Initial characterization of two novel spindle proteins, CHICA and HURP [Elektronische Ressource] : focus on the role of the Ran-importin β-regulated HURP protein in kinetochore-fiber stabilization / vorgelegt von Susanna Nagel

Susanna Nagel

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 30 Mo

Extrait

INITIAL CHARACTERIZATION OF TWO NOVEL
SPINDLE PROTEINS, CHICA AND HURP:
Focus on the role of the Ran-importin β-regulated
HURP protein in kinetochore-fiber stabilization.
Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Fakultät für Biologie der
Ludwig-Maximilians Universität München
vorgelegt von
Susanna Nagel
Martinsried / München 2008




Initial Characterization of two novel spindle
proteins, CHICA and HURP:
Focus on the role of the Ran-importin β-regulated
HURP protein in kinetochore-fiber stabilization.





Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians Universität München


vorgelegt von
Susanna Nagel

Martinsried /München 2008



















Eingereicht am:
Tag des Promotionskolloquiums: 19. Juni 2008

Gutachter
Erstgutachter: Prof. Dr. Erich A. Nigg
Zweitgutachter: Prof. Dr. Peter B. Becker




Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig angefertigt habe
und keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt
habe. Sämtliche Experimente sind von mir selbständig durchgeführt worden, falls
nicht explizit auf Dritte verwiesen wird. Ich versichere, dass ich weder versucht habe
diese Dissertation oder Teile davon an einer anderen Stelle einzureichen, noch eine
Doktorprüfung durchzuführen.





Susanna Nagel Martinsried, den 14. 03. 2008 TABLE OF CONTENTS
Table of contents
SUMMARY....................................................................................................................................................................1
ZUSAMMENFASSUNG..............................................................................................................................................2
1 INTRODUCTION .................................................................................................................................................4
1.1 MITOSIS ............................................................................................................................................................4
1.2 THE MITOTIC SPINDLE – A DYNAMIC ASSEMBLY OF MICROTUBULES AND MOTORS......................................5
1.2.1 MT dynamics............................................................................................................................................7
1.2.2 K-fiber formation .....................................................................................................................................8
1.3 SPINDLE ASSEMBLY PATHWAYS ......................................................................................................................9
1.3.1 Centrosome-mediated “search-and-capture” hypothesis ....................................................................9
1.3.2 Chromosome-induced Ran-regulated spindle formation ....................................................................11
1.4 STABILIZING AND DESTABILIZING MICROTUBULE-ASSOCIATED PROTEINS .................................................13
1.5 MOTOR PROTEINS ORGANIZE THE SPINDLE ...................................................................................................15
1.5.1 Poleward forces .....................................................................................................................................17
1.5.2 Polar ejection force ...............................................................................................................................17
1.6 REGULATION BY PHOSPHORYLATION ............................................................................................................19
1.6.1 Mitotic kinases .......................................................................................................................................19
2 AIM OF THE WORK.........................................................................................................................................21
3 RESULTS..............................................................................................................................................................22
3.1 INITIAL CHARACTERIZATION OF THE NEW SPINDLE PROTEIN C20ORF129 (CHICA) .................................22
3.1.1 CHICA localizes to the proximity of the spindle poles........................................................................23
3.2 CHICA DOES NOT PLAY A ROLE IN K-FIBER STABILIZATION.......................................................................24
3.3 HURP A NEW TARGET OF THE RAN-IMPORTIN β-REGULATED SPINDLE ASSEMBLY PATHWAY ..................26
3.3.1 HURP localizes to microtubules in the vicinity of chromosomes.......................................................27
3.3.2 HURP localizes predominantly to kinetochore microtubules.............................................................29
3.3.3 HURP is required for stabilization of K-fibers....................................................................................32
3.3.4 HURP binds, bundles, and stabilizes microtubules in vitro ...............................................................36
3.3.5 HURP interacts with importin β and shuttles between the cytoplasm and nucleus ..........................37
3.3.6 Importin β regulates the mitotic spindle localization and function of HURP ...................................40
3.4 HOW IS HURP SPECIFICALLY LOCALIZED TO K-FIBERS?.............................................................................44
3.4.1 Structure-function analysis of different HURP-domains ....................................................................44
3.4.2 Identification of the importin β binding site within the HURP N-terminus.......................................52
3.4.3 Regulation of HURP by phosphorylation ............................................................................................56
3.4.4 Search for a recruitment factor targeting HURP to the KMT plus ends ...........................................60
4 DISCUSSION .......................................................................................................................................................66
4.1 CHARACTERIZATION OF NEW SPINDLE COMPONENTS...................................................................................66
4.1.1 CHICA is required for loading the chromokinesin Kid onto the mitotic spindle ..............................66
I TABLE OF CONTENTS
4.1.2 HURP, is a novel target of the Ran-regulated spindle assembly pathway........................................ 67
4.2 RAN-REGULATED K-FIBER STABILIZATION BY HURP ................................................................................. 68
4.3 HOW IS HURP SPECIFICALLY RECRUITED TO K-FIBERS?............................................................................. 70
4.3.1 Structure-function analysis of HURP domains ................................................................................... 71
4.3.2 Regulation of HURP by phosphorylation ............................................................................................ 75
4.3.3 Search for new HURP interaction partners ........................................................................................ 76
5 MATERIAL AND METHODS ......................................................................................................................... 78
5.1 CHEMICALS AND MATERIALS......................................................................................................................... 78
5.2 PLASMID PREPARATION AND SITE DIRECTED MUTAGENESIS........................................................................ 78
5.3 RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION ........................................................................... 79
5.4 ANTIBODY PRODUCTION AND TESTING ......................................................................................................... 80
5.5 CELL CULTURE AND SYNCHRONIZATION....................................................................................................... 81
5.6 TRANSIENT TRANSFECTIONS AND SIRNA ..................................................................................................... 81
5.7 CELL EXTRACTS, WESTERN BLOTS, AND IMMUNOPRECIPITATIONS........................................................... 81
5.8 IMMUNOFLUORESCENCE MICROSCOPY.......................................................................................................... 82
5.9 LIVE-CELL IMAGING....................................................................................................................................... 83
5.10 IN-VITRO MICROTUBULE CO-SEDIMENTATION AND BUNDLING ASSAYS.................................................... 83
5.11 YEAST TWO-HYBRID ANALYSIS ................................................................................................................... 84
5.12 IN VITRO COUPLED TRANSCRIPTION TRANSLATION .................................................................................... 84
5.13 GELFILTRATION OF CELL EXTRACTS ....