Study of the chromosome structure of saccharomyces cerevisiae under stress response

Resumen

Chromosome structuring and condensation are the main characteristics of mitosis, in which the condensed chromosomes migrate to the equatorial plane of the cell, in order to segregate into two nuclei before the cytokinesis or cell division. Historically, several experimental model organisms have been used to study mitosis. In particular, the chromosome structure in the yeast Saccharomyces cerevisiae, only visible at the microscopic level on chromosome XII, has served for this purpose. The ribosomal DNA (rDNA) located in the long arm of this chromosome has been used to observed different phenomena of compaction, segregation and chromosomal structure at different phases of the cell cycle. The metaphase structure (“loop”) depends, among others, on the condensin complex; and its segregation during anaphase depends on that complex and on the cell cycle phosphatase Cdc14, which are essentials for it. This study aims to elucidate chromosome and nucleolar remodelling under different conditions (mid-Mitosis arrest (mid-M), thermal stress or Heat Shock (HS), and rapamycin treatment, among others, and the influence of the condensin complex, Cdc14 and the TOR pathway in this process. For this, different tagged proteins that bind to the rDNA locus were used for their monitoring and visualisation with fluorescent and confocal microscopy, besides, several molecular and immunological techniques were employed. An auxin based degron system to degrade proteins by the proteasome was applied, in order to study the role of specific proteins in this process. Here, I show that the rDNA loop is dramatically changed upon incubation at 37ºC in wild type yeast strains. Hence, I have checked again the roles of condensin, Cdc5, Cdc14 and Cdc15, but making use of the novel auxin-mediated degradation of aid alleles as an alternative to temperature sensitive (ts) alleles, showing that the classical findings of ts alleles for these four players still prevail. Also, I demonstrate that this novel mechanism of rDNA condensation in metaphase is dependent on TORC1 but independent of Cdc14. Furthermore, I show that the vacuole, which occupies a large proportion of the cell volume, serves as a template to reconfigure the nuclear morphology during nuclear envelope (NE) expansion in mid-M, and thus emerges as a major determinant in the morphology of the rDNA loop. The NE often acquires projections that contain the rDNA and distal parts of chromosome XII. These projections often bend themselves around the vacuole and reshape the nucleus towards a bilobed morphology, with one lobe containing most of the nuclear mass and with the nucleoplasmic handle that connects both lobes forming the rDNA loop. Alternatively, the rDNA in the projection opens up and blossoms into a horseshoe loop, leaving a NE ladle underneath. I further show that this reorganisation of the nuclear shape requires an active TORC1 and new membrane synthesis but is independent of reported nuclear-vacuole and rDNA-NE contacts. I propose here that the apparently separated mechanisms and players that have an impact on the morphology of the rDNA, can be united and reconciled by these findings. Moreover, it has been suggested that the reorganisation and size of the nucleolus, the ribosomal biogenesis and nucleolar stress could be novel targets in cancer therapy. The results of this work were published in a series of articles in peer-reviewed scientific journals.