Parallel and miniaturised analysis of protein-protein interactions in T-cell signal transduction by fluorescence cross-correlation spectroscopy and peptide microarrays [Elektronische Ressource] = Parallele und miniaturisierte Analyse von Protein-Protein-Interaktionen in der T-Zell-Signaltransduktion mittels Fluoreszenz-Kreuzkorrelations-Spektroskopie und Peptidmikroarrays / vorgelegt von Oda Stoevesandt

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• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2006
• Nombre de lectures: 36
• Poids de l'ouvrage: 3 Mo

Extrait

Parallel and miniaturised Analysis
of
Protein-Protein Interactions in T-Cell Signal Transduction
by
Fluorescence Cross-Correlation Spectroscopy
and
Peptide Microarrays



Parallele und miniaturisierte Analyse von
Protein-Protein-Interaktionen in der T-Zell-Signaltransduktion
mittels Fluoreszenz-Kreuzkorrelations-Spektroskopie
und Peptidmikroarrays




DISSERTATION

der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen
zur Erlangung des Grades eines Doktors
der Naturwissenschaften



2006


vorgelegt von

ODA STOEVESANDT





























Tag der mündlichen Prüfung: 1. 12. 2006

Dekan: Prof. Dr. L. Wesemann
1. Berichterstatter: Prof. Dr. H.-G. Rammensee
2. Berichterstatter: PD Dr. R. Brock
3. Berichterstatter: Prof. Dr. W. Kolanus














für meine Eltern!




Table of Contents







1 Introduction....................................................................................................................... 1
2 Background........................................................................................................................ 2
2.1 Signal transduction in T-lymphocytes.................................................................... 2
2.1.1 SH2 and SH3 domains ....................................................................................... 2
2.1.2 Upstream events and molecular players in T-cell signalling....................... 4
2.2 Fluorescence and confocal fluorescence detection................................................ 8
2.2.1 Fluorescence ........................................................................................................ 8
2.2.2 Confocal fluorescence detection....................................................................... 9
2.3 Fluorescence correlation and cross correlation spectroscopy ........................... 11
2.3.1 Basics of fluorescence auto- and cross-correlation spectroscopy .............. 11
2.3.2 Intracellular applications of FCS and FCCS ................................................. 17
2.4 Microarrays for the parallelised analysis of proteins ......................................... 18
2.4.1 Basic concepts and different formats of arrays ............................................ 18
2.4.2 Brief overview of applications........................................................................ 20
3 General Materials and Methods.................................................................................. 22
3.1 Basic buffers.............................................................................................................. 22
3.2 Peptides ..................................................................................................................... 23
3.3 Antibodies ................................................................................................................. 25
3.4 Cell culture, stimulation and lysis......................................................................... 26
3.5 Immunoprecipitation, SDS-page and western blotting...................................... 27
3.6 FCS of ZAP70-YFP in cell lysates........................................................................... 27
3.7 Cellular FCS measurements ................................................................................... 28
4 Methods developed during the thesis........................................................................ 29
4.1 Protein complex detection by mass-tag FCS and FCCS ..................................... 29
4.1.1 Sample preparation .......................................................................................... 29
4.1.2 FCS and FCCS measurement and data evaluation...................................... 31
4.1.3 Anticipated results for FCCS .......................................................................... 33
4.2 Protein complex detection on peptide microarrays............................................ 35
4.2.1 Generation of peptide arrays 35
4.2.2 Incubation and scanning of peptide arrays .................................................. 36
4.2.3 Data analysis ..................................................................................................... 37
I 5 Results and Discussion ................................................................................................. 40
5.1 Peptide microarrays for the detection of protein interactions in cellular signal
transduction ........................................................................................................ 40
5.1.1 Pilot experiments .............................................................................................. 40
5.1.1.1 Detection of changes in the availability of binding domains............... 40
5.1.1.2 Determination of free and occupied binding sites of ZAP70 45
5.1.1.3 Discussion.................................................................................................... 49
5.1.2 Additional interactions, 2-Colour detection ................................................. 50
5.1.3 Dissection of signalling complexes on peptide microarrays...................... 53
5.1.3.1 Phosphorylation-dependent changes in interaction profiles............... 53
5.1.3.2 Dissection of complex architectures by peptide competition and
titration 55
5.1.3.3 Effect of single protein deficiencies on the interactions network........ 58
5.1.3.4 Stimulus-dependence and time course of protein interactions in T-cell
activation.................................................................................................................. 60
5.1.3.5 Discussion.................................................................................................... 62
5.2 FCS and FCCS for the detection of signalling complexes .................................. 65
5.2.1 FCS of ZAP70-YFP in cells and lysates.......................................................... 66
5.2.1.1 Cellular measurements .............................................................................. 66
5.2.1.2 Measurements in lysates ........................................................................... 68
5.2.2 Detection of signalling complexes in cell lysates by indirect
immunolabelling............................................................................................... 70
5.2.2.1 Mass-tag FCS 72
5.2.2.2 Fluorescence Cross Correlation Spectroscopy ....................................... 76
5.2.2.3 Dissection of the LAT-signalosome with interacting peptides............ 79
5.2.2.4 Discussion.................................................................................................... 83
6 Summary .......................................................................................................................... 88
7 Zusammenfassung ......................................................................................................... 91
8 References ........................................................................................................................ 94
9 Publications ................................................................................................................... 108
II

Abbreviations

For some of the proteins, the origin of the abbreviation could not be deduced. In
these cases, or if the full designation held little information on the function of the
protein, some additional information is given.

cCBL casitas B-lineage lymphoma proto-oncogene, E3 ubiquitin-protein
ligase
CD28 T-cell-specific surface glycoprotein CD28
CD3ε T-cell surface glycoprotein CD3ε-chain
CD3ζ ζ
CFP cyan fluorescent protein
CSK cSRK kinase
FCCS fluorescence cross-correlation spectroscopy
FCS fluorescence correlation spectroscopy
FYB FYN-binding protein;
synonym: SLAP, SLP76 associated phosphoprotein
FYN proto-oncogene tyrosine-protein kinase
GAB2 GRB2-associated binding protein 2
GADS GRB2-related adapter protein downstream of SHC
GEM glycosphingolipid-enriched membrane microdomain
GFP green fluorescent protein
GRB2 growth factor receptor bound protein
ITAM immunoreceptor tyrosine-based activation motif
ITK synonym: TSK, T-cell-specific kinase
LAT linker for activation of T-cells
LCK lymphocyte cell-specific protein-tyrosine kinase
MHC major histocompatibility complex
NCK SH2/SH3 adapter protein
PAG phosphoprotein associated with GEMs
PAK1 p21-activated kinase1, serine/threonine-protein kinase
PI3K p85 phosphatidyl inositol 3-kinase, regulatory subunit of 85 kDa
PLCγ1 phospolipase C gamma 1,
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma 1
PSF point spread function
PTB phosphotyrosine-binding domain
polyP proline-rich sequence motif
PV pervanadate
pY phosphotyrosyl residue
III SH2