Interacción funcional entre la maquinaria que regula el crecimiento polarizado, la citocinesis y las rutas de MAP quinasas de estrés e integridad celular

Resumo

Cytokinesis, the process that allows the physical separation of cells after mitosis is complete, involves the formation of a contractile ring (CAR) consisting of actin and myosin II filaments, whose activity is finely regulated by phosphorylation of their regulatory light chain (RLC). In eukaryotic cells, MAPK signalling pathways play a key role during the adaptive response to environmental signals. The correct assembly and organisation of actin filaments depends critically on the activation and/or inhibition of several signalling pathways, including MAPKs. The fission yeast Schizosaccharomyces pombe has established itself as an excellent model organism to study various mechanisms linked to MAPK signalling pathways and cytokinesis, due to its high degree of evolutionary conservation. In addition, the dimorphic fission yeast Schizosaccharomyces japonicus is an attractive model for exploring evolutionary divergence within the genus Schizosaccharomyces. S. japonicus has the additional features of defective respiration, a highly dynamic actin cytoskeleton and semi-open mitosis. Thus, they have positioned themselves as simple but exceptional models for evolutionary biology studies. Based on this background, in this PhD Thesis we have focused on the study of the functional interaction between the machinery regulating polarised growth, cytokinesis and the MAPK stress response pathway in fission yeast in order to analyse evolutionary divergence in two model organisms of the genus Schizosaccharomyces. To this end, I have addressed three main objectives: 1. Study of the mRNA-binding protein Rnc1 as a possible regulator of the activity of the MAPKs stress response pathway. 2. Functional relationship between the activity of myosin II by phosphorylation of its regulatory light chain and the availability of actin cables in response to changes in metabolism mediated by the activity of the SAPK pathway. 3. Evolutionary divergence of the control of cytokinesis and dimorphism mediated by myosin II activity through phosphorylation of its regulatory light chain. Both wild-type strains and different mutants of S. pombe and S. japonicus obtained by genetic manipulation or from other sources (research groups and strain collections), as well as different experimental techniques, have been used to achieve the objectives described above. These include: i- The construction of yeast mutant strains by genetic manipulation (deletions; targeted mutagenesis; genomic fusions). ii- Molecular biology techniques (PCR; qPCR) iii- Protein analysis (protein purification; SDS-PAGE; detection of phosphorylation status and protein levels by Western blotting; immunoprecipitation; in vivo/in vitro phosphorylation assays; PhosTag) iv- Microscopy techniques (F-actin staining and confocal fluorescence time-lapse microscopy). The results as a whole allow us to draw the following conclusions: - The mRNA-binding proteins constitute another foothold as regulators to modulate the magnitude of the central MAPK response of diverse MAPK pathways by positively or negatively regulating the stability of mRNAs involved in MAPK activation or inhibition. - Cytokinesis is a process that is highly influenced by nutritional conditions. It is therefore a perfectly coordinated and regulated process where the regulation of myosin II activity by phosphorylation of its RLC is critical for cytokinesis to be carried out properly. - Evolutionary divergence in Schizosaccharomyces species has resulted in the development of two cytokinesis control mechanisms dependent and independent of RLC phosphorylation as a modulator of myosin II activity.