Plk1 regulates PICH, a centromere-associated SNF2 family translocase that is required for the spindle checkpoint [Elektronische Ressource] / vorgelegt von Christoph Baumann

Christoph Baumann

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 46
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Plk1 regulates PICH, a centromere-
associated SNF2 family translocase that
is required for the spindle checkpoint



Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät für Biologie der Ludwig-Maximilian Universität
München

Vorgelegt von

Christoph Baumann


München 09.02.2007











































Datum der mündlichen Prüfung: 30.03.2007

Erstgutachter: Prof. Dr. Erich A. Nigg

Zweitgutachter: Prof. Dr. Peter B. Becker





Ehrenwörtliche Erklärung und Erklärung über frühere Promotionsversuche



Hiermit erkläre ich, dass ich, Christoph Baumann, die vorliegende Dissertation
selbständig und ohne unerlaubte Hilfe angefertigt habe. Sämtliche Experimente
sind von mir selbst durchgeführt, ausser wenn explizit auf Dritte verwiesen wird.
Ich habe weder anderweitig versucht, eine Dissertation einzureichen oder eine
Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile
derselben einer anderen Prüfungskommission vorgelegt.




München, den 06. Februar 2007

Table of contents_________________________________________________________________________

Table of contents


Acknowledgements ........................................................................................ 1
Zusammenfassung......................................................................................... 3
Summary .......................................................................................................... 4
Introduction ...................................................................................................... 5
Cell division......................................................................................................... 5
The spindle assembly checkpoint ....................................................................... 6
Regulation of sister chromatid separation........................................................... 9
Polo-like kinase 1 (Plk1) ................................................................................... 11
The centromere, the kinetochore and the Ndc80 complex ............................... 13
SNF2 type helicases......................................................................................... 15
Topoisomerase II.............................................................................................. 17

Aim of this work............................................................................................. 19

Results part I.................................................................................................. 20
Studies on the Ndc80 complex ......................................................................... 20
Assembly of the Ndc80 complex, a structural component of the kinetochore... 20
Interaction partners of the Ndc80 complex ....................................................... 23
A monoclonal antibody specific for human Nuf2............................................... 26
The Ndc80 complex is required for Plk1 kinetochore localization..................... 28

Conclusions I ................................................................................................. 30




iTable of contents_________________________________________________________________________

Results part II ................................................................................................ 31
Studies on Plk1................................................................................................. 31
Identification of PICH, a novel Plk1 interacting protein ..................................... 31
Detection of endogenous PICH ........................................................................ 35
The Plk1-PICH interaction is mediated by Polo-box binding to phosphorylated
threonine 1063.................................................................................................. 36
PICH colocalizes with Plk1 at kinetochores ...................................................... 43
Plk1 phosphorylation removes PICH from chromatid arms............................... 45
Structural requirements for PICH localization ................................................... 50
PICH reveals inter-kinetochore threads in anaphase........................................ 53
PICH positive threads are catenated, centromere-related, sister kinetochore
connecting chromatin........................................................................................ 57
PICH is required for the spindle checkpoint...................................................... 65
PICH depleted cells have intact kinetochores and functional microtubule
attachment........................................................................................................ 71

Conclusions II................................................................................................ 75

Discussion ...................................................................................................... 77
The Ndc80 complex.......................................................................................... 77
PICH – a novel mitotic target of Plk1 ................................................................ 78
PICH defines a novel subclass of the SNF2 ‘helicase’ family ........................... 81
Evidence for PICH localization to catenated centromeric DNA......................... 81
Decatenation of PICH threads in anaphase...................................................... 82
A regulatory network around PICH and Topoisomerase II................................ 83
Human PICH is an essential component of the SAC ........................................ 84
PICH in other organisms................................................................................... 85
A working model for PICH function as tension sensor ...................................... 86


iiTable of contents_________________________________________________________________________

Material and Methods .................................................................................. 89
Chemicals and materials................................................................................... 89
Plasmids and antibodies 89
Immunofluorescence microscopy and live cell imaging .................................... 94
Transient transfections and siRNA ................................................................... 95
Biochemical assays .......................................................................................... 95
Co-immunoprecipitations .................................................................................. 96
Miscellaneous reagents .................................................................................... 97

Abbreviations................................................................................................. 98
References................................................................................................... 100
Publications.................................................................................................. 114
Curriculum Vitae ......................................................................................... 115

iiiAcknowledgements _________________________________________________________________________


I would like to thank Prof. Erich Nigg for the possibility to work in his lab on that
wonderful subject and for all his support.

I am thankful to Prof. Peter Becker for reviewing this manuscript.

I am grateful to Prof. Matthias Wabl for his continuous support, for being a mentor
and friend over the recent years.

I would like to thank Francis Barr, Thomas Mayer, Herman Sillje and Olaf
Stemmann for their support and many helpful discussions. Thanks to Susan
Gasser, Ulrike Grüneberg, Anja Hanisch, Kay Hofmann, Karl-Peter Hopfner,
Andrea Musacchio, Rüdiger Neef and Frank Uhlmann for discussion and/or
reagents.

Thanks to my colleagues and friends: Sabine Elowe, Tom and Louise Gaitanos,
Stefan Hümmer (thanks man!), Mikael LeClech, Anna Santamaria, Andreas
Schmidt and Volker Stucke for support, reagents, discussions and a good time.
I am happy that Lily Wang and Manuel Kaulich (the new “PICH folks”) are
following up on my project.

Having mentioned the perfect organization of the lab I want to highlight that the
permanent warm atmosphere and perfect setup of the lab is largely achieved by
Alicja Baskaja, Alison Dalfovo, Elena Nigg, Lidia Pinto, Albert Ries, Marianne
Siebert and Klaus Weber. Thank you!

Thanks to Roman Körner not only for excellent MS-data but also for all the
Caipirinha.

Thank you Ulla (Ulf Klein) for your fri