Control ambiental de la división y diferenciación celular en levaduras con fisión

Abstract

In eukaryotic cells the evolutionarily conserved mitogen-activated protein kinase (MAPKs) signaling pathways play an essential role during the adaptive response to environmental cues. The actin cytoskeleton is a complex network of polymers that plays a fundamental role in the regulation of cellular processes such as motility, endocytosis, polarity, and cell division. The correct assembly and organization of actin polymers depends to a large extent on the coordinated activation and/or inhibition of many conserved proteins through different signaling cascades, including MAPKs. The rod-shaped fission yeast Schizosaccharomyces pombe has been established as an excellent model organism to study the mechanisms that activate MAPKs pathways and other cellular processes, since its genome is evolutionarily conserved as compared to higher eukaryotes, and it is also highly versatile for genomic, biochemical, and cell biology analyses. Nevertheless, although S. pombe has traditionally been used as the prototype fission yeast model, Schizosaccharomyces japonicus, another of the four species in this genus, is gradually becoming an attractive model in recent years. S. japonicus was the first species to diverge evolutionarily from the remaining species in the genus and, despite being non-pathogenic, it is a dimorphic organism that can grow as a yeast or differentiate to form hyphae in response to different environmental cues. S. pombe and S. japonicus also show distinctive features in key biological processes such as nuclear envelope remodeling during mitosis, division plane positioning, or cell polarity regulation. Based on these and other differences, both yeast species are optimal models for evolutionarily-related cell biology studies. Thus, the present Thesis has focused on the functional characterization of the stress-activated (SAPK) and cell integrity (CIP) MAPKs pathways in both fission yeasts from an evolutionary point of view, and their putative role as regulators of morphogenesis. To this end, I have addressed two essential objectives: 1. To study the evolutionary conservation of the stress-activated and cell integrity MAP kinase signaling pathways within the genus Schizosaccharomyces and their role during dimorphism in S. japonicus. 2. To investigate the role of stress-activated MAPKs as modulators of the integrity of the actin cytoskeleton and cytokinesis within the genus Schizosaccharomyces. To achieve the above objectives, I have employed both wild type and mutant S. pombe and S. japonicus strains obtained either by genetic manipulation or from alternative sources (i.e. from another research groups or strains collections), as well as different experimental techniques. These included: i-the construction of mutant yeast strains by genetic manipulation (gene deletion; site-directed mutagenesis; gene fusion to specific epitope tags), ii- molecular biology techniques (PCR; qPCR; RNAseq analysis), iii- protein analysis (protein tagging and purification; SDS-PAGE; detection of protein phosphorylation status and levels by Western blot; immunoprecipitation; in vivo/in vitro phosphorylation assays; PhosTag technology), and iv- microscopic techniques (confocal and wide-field fluorescence time-lapse microscopy). From the results obtained in this Thesis, the following general conclusions can be proposed: - The cell integrity MAPK module architecture is strongly conserved in S. pombe and S. japonicus, while the upstream activators Pck1 and Pck2 antagonistically regulate S. japonicus dimorphic switch through the precise control of the activity of MAPK Pmk1. Moreover, S. japonicus Pmk1 possesses a unique and evolutionarily distinct secondary structure that allows CIP signaling to comply with the specific developmental requirements of this fission yeast species. - S. japonicus features a cell density-depending quorum sensing mechanism mediated by aromatic alcohols that, together with the stress-activated MAPK pathway, negatively modulates the yeast to hyphae transition dimorphic switch in response to environmental changes. - The SAPK pathway negatively regulates the assembly of the contractile actomyosin ring (CAR) in S. pombe by decreasing the protein levels of the formin For3 in response to cytoskeletal damage induced with Latrunculin A (LatA). On the contrary, SAPK signaling positively contributes to CAR assembly in S. japonicus, thus reflecting the evolutionary adaptation of this signaling cascade to the marked differences in the execution of cytokinesis in both fission yeast species.