Molecular dynamics of the neuronal Ca_1hn2_1hn+ -binding proteins Caldendrin and Calneurons [Elektronische Ressource] / vorgelegt von Marina Gennadievna Mikhaylova

Marina Marcout

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 107
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

2+Molecular dynamics of the neuronal Ca -binding proteins
Caldendrin and Calneurons


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 Diplom-Biologin Marina Gennadievna Mikhaylova
geb. am 1 März 1981 in Ufa, Russland

Gutachter:
Prof. Dr. E.D. Gundelfinger


am: 30.07.2009






vorgelegt von: Diplom-Biologin Marina Gennadievna Mikhaylova Acknowledgements


This thesis is the account of almost four years of devoted work at the Leibniz Institute for
Neurobiology Magdeburg in the group of Michael R. Kreutz (Project Group Neuroplasticity)
which would not have been possible without the help of many friends and colleagues.

First of all, I would like to thank Michael for being a great advisor. His ideas and
tremendous support had a major influence on this thesis.

I want to thank Anna Karpova for being my friend and colleague for many years. I enjoyed
to do research together with her as well as the nice discussions we daily had.

Many thanks also to Prof. Eckart D Gundelfinger for his encouraging discussions and help
with corrections of the papers.

My thanks to Thomas Munsch, Peter Landgraf, Karl-Heinz Smalla, Ulrich Thomas, Oliver
Kobler, Yogendra Sharma and Thomas Behnisch for the great collaboration over the years.
It was a pleasure to work with all these people and to benefit from their knowledge.

My thanks to Paramesh Pasham Reddy, Anne-Christin Lehman, Johannes Hradsky and
Philipp Bethge for their collaboration, support and friendship while doing their Ph.D studies,
diploma theses or internships in our group.

My thanks to my Nplast colleagues Christina Spilker, Sujoy Bera, Jale Sahin, Rahul Kashuk,
and Vivian Dambeck for the great time I had with them in our group.

I am particularly grateful to our technicians Corinna Borutzki, Stefanie Hochmuth and
Monika Marunde for preparing primary neuronal culture and also taking care of our
everyday lab work.

I would like to thank P. Aravind for reviewing my thesis and giving valuable suggestions on
my future projects.

Thanks a lot to people from the Department of Neurochemistry and Molecular Biology at
the IfN for providing a “training ground” and for the discussions we had on the seminars.

And at the end I would like to thank my family and my friends in Russia for the emotional
support they gave me over these years.
2 Erklärung

Hiermit erkläre ich, dass ich die von mir eingereichte Dissertation zum dem Thema
2+”Molecular dynamics of the neuronal Ca -binding proteins Caldendrin and
Calneurons” selbständig verfasst, nicht schon als Dissertation verwendet habe und die
benutzten Hilfsmittel und Quellen vollständig angegeben wurden.

Weiterhin erkläre ich, dass ich weder diese noch eine andere Arbeit zur Erlangung des
akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an anderen Einrichtungen
eingereicht habe.



