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In depth analysis of the role of essential kinases in regulation of meiotic chromosome segregation in Schizosaccharomyces pombe

Final Report Summary - AEKSP (In depth analysis of the role of essential kinases in regulation of meiotic chromosome segregation in Schizosaccharomyces pombe)


Executive Summary:

Meiosis is a specialized cell cycle that enables diploid organisms to reproduce sexually by generating haploid gametes through two successive divisions. To depict the detailed regulatory mechanism of meiotic chromosome segregation, the relevant proteins need to be identified and characterized. It is widely accepted, that protein kinases play important role and regulate multiple biological processes, including meiosis. Up to now, several protein kinases have been shown to be implicated in regulation of meiotic chromosome segregation. However, some protein kinases are essential for mitotic growth, a fact that precluded their detailed analysis.

The proposed project was aimed to analyse in more detail the molecular functions of essential protein kinases and define how these protein kinases regulate meiotic chromosome segregation. As an reintegration Marie Curie fellowship (ERG-AEKSP-268167; 2010-2013), the grant was intended to facilitate the consolidation of the fellow upon the completion of his postdoctoral stay at the Max F. Perutz Laboratories, University of Vienna, Austria supported by a previous Intra-European Marie Curie fellowship (IEF-NPKM-220518; 2008-2010). As a straightforward continuation of the previous Marie Curie fellowship, the project was successfully implemented by using the skills and knowledge gained during the previous training. The proposed project was implemented in parallel with another competitive projects with shared objectives funded by the Austrian Science Fund (P21437, P23609 and F34).

The main achievements of the project includes:

- relevant scientific publications
- strengthening of existing research networks and starting new ones between the fellow group and other groups in Europe and the world
- successful consolidation of the researcher as an independent and mature group leader in Europe

Protein phosphorylation has been demonstrated to be crucial in the proper regulation of nearly all cellular processes. It supplies irreversible unidirectional means of protein synthesis and degradation. Until recently, the role of essential protein kinases was scarcely studied due to unavailability of suitable mutants. The progress in the field of essential protein kinases required the development of new alleles which will enable to decrease/turn off the activity of selected protein kinase and recover/turn it on if needed. Only by having such tools, the real scientific significance of our understanding of molecular function of essential protein kinases would be unveiled. During IEF Marie Curie and reintegration Marie Curie fellowships, we developed and applied the chemical genetic strategy to create the conditional analog-sensitive alleles of essential protein kinases of Schizosaccharomyces pombe (Cipak et al., Cell Cycle, 2011, doi: 10.4161/cc.10.20.17792). Inhibition of particular protein kinases during meiosis and analysis of meiotic chromosome segregation under such condition revealed that seven of essential protein kinases play important role for faithful meiotic chromosome segregation. To elucidate the regulatory functions of these kinases, their role in meiotic recombination, in mono-orientation of sister kinetochores and in protection of centromeric cohesion were analysed. In addition, the proteomic approach was applied to gain further insights into the kinases functions through identification of kinases targets and their posttranslational modifications. Currently, we continue in molecular analysis of biological role of essential protein kinases by analysing substrate specificity of these protein kinases towards proteins co-purified and in analysis of which stages of meiosis are affected after decreasing the activity of particular essential protein kinases by applying ImageStream and live cell approaches (experiments are part of recently approved follow-up grants of ERG fellow).

Importantly, we develop a novel conditional analog-sensitive allele of Pat1 protein kinase (pat1-as2, L95A) as an improved tool to induce synchronous meiotic cultures at optimal temperature (Cipak et al., Cell Cycle, 2012, doi: 10.4161/cc.10.20.17792; Cipak et al., Nature Protocols, in press). This allele eliminates some abnormalities observed in pat1-114-induced meiosis at 34°C. Further improvement of the pat1-as2-induced meiosis by introducing ectopic expression of genes from both mat loci activated mating pheromone-signalling pathway, which in turn improved the fidelity of chromosome segregation and spore viability. Detailed molecular analysis of DNA intermediates of recombination in pat1-as2 mutant at 25°C revealed that DNA double-strand breaks at most hotspots are similarly abundant as at 34°C, except of a few hotspots which are distinctly deficient at 25°C. Similarly as at 34°C, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of pat1-as2 system for assaying of recombination intermediates in S. pombe at 25°C and provide new information on the mechanism of meiotic recombination (Hyppa et al., Nucleic Acid Research, 2013, doi:). Newly introduced pat1-as2 allele provides a boost to dissecting the special features of meiosis.

In regard to ERG project and evoked follow up projects, the results will help us to better understand the molecular mechanism how chromosome segregation is regulated during meiosis and in the long-run also the principles of gametogenesis as well as the reason of genetic disorders resulting from chromosome missegregation.

Currently, the Marie Curie ERG fellow holds permanent group leader position at Cancer Research Institute of Slovak Academy of Sciences in Bratislava, Slovakia (starting October 2013). He obtained sufficient financial support to continue work on the role of essential protein kinases in regulation of chromosome segregation during meiosis and mitosis. By the time this report was prepared (November 2013), the fellow was awaiting the final decision on three additional grants (one national a two FP-7 projects). The fellow´s group consists of 5 postdoctoral scientists, one PhD student and a lab technician.