Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Mark Winey

Second Advisor

Andreas Hoenger

Third Advisor

Thomas Perkins

Fourth Advisor

Gia Voeltz

Fifth Advisor

Ernst-Ludwig Florin

Abstract

Many cells and organisms react to depletion of nutrients with an energy saving program. Here we studied Schizosaccharomyces pombe (S. pombe; fission yeast) cells and how they respond to nutrient starvation by entering a quiescent state that is characterized by a substantial viscosity increase of the cytoplasm, which we term "cytoplasmic freezing". Recently we found evidence that the transition in viscosity of the cell cytoplasm could be reliably reproduced by starving S. pombe cells of glucose. Also, there is evidence that septins, a GTP binding protein family, might be involved in generating and maintaining the frozen cytoplasmic state. Here I have confirmed by light microscopy that in the absence of Spn2p the phenomenon of “cytoplasmic freezing” did not happen or occurred much later (Florin, unpublished). Taking advantage of confocal fluorescence microscopy, I have identified the relocation of Spn3p in wild type and Spn2 deleted S. pombe cells during exponential growth conditions and compared them to the situation upon up to seven days of glucose starvation. In addition using conventional electron microscopy and cryo-electron microscopy, I have imaged for the first time the formation of filamentous septin in starved S. pombe cells. Similar septin bundles have been reported for different conditions, but not during the kind of starvation conditions reported here (An et al., 2004). I have further confirmed that absence of Spn2 relocates Spn3p upon starvation in S. pombe by immuno-labeling combined with conventional and cryo-electron microscopy. Also, I have observed by confocal microscopy that neither the deletion of Spn2 nor the "cytoplasmic freezing" state in starved cells affect actin location in S. pombe. However, actin behaves substantially different before versus after starvation, and during exponential growth its distribution is radically different between wildtype and Spn2 deletion strains. These significant details can help our understanding of the role of septin during glucose starvation in S. pombe or even in other organisms in the future. Furthermore, by conventional electron microscopy and cryo-electron microscopy I have demonstrated the shape change and fission of mitochondria from elongating tubular structures to separated small ovals upon glucose starvation in S. pombe. I have also directly observed that during glucose starvation ribosomes tightly assemble at the outer membrane of mitochondria, which is a phenomenon that has not been shown in detail anywhere and its relation to glucose starvation is not well understood. During all the structural studies, I have also tested conditions of a new method that we named cryo-pickling, loosely in association of pickling vegetables etc. to make them tender and/or to preserve them by incubation with a brine solution. Here we tested whether it will be possible to make frozen-hydrated specimens accessible (e.g. vitrified sections) for antibodies or staining solution delivered to the frozen-hydrated specimen by a low-freezing (mix of) solvent(s). Cryo-pickling is a potential freeze substitution method designed to stain or label samples for vitrified sectioning and cryo-EM. This method might provide important information for future cryo-sample preparation improvement.

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