Biochemical and functional analysis of {γ-protocadherin [gamma-protocadherin] intracellular signaling pathways [Elektronische Ressource] / presented by Noam Pilpel

Yair

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences





















presented by

M.Sc. Noam Pilpel
born in Jerusalem, Israel
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences





















presented by

M.Sc. Noam Pilpel
born in Jerusalem, Israel
thOral-examination: March 27 , 2009


Biochemical and Functional Analysis of -Protocadherin
Intracellular Signaling Pathways



























Referees: Prof. Dr. Peter H. Seeburg
Priv.-Doz. Dr. Matthias Klugmann






















Erklärung gemäß § 8 (3) b) und c) der Promotionsordnung:

Ich erkläre hiermit, daß ich die vorgelegte Dissertation selbst verfaßt und mich dabei
keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient
habe. Desweiteren erkläre ich hiermit, daß ich an keiner anderen Stelle ein
Prüfungsverfahren beantragt bzw. die Dissertation in dieser oder anderer Form bereits
anderweitig als Prüfungsarbeit verwendet oder einer anderen Fakultät als Dissertation
vorgelegt habe.

Heidelberg, den 27 Januar 2009














For Eden and Yair






Index
SUMMARY ...........................................................................................................................................................1
ZUSAMMENFASSUNG .....................................................................................................................................2
1. INTRODUCTION............................3
1.1 THE CADHERIN SUPERFAMILY.....................................................................................................................3
1.2 PROTOCADHERINS .......................................................................................................................................4
1.3 EXPRESSION PATTERN OF CLUSTERED PCDHS IN THE BRAIN .....................................................................6
1.4 POST-TRANSLATIONAL PROCESSING OF CLUSTERED PCDHS......................................................................7
1.5 TRANSGENIC MOUSE MODELS FOR THE RESEARCH OF -PCDHS ................................................................9
1.6 RECENT DEVELOPMENTS IN -PCDHS RESEARCH; THE USE OF CONDITIONAL KNOCK-OUTS..................10
1.7 -PCDH INTERACTION PARTNERS ..............................................................................................................11
1.8 VIRUS-MEDIATED TRANSDUCTION OF NEURONS IN NEONATAL BRAIN....................................................11
1.9 THESIS OBJECTIVES....................................................................................................................................12
2. MATERIALS AND METHODS .................................................................................................................14
2.1 ANTIBODY PURIFICATION ..........................................................................................................................14
2.2 ANTIBODY CROSS-LINKING TO PROTEIN-A AGAROSE BEADS ..................................................................14
2.3 IMMUNOPRECIPITATION.............................................................................................................................15
2.4 WESTERN BLOTTING...16
2.5 SLICING AND IMMUNOSTAINING ...............................................................................................................17
2.6 X-GAL STAINING ........................................................................................................................................17
2.7 NISSL STAINING..........18
2.8 VIRUS PURIFICATION..18
2.9 PLASMIDS....................19
2.10 ELECTROPHYSIOLOGICAL RECORDINGS OF MINIATURE SYNAPTIC CURRENTS......................................20
3.RESULTS .........................................................................................................................................................21
3.1 ANALYSIS OF -PCDH INTERACTION PARTNERS .......................................................................................21
3.1.1 Optimization of Western blot conditions .........................................................................................21
3.1.2 Optimization of immunoprecipitation conditions ...........................................................................22
3.1.3 Mass spectrometry analysis of co-immunoprecipitated proteins...................................................24
3.1.4 Verification of mass spectrometry results using co-immunoprecipitation...................................30
3.2 ESTABLISHMENT OF A METHOD FOR TRANSDUCTION OF NEURONS BY TARGETED RECOMBINANT VIRUS
INJECTIONS INTO NEONATAL MOUSE BRAINS..................................................................................................33
3.2.1 Establishment of targeted, reproducible injections into the neonatal mouse brain .....................33
3.2.2 Characterization of virus-infected brain regions ...........................................................................35
3.2.3 Onset and duration of fluorescent protein expression....................................................................38
3.2.4 Virus distribution after parenchymal injection into the neonatal brain........................................39
3.2.5 Expression of full-length -Pcdh in the neonatal mouse brain ......................................................42
3.2.6 -ICD injection and staining in brain using specific monoclonal antibodies...............................45
3.2.7 Successful overexpression of the C-terminal domain -Pcdh in P0 mice .....................................47
3.2.8 Physiological effects of -ICD overexpression in principal cortical neurons ..............................48
4. DISCUSSION..................................................................................................................................................52
4.1 -PCDH STRUCTURE AND PROCESSING SUGGESTS A ROLE IN SIGNAL TRANSDUCTION ...........................52
4.2 -PCDHS AND BRAIN COMPLEXITY: THE SEARCH FOR INTERACTION PARTNERS.....................................53
4.3 SAM68 AND SLM-2: -PCDHS INTERACTING PROTEINS REVEAL POSSIBLE FUNCTIONS OF -PCDHS ....55
4.4 OTHER -PCDHS INTERACTING PROTEINS .................................................................................................56
4.5 CURRENT MODELS FOR -PCDHS RESEARCH.............................................................................................58
4.6 EXPRESSION OF TRANSGENES BY VIRUS-MEDIATED GENE TRANSFER INTO NEONATAL MOUSE BRAINS59
4.7 PHYSIOLOGICAL PHENOTYPES DETECTED USING NEONATAL OVEREXPRESSION OF -ICD IN
FOREBRAIN; FUTURE PROSPECTS FOR -PCDHS RESEARCH ............................................................................60
5. ABBREVIATIONS ........................................................................................................................................62

Index
6. ACKNOWLEDGEMENTS ..........................................................................................................................63
7. REFERENCES ...............................................................................................................................................64


Summary
Summary
The precisely organized complexity of the central nervous system (CNS) requires an
enormous number of specific cell-cell interactions, presumably mediated by a diverse
array of membrane associated proteins. The classic cadherins are known to play essential
roles in the maintenance of neuronal connectivity and synaptic plasticity. Three complex
genomic loci encoding proteins of the cadherin superfamily, the -, -, and - clustered
protocadherins (Pcdhs), have been hypothesized to take part in this task. Their large
number, diverse expression pattern during neurogenesis, and partial synaptic localization
suggest a role in synaptogenesis. The genomic architecture of the clustered Pcdhs is
reminiscent of the immunoglobulin and T-cell receptor clusters which confer the huge
variety of antibody molecules: within each cluster, exons encoding variable extracellular
and transmembrane domains are alternatively spliced onto a cluster-specific conserved
intracellular domain. Thus, Pcdhs confer molecular diversity on the cell surface, with
conserved signaling mechanisms in the cytoplasm.