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Titel:Conditional Degrons to Study Gene Functions During Saccharomyces cerevisiae Gametogenesis and Proliferation
Autor:Renicke, Christian
Weitere Beteiligte: Taxis, Christof (PD Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0059
URN: urn:nbn:de:hebis:04-z2017-00592
DOI: https://doi.org/10.17192/z2017.0059
DDC: Biowissenschaften, Biologie
Titel(trans.):Konditionale Degrons zur Untersuchung von Genfunktionen während der Gametogenese und Proliferation von Saccharomyces cerevisiae
Publikationsdatum:2017-01-24
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Saccharomyces cerevisiae, Signaltransduktion, Mitotic Exit Network, Sporenbildung, Biowissenschaften, Synthetische Biologie, Genetik, Proteolyse, Cytologie, LOV2, Meiose, Conditional Degrons, TEV Protease, Spindle Polarity

Summary:
Diploid cells of Saccharomyces cerevisiae can form stable spores to ensure survival under poor nutritional conditions. Sporulation is a coupled developmental program of meiotic divisions and spore formation. The latter process is initiated at onset of meiosis II at the spindle pole bodies (SPBs), the yeast centrosome equivalents. The SPBs are embedded in the nuclear envelope and duplicate twice during meiosis in a mostly conservative fashion. Thus, three generations of SPBs are present in meiosis II. The first SPB inherited from mitosis, the second formed in meiotic pro-phase and the two youngest SPBs generated prior to meiosis II. At the onset of meiosis II the cytoplasmic faces of the SPBs are modified by meiotic plaques. They serve as nucleation platform for the prospore membranes, which grow around the nuclear lobes and close after meiosis II spindle breakdown. The spore wall is then formed in the lumen of the double-layered prospore membrane. Finally, the former mother cell collapses and forms the spore-containing ascus. Cells are able to adjust the spore numbers according to the available nutrients by reducing meiotic plaque protein levels to generate asci with less than four spores. This regulation is facilitated by meiosis II spindle polarity, which directs meiotic plaque formation towards the younger SPBs. Yet, the underlying mechanisms are poorly understood, although this process significantly contributes to preservation of genetic variability and population fitness by ensuring encapsulation of non-sister chromosomes in asci with only two spores. Here, I developed different synthetic tools to study the role of the mitotic exit network (MEN) in meiotic spindle polarity and spore number control of S. cerevisiae. The MEN is a conserved signaling cascade essential for vegetative growth. It coordinates mitotic exit with genome segregation and cytokinesis and establishes mitotic spindly polarity in metaphase. However, the meiotic functions of this network are mainly unknown due to the lack of reliable methods for creation of meiosis-specific mutants of the mainly essential proteins of the MEN. To overcome this obstacle, I pursued two different approaches to control the abundance of a protein with sequences inducing conditional degradation (degrons). 1. I established a photo-sensitive degron module which combines the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin 1 attached to a synthetic C-terminal degron. In the dark, this degron is sterically inaccessible. Upon blue-light illumination, structural rearrangements of the LOV2 domain lead to activation of the degron and degradation of the target protein it is fused to. 2. I improved an established system for protein destabilization, which employs tobacco etch virus (TEV) protease to activate a cryptic degron. Control of protease production by a meiosis-specific promoter has been used previously to study protein functions during sporulation. To develop a more efficient system, I followed two strategies in parallel: by directed evolution, I created a TEV protease variant with a higher substrate tolerance, allowing usage of stronger degrons. Independently, I combined transcriptional shut-off of the target gene upon initiation of meiosis with elevated protease levels during sporulation. The latter approach was used successfully to create meiosis-specific mutants of all core MEN components. I could demonstrate a role of the MEN in age-based selection of SPBs for meiotic plaque modification. Moreover, I found functional diversification of MEN components during sporulation. The upstream kinase Cdc15 is involved in regulation of meiotic plaque numbers and prospore membrane closure, while Cdc15 and the downstream kinase complexes consisting of Dbf2/20-Mob1 are all necessary for SPB selection at the onset of meiosis II. After the meiotic divisions, efficient genome inheritance requires Dbf2/20-Mob1 during subsequent spore wall formation. Together, these data reveal a developmental-specific plasticity of the signaling network. In contrast to mitosis, execution of meiosis does not require the MEN but faithful genome inheritance requires concerted action of different MEN components at distinct steps of spore formation.

