WWW.ABSTRACT.XLIBX.INFO
FREE ELECTRONIC LIBRARY - Abstract, dissertation, book
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 | 5 |   ...   | 12 |

«Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der ...»

-- [ Page 1 ] --

Control of Centriole Numbers by Plk4

Autophosphorylation and βTrCP-mediated

Degradation

Inauguraldissertation

zur Erlangung der Würde eines Doktors der Philosophie

vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät

der Universität Basel

von

Gernot Guderian

aus Koblenz, Deutschland

Basel, 2010

Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel

edoc.unibas.ch

Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle NutzungKeine Bearbeitung 2.5 Schweiz“ lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licences/by-nc-nd/2.5/ch eingesehen werden.

Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz

Sie dürfen:

das Werk vervielfältigen, verbreiten und öffentlich zugänglich machen

Zu den folgenden Bedingungen:

Namensnennung. Sie müssen den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen (wodurch aber nicht der Eindruck entstehen darf, Sie oder die Nutzung des Werkes durch Sie würden entlohnt).

Keine kommerzielle Nutzung. Dieses Werk darf nicht für kommerzielle Zwecke verwendet werden.

Keine Bearbeitung. Dieses Werk darf nicht bearbeitet oder in anderer Weise verändert werden.

Im Falle einer Verbreitung müssen Sie anderen die Lizenzbedingungen, unter welche dieses Werk fällt, • mitteilen. Am Einfachsten ist es, einen Link auf diese Seite einzubinden.

Jede der vorgenannten Bedingungen kann aufgehoben werden, sofern Sie die Einwilligung des • Rechteinhabers dazu erhalten.

Diese Lizenz lässt die Urheberpersönlichkeitsrechte unberührt.

• Die gesetzlichen Schranken des Urheberrechts bleiben hiervon unberührt.

Die Commons Deed ist eine Zusammenfassung des Lizenzvertrags in allgemeinverständlicher Sprache:

http://creativecommons.org/licenses/by-nc-nd/2.5/ch/legalcode.de

Haftungsausschluss:

Die Commons Deed ist kein Lizenzvertrag. Sie ist lediglich ein Referenztext, der den zugrundeliegenden Lizenzvertrag übersichtlich und in allgemeinverständlicher Sprache wiedergibt. Die Deed selbst entfaltet keine juristische Wirkung und erscheint im eigentlichen Lizenzvertrag nicht. Creative Commons ist keine Rechtsanwaltsgesellschaft und leistet keine Rechtsberatung. Die Weitergabe und Verlinkung des Commons Deeds führt zu keinem Mandatsverhältnis.

Quelle: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/ Datum: 3.4.2009 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Erich A. Nigg Prof. Anne Spang Prof. Brian Hemmings Basel, den 19.10.2010

–  –  –

Table of Contents 1 SUMMARY

2 INTRODUCTION

2.1 Structure and Function of the Centrosome

2.1.1 Structure of the Centrosome

2.1.2 The Centrosome as the Microtubule-organizing Center (MTOC)

2.1.3 The Centriole as Template for Cilia and Flagella

2.2 The Centrosome Cycle

2.2.1 Centriole Biogenesis in Caenorhabditis elegans

2.2.2 Centriole Biogenesis in Human Cells

2.2.3 Regulation of Centriole Duplication

2.2.3.1 Cell-Cycle Control

2.2.3.2 Copy-Number Control

2.2.3.3 Canonical versus de novo Centriole Duplication

2.3 Polo-like Kinase 4 (Plk4)

2.4 The Centrosome and Cancer

2.5 The Ubiquitin-Proteasome System

2.5.1 Ubiquitin-dependent Protein Degradation

2.5.2 The SCFβTrCP Complex

2.5.2.1 Structure of SCF complexes

2.5.2.2 Regulation of βTrCP-mediated Degradation

2.5.2.3 The SCFβTrCP Complex at the Centrosome

2.5.2.4 Regulation of Plk4 Expression

3 AIM OF THIS PROJECT

4 RESULTS

4.1 Generation and Characterization of anti-Plk4 Antibodies

–  –  –

4.2 Kinase-dead Plk4 Causes Centriole Overduplication

4.2.1 Plk4-WT and Plk4-KD Trigger Centriole Overduplication

4.2.2 Endogenous Plk4 is Required for Plk4-KD-induced Centriole Overduplication.... 28 βTrCP-dependent Degradation of Plk4

