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Conditional Degrons to Study Gene Functions During Saccharomyces cerevisiae Gametogenesis and Proliferation
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), th...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2016
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| Zusammenfassung: | 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. |
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| DOI: | 10.17192/z2017.0059 |