______________ ______________
(Ort, Datum) (Marina Mikhaylova)

3 Index

Summary 7
1 Introduction 9
1.1 Neuronal calcium signaling 9
1.1.1 The role of calcium as a second messenger 9
2+1.1.2 Sources and processed regulated by Ca 9
1.1.2.1 Endoplasmic reticulum 10
1.1.2.2 The Golgi apparatus 11
2+1.1.2.3 Ca is a regulator of secretory processes 12
1.1.2.4 Synaptic plasticity 13
1.1.2.5 Gene transcription 14
1.2 Calcium binding proteins 15
1.2.1 Types of calcium binding proteins 15
1.2.2 Neuronal calcium sensor pr 17
1.2.2.1 NCS-1/Frequinin: Its role for regulation of PI-4K
activity and Golgi trafficking 18
1.2.2.3 The Caldendrin/CaBP1-5 gene family 20
1.2.2.4 Jacob is a Caldendrin interaction partner in the synapse 22
1.2.2.5 The identification of Calneurons 23
1.2.3 Aims of the study 24
2 Materials and methods 25
2.1 Materials and production of materials 25
2.1.1 Chemicals 25
2.1.2 Enzymes and kits 25
2.1.3 cDNA constructs 25
2.1.4 Cloning of Jacob constructs into a Semliki Forest Virus vector
and production of viral particles 26
2.1.5 In situ hybridization (oligonucleotides) 27
2.1.6 Generation of Calneuron-specific antibodies 27
2.1.7 Antibodies used for IB, IF and IPs 28
2.1.8 Common buffers and cell culture media 29
2.1.9 Prokaryotic and eukaryotic cell lines 29
2.1.10 Animals 30
2.2 Methods 30
2.2.1 Cell culture, transfections and immunocytochemistry 30
2.2.2 Stimulation of primary hippocampal neurons 31
2.2.3 Confocal laserscan microscopy 31
2.2.4 Immunoblotting 31
2.2.5 Subcellular fractionation and microsomal preparation 32
2.2.6 Gel filtration 32
2.2.7 Co-immunoprecipitation 33
2.2.8 Bacterial expression and purification of recombinant proteins
2.2.9 Isothermal titration calorimetry (ITC) 35
2.2.10 Steady-state fluorescence studies 36
2.2.11 8-Anilino-1-naphthalene sulfonic acid binding 36
2.2.12 Pull-down assays 36
2.2.13 Competition pull-down assays 37
4
2.2.14 Surface plasmon resonance analysis 37
2.2.15 PI-4K activity assays 38
2.2.16 PI(4)P assay 39
2.2.17 hGH release assay 39
2.2.18 VSV-G trafficking assay 40
2+2.2.19 Ca imaging 40
2.2.20 FRAP experiments 41
2.2.21 Life imaging experiments 42
2.2.22 Analysis of Golgi complexes and PTVs in primary neurons 42
2.2.23 Structural modeling 43
2.2.24 Statistical analysis 43
3 Results 44
3.1 Jacob is a Caldendrin binding partner in brain and competes with
Importin- for an overlapping binding site 44
3.2 Importin-bound Jacob translocates to the nucleus after stimulation
of NR2B- containg NMDARs 46
3.3 Caldendrin binding targets Jacob outside the nucleus only after
synaptic NMDAR stimulation 49
3.4 Jacob is part of the CREB shut-off pathway 53
3.5 The primary structure of Caldendrin and Calneurons 54
3.6 Comparative modeling of Cain and Calneurons EF-hand
structures 56
3.7 Caldendrin and Calneurons are abundant in brain 59
3.8 Calneurons are localized at the Golgi apparatus and associate
with PI-4K in vivo 60
3.9 Calneurons physically interact with PI-4K and compete with
2+NCS-1 binding in a Ca -dependent manner 65
2+ 3.10 The Ca binding affinity of NCS-1 but not of Calneurons is
2+regulated by Mg 69
2+3.11 Calneuron-1 regulates PI-4K activity in a Ca -dependent manner
and opposing to NCS-1 72
3.12 Calneurons regulate vesicular trafficking of the VSV-G protein 74
3.13 Calneuron over-expression inhibits hGH release in PC12 cells 76
3.14 Overexpression of Calneurons in cortical neurons induces a
prominent enlargement of TGN 77
3.15 Calneurons regulate vesicle trafficking at neuronal Golgi 80
3.16 Calneurons regulate the number of PTVs in axon at early
developmental stages 83
4 Discussion 86
4.1 Caldendrin and Calneurons are close but still rather different
‘relatives’ in the brain 87
4.2 Caldendrin and Jacob are key molecules on a novel pathway from
the synapse to the nucleus 88
4.2.1 Jacob is a synaptic binding partner of Caldendrin 88
4.2.2 Caldendrin is a first example when interaction of a
2+Ca -sensor with a NLS can mask the site and prevents nuclear
translocation by competing with Importin- –binding 90
4.2.3 The Caldendrin - Jacob interaction in the frame of
neuronal function 91
5
4.2.4 Nuclear Jacob induces pleiotropic negative effects
on synapto-dendritic cytoarchitecture and induces CREB shut-off 91
4.2.5 What is the physiological role of Jacob in the nucleus? 92
2+4.3 Calneurons provide a Ca threshold for trans-Golgi network
to plasma membrane trafficking 94
4.3.1 PI-4K is an interaction partner of Calneurons in the Golgi 94
2+4.3.2 Calneurons are setting a ‘Ca threshold’ for PI-4K activation 95
4.3.3 The competitive binding of Calneurons and NCS-1 to
2+PI-4K is regulated by Mg 97
5 References 100
6 Supplementary information 110
6.1 Supplementary Table 1. List of the constructs produced in the lab 110
6.2 y Table 2. List of the constructs obtained from the
collaborators 111
6.3 Supplementary Table 3. Plasmids used for the cloning and protein
expression
6.4 y Table 4. Common buffers 112
6.5 Supplementary Table 5. Common media 112
6.6 y Table 6. Solutions for PSD preparation 113
6.7 y Scheme 1. PSD preparation 114
7 Abbreviations 116

Curriculum Vitae
Scientific publications

6
Summary

The calcium sensor protein Caldendrin is an EF-hand protein with a high homology of
Calmodulin (CaM). Calneurons are two novel calcium binding proteins that apart from CaM
represent the closest homologues of Caldendrin in brain. Caldendrin and Calneuron-1 and -2
are abundantly expressed in neurons and retina