4.3 4.3.1 Centrosomal Plk4 Protein Levels are Regulated by the Proteasome

4.3.2 βTrCP is Required for Control of Plk4 Protein Levels and Centriole Number....... 32 4.3.3 Plk4 Autophosphorylation Controls Its Degradation

4.3.3.1 Plk4 and βTrCP Interact Directly

4.3.3.2 The Interaction of Plk4 and βTrCP Requires an Intact DSG Motif

4.3.3.3 Plk4 Autophosphorylation is Required for its Interaction with βTrCP............... 36 4.3.3.4 Plk4 Autophosphorylation is Required for its Ubiquitination and Degradation. 37 4.3.4 Plk4 trans-Autophosphorylation Controls its Degradation and Centriole Number. 39 4.3.4.1 Plk4 Autophosphorylates Itself in trans

4.3.4.2 An N-terminal Truncation of Plk4 Causes Centriole Overduplication............... 40 4.3.4.3 Plk4 Autophosphorylation in trans Restores βTrCP Binding to Plk4-KD.......... 42 4.3.4.4 Plk4 Autophosphorylation is Not Sufficient for βTrCP Binding

4.3.5 Does p38 Control the Interaction of Plk4 and βTrCP in vitro?

4.3.5.1 Inhibition of p38 Disrupts the Interaction of Plk4 and βTrCP





4.3.5.2 p38 Inhibitors Do Not Perturb Plk4 Autophosphorylation

4.3.5.3 Absence of p38 Activity Does Not Lead to Centriole Overduplication in vivo.. 48 5 DISCUSSION

5.1 Plk4 Kinase Activity is Essential for Centriole Duplication

βTrCP Controls Centriole Numbers through Degradation of Plk4

5.2 Plk4 trans-Autophosphorylation Regulates its βTrCP-mediated Degradation............. 55 5.3 Plk4 Kinase Activity is Not Sufficient for its βTrCP-mediated Degradation............... 58 5.4 6 MATERIALS AND METHODS

7 ABBREVIATIONS

–  –  –

8 REFERENCES

9 PUBLICATIONS

10 CURRICULUM VITAE

11 ACKNOWLEDGEMENTS

–  –  –

1 SUMMARY Proper centrosome numbers are imperative for faithful cell division, as aberrant centrosome numbers can lead to chromosomal instability, a hallmark of cancer development (Nigg 2002; Ganem et al., 2009). Hence, initiation of centriole duplication has to be tightly regulated. Recently, we and others demonstrated that Polo-like kinase 4 (Plk4) fulfills a pivotal role in regulating this process (Bettencourt-Dias et al., 2005;

Habedanck et al., 2005). Plk4 protein levels and its activity directly correlate with centriole numbers: depletion of Plk4 leads to sequential loss of centrioles in successive cell divisions (Bettencourt-Dias et al., 2005; Habedanck et al., 2005) and its overexpression promotes bona fide overduplication of centrioles (Habedanck et al., 2005;

Kleylein-Sohn et al., 2007), while both lead to progressive increase in abnormal spindle formation (reviewed in Nigg 2007). Even though Plk4 is a key regulator of centriole biogenesis and is crucial for maintaining constant centriole number, the mechanisms regulating its activity and expression are only beginning to emerge.

Here, we show that human Plk4 is subject to βTrCP-dependent proteasomal degradation, indicating that this pathway is conserved from Drosophila to human (CunhaFerreira et al., 2009; Rogers et al., 2009). Unexpectedly, we found that stable overexpression of kinase-dead Plk4 leads to centriole overduplication. Our data indicate that this phenotype depends on the presence of endogenous wild-type Plk4 and that centriole overduplication results from disruption of Plk4 trans-autophosphorylation by kinase-dead Plk4, which then shields endogenous Plk4 from recognition by βTrCP. We conclude that active Plk4 promotes its own degradation by catalyzing βTrCP binding through trans-autophosphorylation within homodimers which has been independently confirmed by others (Holland et al., 2010). Additionally, we propose that Plk4 autophosphorylation is not sufficient for its degradation and that instead an additional kinase is required for this process.

