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Characterization of Drosophila Cyclin J and Mps1

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Characterization of Drosophila Cyclin J and Mps1 Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2011 Characterization of Drosophila Cyclin J and Mps1 Althoff, Friederike Erika Andrea Abstract: Der fehlerfreie Ablauf des Zellzyklus erfordert eine genaue Regulation an bestimmten Kon- trollpunkten. Die Steuerung stellt sicher, dass der Wechsel von der jeweiligen Zellzyklusphase in die nächste nur möglich ist, wenn die zellulären Vorgänge, die für die vorangehende Zellzyklusphase charak- teristisch sind, sämtlich ohne Fehler abgeschlossen wurden. Cycline, die mit Cyclin-abhängigen Kinasen (Cdks) interagieren, sind für diese Steuerung wesentlich. Die Aktivitäten der verschiedenen Cyclin-Cdk- Komplexe begünstigen bestimmte Übergänge. Sie werden durch komplexe Kontrollpunkt-Signalwege gesteuert, die aktiv bleiben, solange die jeweilige Zellzyklusphase unvollständig oder fehlerbehaftet ist. So können Kontrollpunkt-Signalwege den Ablauf des Zellzyklus unterbrechen und dadurch Zeit zum Nachbessern oder zum Abschluss der zellulären Vorgänge zur Verfügung stellen. Der erste Teil dieser Arbeit beschreibt die Charakterisierung eines bisher wenig untersuchten Cyclins – Cyclin J. Nach seiner ursprünglichen Identifizierung in einem Yeast-two-Hybrid- Screen, wurde angenommen, dass Drosophila Cyclin J an der Kontrolle der spezialisierten früh- embryonalen Mitosen beteiligt ist. Nachträglich wurde jedoch entdeckt, dass es im gesamten Reich der mehrzelligen Tiere konserviert ist. Paraloge kommen daher in vielen Spezies vor, die nicht durch syncytiale Teilungszyklen, welche charakteristischerweise nur in der frühen Embryogenese bei Insekten vorkommen, gehen. Cyclin J muss daher eine allgemeinere Funktion jenseits der spezialisierten Zyklen von Insektenembryos haben. Sein Expressionsmuster in Drosophila zu- grundelegend, schien eine meiotische Funktion wahrscheinlich. Allerdings zeigten genaue Fertilitätstests und meiotische Missegregationstests an Fliegen ohne Cyclin J-Funktion keine Defekte. Des weiteren und im Gegensatz zu früheren Behauptungen beeinflusste der Verlust von Cyclin J den Ablauf der frühen em- bryonalen Zyklen nicht. Biochemische Analysen identifizierten Cyclin J als Interaktionspartner von Cdk1. Interessanterweise wird Cyclin J jedoch nicht während der Mitose degradiert wie die anderen bekannten Cdk1-Interaktionspartner, die mitotischen Cycline A, B und B3. Der zweite Teil dieser Arbeit unter- sucht die Funktion von Mps1, einer Proteinkinase, die bekanntermaßen am Spindelkontrollpunkt (SAC) beteiligt ist. Der SAC verhindert Segregationsfehler der Chromosomen während der Mitose, indem er auf fehlerhafte Verknüpfungen zwischen Kinetochoren und mitotischer Spindel anspricht. Die vorüberge- hende Anreicherung von Mps1 an Kinetochoren von Prophase bis Metaphase korreliert mit SAC-Aktivität. Daher wurden verschiedene Aspekte der Kinetochor-Lokalisierung von Mps1 in Drosophila untersucht. Es wurde gezeigt, dass Mps1-Rekrutierung an die Kinetochore von dessen eigener Kinaseaktivität und teilweise von der SAC-Komponente Mad1, welche in einem Komplex mit Mps1 vorliegt, abhängt. Das Verschwinden von Mps1 von den Kinetochoren vor der Metaphasen-zu-Anaphasen-Transition korreliert nicht mit substantieller Mps1-Degradation oder Veränderungen in dessen N-terminaler Phosphorylierung. Allerdings wurde gezeigt, dass die präzise Kontrolle von Lokalisierung und Menge an Mps1 entscheidend für eine erfolgreiche Mitose ist. Weiterhin deuten meine Untersuchungen darauf hin, dass Mps1 eine bisher unerkannte Funktion haben könnte, die die Schwester-Chromatiden-Trennung in einer SAC-unabhängigen Art und Weise inhibiert. Summary Error-free progression through the cell division cycle requires careful regulation at several distinct control points. The regulation assures that the transition from a given cell cycle phase into the subsequent phase is only permitted when the cellular processes characteristic of the preceding phase have all been completed without mistakes. Cyclins which interact with Cyclin-dependent kinases (Cdks) are central for this regulation. The activities of different Cyclin-Cdk complexes promote distinct transitions. They are inhibited by intricate checkpoint pathways that remain active as long as cell cycle steps remain incomplete or afflicted by mistakes. Thereby checkpoint pathways can pause cell cycle progression and provide time for repair or completion of cellular processes. The first part of this work describes the characterization of a poorly analyzed Cyclin - Cyclin J. After its original identification in a yeast two-hybrid screen, Drosophila Cyclin J was implicated in