Zusammenfassung:
Diploide Saccharomyces cerevisiae Zellen können Sporen bilden, um Mangelbedingungen zu überdauern. Sporulation bezeichnet eine Zelldifferenzierung, bei der Meiose und Sporenbildung verknüpft sind. Eingeleitet wird die Sporenbildung beim Übergang in Meiose II an den Spindelpolkörpern, den Zentrosomenäquivalenten der Hefe. Eingebettet in die Kernmembran werden diese während der Meiose zweimal durch einen vorwiegend konservativen Mechanismus verdoppelt. Daraus resultieren drei Generationen von Spindelpolkörpern in Meiose II: Der erste stammt aus dem vorangegangenen Zellzyklus, der zweite wird während der meiotischen Prophase gebildet und die dritte Generation entsteht vor Eintritt in die zweite meiotische Teilung. Zu Beginn der Meiose II formen sich an den zytoplasmatischen Plaques der Spindelpolkörper die sogenannten meiotischen Platten. Diese ermöglichen die Bildung der Prosporenmembranen, welche um die Ausstülpungen des Kerns herum wachsen und sich nach Zusammenbruch der Meiose II-Spindeln schließen. Die Sporenwand wird anschließend im Lumen der entstandenen Doppelmembran aufgebaut. Zuletzt kollabiert die Mutterzelle um die Sporen und formt den Ascus. Hefezellen sind in der Lage entsprechend der Nahrungsbedingungen Asci mit weniger als vier Sporen zu bilden indem sie weniger Proteine der meiotischen Platten produzieren. Ermöglicht wird diese Regulation durch die Polarität der Meiose II-Spindeln, durch welche meiotische Platten bevorzugt an den jüngeren Spindelpolkörpern geformt werden. Dieser Prozess stellt sicher, dass Asci mit nur zwei Sporen keine Schwesterchromatiden enthalten und trägt damit entscheidend zur Aufrechterhaltung der genetischen Vielfalt und Überlebensfähigkeit einer Population bei. Dennoch ist wenig über den zugrundeliegenden Mechanismus bekannt. In dieser Arbeit habe ich verschiedene synthetische Methoden entwickelt, um den Einfluss des „Mitotic Exit Networks“ (MEN) auf meiotische Spindelpolarität und Sporenzahlkontrolle zu erforschen. Das MEN ist ein konservierter Signaltransduktionsweg, der essentielle Funktionen während des vegetativen Wachstums erfüllt indem er die Aufteilung des Genoms mit der Zytokinese koordiniert. Außerdem kontrolliert er die Ausbildung der mitotischen Spindelpolarität während der Metaphase. Die Funktionen des MEN in der Meiose sind weitgehend unbekannt, da es an verlässlichen Methoden zur Herstellung von sporulationsspezifischen Mutanten mangelte, die nötig gewesen wären, um die für den Zellzyklus essentiellen Komponenten des MEN zu untersuchen. Zur Lösung dieses Problems habe ich zwei verschiedene Ansätze zur Kontrolle der Proteinmengen durch konditionale Degradationssequenzen (Degrons) gewählt. Erstens wurde ein photosensitives Degron etabliert, welches auf der Fusion eines synthetischen C-terminalen Degrons an die LOV2 Photosensor-Domäne des Phototropin 1 aus Arabidopsis thaliana basiert. Im Dunkeln ist das Degron maskiert während konformationelle Änderungen der LOV2-Domäne unter Blaulicht zur Aktivierung des Degrons und Abbau des markierten Zielproteins führen. Zweitens wurde ein bestehendes System zur Protein-Destabilisierung weiterentwickelt, welches die Tabak-Ätz-Virus-Protease verwendet, um ein geschütztes Degron zu aktivieren. In vorangegangenen Arbeiten ermöglichte die Regulation der Biosynthese dieser Protease durch einen meiosespezifischen Promotor die Aufklärung von Proteinfunktionen während der Sporulation. Zur Entwicklung eines effizienteren Systems habe ich zwei parallele Strategien verfolgt: Durch gerichtete Evolution wurde eine Variante der Protease erzeugt, welche durch eine verringerte Substratspezifität die Verwendung potenterer Degrons zulässt. Davon unabhängig habe ich eine meiosespezifische Deaktivierung der Zielgenexpression kombiniert mit einer Steigerung der Proteaseproduktion während der Sporulation. Dieser Ansatz konnte erfolgreich genutzt werden, um meiosespezifische Mutanten aller wesentlichen Komponenten des MEN zu erzeugen. So konnte gezeigt werden, dass dieser Signalweg die altersabhängige Entscheidung beeinflusst, an welchen Spindelpolkörpern meiotische Platten gebildet werden. Außerdem fand ich eine funktionelle Diversifizierung der MEN Komponenten während der Sporulation. Die vorgelagerte Kinase Cdc15 trägt zur Regulation der Anzahl von meiotischen Platten und der Schließung der Prosporenmembranen bei. Für die Spindelpolkörper-Auswahl zu Beginn der Meiose II sind sowohl Cdc15 als auch die nachgelagerten Komplexe aus Dbf2 bzw. Dbf20 und Mob1 notwendig. Nach den meiotischen Teilungen werden diese Komplexe für den Aufbau der Sporenwände und damit eine zuverlässige Weitergabe der haploiden Genome benötigt. Zusammengefasst zeigen diese Ergebnisse eine entwicklungsspezifische Plastizität des MEN: Das Signalnetzwerk wird nicht für das Durchlaufen der meiotischen Teilungen benötigt, verschiedene Komponenten wirken aber während bestimmter Schritte in der Sporenbildung und sichern so die erfolgreiche Weitergabe des Erbguts.

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