–  –  –

2 INTRODUCTION

The centrosome, Latin for “central body”, was first discovered in the late 19th century by Édouard van Beneden in various parasites (van Beneden 1875-6; van Beneden 1883).

While van Beneden discovered centrosomes and described them at a morphological level, it was Theodor Boveri who coined the term centrosome and postulated that the centrosome is self-replicating (Boveri 1887; Boveri 1888). Moreover, he later formulated the hypothesis that centrosome and chromosome aberrations are linked and contribute to tumorigenesis (Boveri 1914). Even though centrosomes are present in almost all eukaryotes, their composition, organization, mode of replication and specific functions have remained elusive until the rediscovery of centrosome biology in the late 20th century.

Today, pivotal functions of the centrosome have been uncovered and described, albeit the details of how these functions are fulfilled and regulated are still under intense investigation. Centrosome function is twofold, as microtubule-organizing center (MTOC) in dividing cells and as scaffold for basal bodies of flagella or cilia in differentiated or quiescent cells. In recent years, centrosome biology has become widely recognized due to the causal link between centrosome aberrations and the development of various human diseases.

2.1 Structure and Function of the Centrosome 2.1.1 Structure of the Centrosome The centrosome is a non-membranous organelle of approximately 1 µm in diameter which is usually located in close proximity to the nucleus (reviewed in Doxsey 2001). It is composed of two interconnected centrioles which are highly stable, barrel-shaped arrays of microtubule triplets arranged in a nine-fold symmetry (Figure 1). The individual microtubules (MTs) of each triplet are referred to as the A-, B- and C-tubule and reach a length of 400 nm during centriole elongation (reviewed in Bornens 2002; BettencourtDias and Glover 2007). In contrast to the A- and B-tubules, which span the complete proximal-distal axis of a fully grown centriole, the C-tubule does not stretch to the distal end of the centriole.

INTRODUCTION

Figure 1. Centrosome and centriole structure. Schematic view of a centrosome containing mother and daughter centrioles. Both centrioles are composed of nine-fold microtubule (MT) triplets. In each triplet, the internal tubule is termed the A-tubule, followed by the B-tubule and C-tubule. The latter does not extend to the distal end of the centriole. The two centrioles are surrounded by the pericentriolar material (PCM), depicted in blue, and interconnected by an unknown linker (centriole engagement fibers) until disengagement at the exit from mitosis. The mature centriole carries subdistal and distal appendages, which dock cytoplasmic MTs and anchor the centriole at the plasma membrane to serve as basal body. The cartwheel structure depicted on the right has been suggested to serve as a template for procentriole formation (adapted from Bettencourt-Dias and Glover 2009).

The centrioles are embedded in the electron-dense, amorphous pericentriolar material (PCM), which harbors coiled-coil proteins that mediate protein-protein interactions (Doxsey 2001; Andersen et al., 2003; Azimzadeh and Bornens 2007).

Additionally, within the PCM proteins reside which are required for microtubule nucleation and anchoring as well as various cell cycle regulators (Moritz et al., 1995;

Zheng et al., 1995; Moritz and Agard 2001). Centrioles and the PCM are intimately connected as loss of centrioles leads to dispersal of the PCM (Bobinnec et al., 1998) and the PCM is vice versa required for the formation and stabilization of procentrioles (Dammermann et al., 2004; Loncarek et al., 2008).

