Isolierung neuer PACSIN-Isoformen

und funktionale Charakterisierung

der Protein-Familie

Inaugural-Dissertation zur

Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultt

der Universitt zu Kln

vorgelegt von

Brigitte Ritter aus Aachen

Kln, 2001

Berichterstatter Prof. Dr. Mats Paulsson Priv.-Doz. Dr. Sabine Waffenschmidt Prof. Dr. Reinhard Sterner Tag der mndlichen Prfung 19. Juni 2001

Inhalt

Abkrzungen............................................................................................................................. 1

1 Einleitung............................................................................................................................ 4 1.1 Biologische Membranen und ihre Transportwege ............................................................... 4 1.2 Lipid Rafts und Caveolae ....................................................................................................... 6 1.3 Vesikel mit Protein-Hlle ....................................................................................................... 7 1.3.1 Coatomer Protein II-umhllte Vesikel ......................................................................................7 1.3.2 Coatomer Protein I-umhllte Vesikel........................................................................................8 1.3.3 Clathrin-umhllte Vesikel .........................................................................................................9 1.4 Endocytose und das Actin- und Tubulin-Cytoskelett ........................................................ 16 1.5 Regulation des intrazellulren Vesikeltransports .............................................................. 18 1.6 PACSIN 1............................................................................................................................... 19 1.7 Zielsetzung der Arbeit .......................................................................................................... 21

2 Material und Methoden................................................................................................... 22 2.1 Material .................................................................................................................................. 22 2.1.1 Puffer und Lsungen ...............................................................................................................22 2.1.2 Enzyme....................................................................................................................................23 2.1.3 Biologisches Material..............................................................................................................24

2.1.3.1 E. coli-Bakterien-Stmme......................................................................................................24 2.1.3.2 Hefen .....................................................................................................................................24 2.1.3.3 Eukaryotische Zellen .............................................................................................................24 2.1.3.4 Paraffinschnitte ......................................................................................................................24

2.1.4 Banken.....................................................................................................................................25 2.1.5 Sonden.....................................................................................................................................25 2.1.6 Antikrper ...............................................................................................................................25

2.1.6.1 Erstantikrper ........................................................................................................................25 2.1.6.2 Zweitantikrper .....................................................................................................................26 2.1.6.3 Streptavidin-Konjugate..........................................................................................................27

2.1.7 Vektoren..................................................................................................................................27 2.1.7.1 Klonierungsvektoren.............................................................................................................. 27 2.1.7.2 Expressionsvektoren .............................................................................................................. 27

2.1.8 Primer......................................................................................................................................28 2.1.8.1 Sequenzierungsprimer ........................................................................................................... 28 2.1.8.2 Primer fr gerichtete Mutagenesen........................................................................................ 28

2.1.9 Verbrauchsmaterial .................................................................................................................30 2.1.10 Gerte .....................................................................................................................................30 2.1.11 EST-Klone...............................................................................................................................31

2.1.11.1 EST-Klone fr PACSIN 2.................................................................................................... 31 2.1.11.2 EST-Klone fr PACSIN 3 .................................................................................................... 31

2.1.12 Zellkultur-Medien ...................................................................................................................32 2.2 Methoden................................................................................................................................ 33 2.2.1 Computeranalyse.....................................................................................................................33 2.2.2 Sequenzierung .........................................................................................................................33

2.2.3 Sondensynthese .......................................................................................................................33 2.2.4 Durchmusterung einer embryonalen Maus-cDNA-Bank........................................................33 2.2.5 RNA-Prparation.....................................................................................................................33 2.2.6 Northern Blot...........................................................................................................................34 2.2.7 Human RNA Master Blot........................................................................................................34 2.2.8 Gerichtete Einfhrung von Punktmutationen..........................................................................34 2.2.9 Antiseren und Affinittsreinigung...........................................................................................36 2.2.10 Massenspektrometrie...............................................................................................................37 2.2.11 Immunhistochemie (IH) ..........................................................................................................37 2.2.12 Affinittsprzipitation .............................................................................................................37 2.2.13 Enzym-gekoppelter Immunnachweis (ELISA) .......................................................................38 2.2.14 Hefe Two-Hybrid System .......................................................................................................38 2.2.15 Immunprzipitation .................................................................................................................39 2.2.16 in vitro-Phophorylierung .........................................................................................................39 2.2.17 Zellkultur.................................................................................................................................39

2.2.17.1 Transfektionen ...................................................................................................................... 39 2.2.17.2 Immunfluoreszenz ................................................................................................................ 40 2.2.17.3 In vitro-Differenzierung von C2-Myoblasten ....................................................................... 40 2.2.17.4 Endocytose-Assay ................................................................................................................ 40

3 Ergebnisse......................................................................................................................... 42 3.1 Isolierung neuer PACSIN-Isoformen .................................................................................. 42 3.1.1 Sequenzanalyse der neuen PACSIN-Isoformen......................................................................43 3.1.2 Evolution der PACSINe ..........................................................................................................46 3.2 Herstellung von Antiseren gegen PACSIN 2 und 3............................................................ 48 3.3 Expression der PACSIN-Isoformen..................................................................................... 49 3.3.1 Expression auf mRNA-Ebene .................................................................