5.3 Does Inhibition of Endosomal Function Have an Ef fAecnty oonf the Synaptic
Vesicle Pools?________________________________ ________________________________ _________________ 95
5.4 Do Vesicle and Plasma Membrane Components Intermix during Synaptic Vesicle
Recycling? _____________________ 96
5.5 How Could Endosomal Sorting Maintain Synaptic Vesicle I?d_______________entity 96
5.6 Open Questions _________ 97
Bibliography ________________________________ ______________________________ 99
A. Appendix ___________________________ 109
1. Matlab Routines for pHluorin Analy________________________________sis _______________ 109
2. Matlab Routines foirn vitro Immunoassay Analysis _116
Curriculum Vitae ________________________________ _________________________ 150
III
ACKNOWLEDGEMENTS
First and foremost I watont t hank my supervisor Dr.S ilvio Rizzoli form neroetly being
a supervisor – you are a true mento. Trhank you for teaching me how to “dsmoar t
science” and how to be criticTahla.n k you for yuor endless support, patience and adv. ice
Thank you fort he time you spared for all those instances of “jusmti na ute” and “can I
ask you something ”and thank you for sharing your thoughts and Ij oamk esd.e eply
indebted to y oanud I truly hope you will give me a chance to re tthuer nfavor.
I want to thaPnrk ofessor Reinhard Jahn and Professor Nils Broseb efoinrg on
my PhD committee. Thank you forsh aringy our scientifice xperiencea nd for giving
helpful and critical advic e.
I want to thank all the collaborwahtoo aris ded in performin gexperiments