Both centrioles present in a mammalian G1 phase cell are loosely tethered at their proximal ends by the proteins C-Nap1, rootletin and Cep68 (Fry et al., 1998; Bahe et al., 2005; Graser et al., 2007b). Even though the two centrioles of a single centrosome are similar in their overall architecture, they are structurally and functionally distinct in that only one has fully matured (Piel et al., 2000; Azimzadeh and Bornens 2007). Mature centrioles are characterized by the presence of two sets of appendages (distal and subdistal; Paintrand et al., 1992) at their distal ends where they are attached to each of the nine centriolar MT doublets. Appendages have been shown to be involved in anchoring MTs and the centriole at the plasma membrane during ciliogenesis (Piel et al., 2000;

Azimzadeh and Bornens 2007) through characterization of several appendage proteins,

INTRODUCTION

e.g. as -tubulin, Cep164, Cep170, ninein, and the ODF-2 splice variant hCenexin1 (Mogensen et al., 2000; Chang et al., 2003; Guarguaglini et al., 2005; Ishikawa et al., 2005; Graser et al., 2007a; Soung et al., 2009).

2.1.2 The Centrosome as the Microtubule-organizing Center (MTOC) The most evident function of the centrosome lies in the orchestration of the microtubule network in eukaryotic cells as the microtubule organizing center (MTOC). Herein, the centrosome mediates the nucleation and anchoring of microtubules by the centrosomeassociated γ-tubulin containing multiprotein ring complexes (γTuRCs). At the hub of the microtubule network, the centrosome is involved in the orchestration of cell motility, cell shape, cell adhesion, cell polarity and intracellular transport (reviewed in Doxsey 2001;

Bornens 2002; Nigg 2004; Doxsey et al., 2005; Azimzadeh and Bornens 2007; Bornens 2008). During cell division, the centrosome shapes the bipolar mitotic spindle to ensure faithful chromosome segregation (reviewed in Marshall 2009). The centrosome has also been attributed an essential function in asymmetric cell divisions, e.g. in stem cell divisions (Wang et al., 2009). In contrast to the requirement for centrosomes as the MTOC in most eukaryotic cells, eukaryotes naturally lacking centrosomes have devised alternative mechanisms for spindle formation, as has been observed in higher plants and certain fungi (reviewed in Marshall 2009).

2.1.3 The Centriole as Template for Cilia and Flagella



Pages:   || 2 | 3 | 4 | 5 |   ...   | 12 |


Similar works:

«Die Eroberung der Zeit Grundzüge einer Philosophie verlängerter Lebensspannen Bearbeitet von Sebastian Knell 1. Auflage 2015. Buch. 750 S. Hardcover ISBN 978 3 518 58619 8 Format (B x L): 14,5 x 22 cm Gewicht: 853 g Weitere Fachgebiete Philosophie, Wissenschaftstheorie, Informationswissenschaft Wissenschaften Interdisziplinär Zeit: philosophische, technische, soziale Aspekte schnell und portofrei erhältlich bei Die Online-Fachbuchhandlung beck-shop.de ist spezialisiert auf Fachbücher,...»

«PROVISION OF ADVANCED ANCILLARY SERVICES THROUGH DEMAND SIDE INTEGRATION Miguel Luís Delgado Heleno Dissertation submitted to the Faculty of Engineering of University of Porto in partial fulfilment of the requirements for the degree of Doctor of Philosophy Thesis Supervisor: Manuel António Cerqueira da Costa Matos Thesis Co-supervisor: João Abel Peças Lopes Full Professors at the Department of Electrical and Computer Engineering Faculty of Engineering, University of Porto July 2015 This...»

«LEARNING AND TEACHING GUIDES Guiding Student Dissertations Dr Eileen Kennedy Roehampton University This series of Learning and Teaching Guides has been commissioned and funded by the Higher Education Academy Network for Hospitality, Leisure, Sport and Tourism Learning and Teaching Guide: Guiding Student Dissertations This guide was authored by: Dr Eileen Kennedy Dr Eileen Kennedy Eileen Kennedy is Director of the Centre for Scientific and Cultural Research in Sport in the School of Human and...»

«CHANNEL ALLOCATION FOR BROADBAND FIXED WIRELESS ACCESS NETWORKS A dissertation submitted to the University of Cambridge in partial fulfillment of the requirements for the degree of Doctor of Philosophy Shin Horng Wong, Queens’ College May 2003 Laboratory for Communication Engineering Department of Engineering University of Cambridge DECLARATION I, Shin Horng Wong of Queens’ College hereby declare that this dissertation and the work reported in it are originated entirely by myself in the...»