the control of the specialized early embryonic mitoses. However, Cyclin J was subsequently found to be conserved throughout the metazoan radiation. Paralogs are therefore present in many species that do not progress through syncytial division cycles as characteristically observed during early insect embryogenesis. Cyclin J must therefore have a more general function beyond the control of the specialized cycles of early insect embryos. Based on its expression pattern in Drosophila a meiotic function appeared likely. However, careful fertility and non- disjunction assays with flies completely lacking Cyclin J function did not reveal any defects. Moreover, in contrast to earlier claims, loss of Cyclin J did also not affect progression through the early embryonic cycles. Biochemical analyses identified Cyclin J as an interaction partner of Cdk1. Interestingly Cyclin J is not degraded during mitosis in contrast to the other known Cdk1 interaction partners, the mitotic Cyclins A, B and B3. The second part of this work investigates the function of Mps1, a protein kinase that is known to be involved in the spindle assembly checkpoint (SAC). The SAC prevents chromosome segregation errors during mitosis by responding to incorrect attachments of kinetochores to the mitotic spindle. The transient accumulation of Mps1 at kinetochores from prophase to metaphase correlates with SAC activity. Therefore, several aspects of Mps1 kinetochore localization in Drosophila were ad- dressed. Mps1 recruitment to kinetochores is shown to be dependent on its own kinase activity and in part on the SAC component Mad1, which is present in a complex with Mps1. The disappearance of Mps1 from kinetochores before the metaphase-to-anaphase transition does not correlate with substantial Mps1 degradation or changes in N- terminal phosphorylation. However, the precise control of localization and levels of Mps1 was shown to be crucial for a successful mitosis. Moreover, my analyses suggested that Mps1 might have a previously unrecognized function that inhibits sister chromatid resolution in a SAC-independent manner. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-82286 Dissertation Published Version Originally published at: Althoff, Friederike Erika Andrea. Characterization of Drosophila Cyclin J and Mps1. 2011, University of Zurich, Faculty of Science. 2 Characterization of Drosophila Cyclin J and Mps1 Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Friederike Erika Andrea Althoff aus Deutschland Promotionskomitee Prof. Dr. Christian F. Lehner (Vorsitz) Prof. Dr. Erich A. Nigg Zürich, 2011 Thesis Overview Table of Contents Contents PART 1—Thesis Overview 5 Summary 5 Zusammenfassung 7 Thesis Introduction 9 Cell Cycle Regulation by Cyclin-Cdks and Checkpoints ...............................................................9 Cyclins—Central Regulators of the Cell Cycle ............................................................................10 Drosophila Cell Cycle Regulation by Cyclins...............................................................................10 Cyclin J ........................................................................................................................................11 SAC—Mechanism and Effect ......................................................................................................12 Kinetochore and Mitotic Spindle ..................................................................................................16 The SAC Components Mps1 and Mad1 ......................................................................................18 Spindle Poisons and Their Impact on Research and Clinic .........................................................19 Meiosis ........................................................................................................................................20 Thesis Objectives 22 The Function of Cyclin J during Early Development....................................................................22 The Infl uence of Level, Localization, and Activity of Mps1 on the SAC .......................................23 Thesis Previews 24 The Function of Cyclin J during Early Development....................................................................24 The Infl uence of Level, Localization, and Activity of Mps1 on the SAC .......................................25 References 28 PART 2—Cyclin J 37 Abstract 39 Introduction 39 Materials and Methods 40 Fly Strains and Genetics .............................................................................................................40 Plasmid Constructions .................................................................................................................40
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