«Die Befunde der „Großen Halle“ von Karakorum Die Ausgrabungen im sogenannten Palastbezirk Text und Abbildungen Inaugural-Dissertation zur Erlangung der Doktorwürde der Philosophischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität zu Bonn vorgelegt von Christina Franken aus Bergisch-Gladbach Bonn 2012 Gedruckt mit Genehmigung der Philosophischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Zusammensetzung der Prüfungskommission: Prof. Dr. Peter Schwieger...»

«Robust Multiscale Control-volume Methods for Reservoir Simulation PhD Thesis Andreas Sandvin Department of Mathematics University of Bergen January, 2012 Preface This dissertation is submitted as a partial fulfilment of the requirements for the degree Doctor of Philosophy (PhD) at the University of Bergen. The work has been conducted at the Centre for Integrated Petroleum Research (CIPR) in collaboration with the Department of Mathematics at the University of Bergen. The dissertation is...»

«WALKING THROUGH THE SHADOWS: RUINS, REFLECTIONS, AND RESISTANCE IN THE POSTCOLONIAL GOTHIC NOVEL A Dissertation Submitted to the School of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Sheri Ann Denison Indiana University of Pennsylvania May 2009 Indiana University of Pennsylvania The School of Graduate Studies and Research Department of English We hereby approve the dissertation of Sheri Ann Denison Candidate for the degree of...»

«Video Game Values Play as Human-Computer Interaction by Pippin Barr A thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Doctor of Philosophy in Computer Science. Victoria University of Wellington ii iii Abstract Video games are a form of software and thus an obvious object of study in Human-Computer Interaction (HCI). Interaction with video games differs from the usual understanding of HCI, however, because people play video games...»

«DIPLOMARBEIT Titel der Diplomarbeit OBJEKT-RAUM-HANDLUNG Performanz und Ritualdynamik im Werke Anish Kapoors Verfasserin Elisabeth Viktoria Würzl angestrebter akademischer Grad Magistra der Philosophie (Mag. phil.) Wien, August 2012 Studienkennzahl lt. Studienblatt: A 000 315 Studienrichtung lt. Studienblatt: Kunstgeschichte Betreuerin: Univ. Prof. Dr. Deborah Klimburg-Salter DANKSAGUNG Mein Dank gilt zuallererst Frau Prof. Dr. Klimburg-Salter, die mich in meiner Arbeit ermunterte und...»

«СОЦИОЛОГИЧЕСКИЕ НАУКИ УДК 316.614 Плотников Владимир Валериевич Plotnikov Vladimir Valeriyevich кандидат философских наук, PhD (Philosophy), Assistant Professor, доцент кафедры философии и социологии Philosophy and Social Science Department, Краснодарского университета Krasnodar University of the Ministry of МВД РФ Internal Affairs of Russia...»

«Wer schreibt vor? Ursprünge der moralischen Präskriptivität Kurt Bayertz Preprints of the Centre for Advanced Study in Bioethics Münster 2011/13 Preprints of the Centre for Advanced Study in Bioethics Münster 2011/13 Wer schreibt vor? Ursprünge der moralischen Präskriptivität* Kurt Bayertz Mai 2011 I 1. – Es ist ein Charakteristikum moralischer Normen, dass sie mit einem Sollen verbunden sind. In philosophischer Terminologie ist auch von ihrer „Präskriptivität“ oder...»

«Do-It-Yourself Devices Personal Fabrication of Custom Electronic Products David Adley Mellis SB Mathematics Massachusetts Institute of Technology, June 2003 MA Interaction Design Interaction Design Institute Ivrea, June 2006 SM Media Arts and Sciences Massachusetts Institute of Technology, September 2011 Submitted to the Program in Media Arts and Sciences School of Architecture and Planning in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Media Arts and...»





 
<<  HOME   |    CONTACTS
2016 www.abstract.xlibx.info - Free e-library - Abstract, dissertation, book

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.