Critical to a cell's survival is its ability to deal with stress by making an appropriate response. This response often takes place in the cytoplasm, which is everything contained within the cell's plasma membrane that is not the nucleus. The cytoplasm is a dynamic environment and its ability to reorganize is essential to the cell's function. This dissertation presents a novel, previously undiscovered state of cytoplasm organization for the model system Schizosaccharomyces pombe, also known as fission yeast. Typically the fission yeast cytoplasm is a fluid-like environment in which endogenous lipid granules subject to thermal fluctuations, move freely as they explore their local surroundings through diffusion. When the cell is in a nutrient depleted environment it is exposed to the stress of advanced glucose starvation. As a result, we find that the cytoplasm undergoes drastic reorganization reminiscent of a phase transition; it is now a solid-like environment in which there is no visible motion. Lipid granules throughout the cell appear to be completely immobilized and are unable to move through the cytoplasm, despite the application of force through optical tweezers. We term this cytoplasmic state the cell frozen state. The cell frozen state is a physiological state, one that the cell can recover from with the addition of fresh nutrients. It is characterized by an anomalous diffusion exponent of [alpha] = 0.23 ± 0.01, which is a significant reduction from the anomalous diffusion exponent [alpha] = 0.66 ± 0.01 found for exponentially growing cells in which there is visible motion. To account for the cell wide immobilization of lipid granules, we hypothesize the formation of a polymer network all through the cytoplasm, and identify septins 1-3 as the most likely filament formers. In addition, we find there is an increase in the number of vacuoles in the cytoplasm during starvation, and propose a vacuole-septin model to describe the cytoplasm reorganization for the cell frozen state. / text

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

Cytoplasmic mRNA-containing granules in eukaryotic cells play key roles inthe storage, localisation and degradation of mRNA. In yeast, depletion of glucoseleads to the rapid inhibition of translation initiation and consequent appearance of Pbodiesand EGP-bodies. P-bodies contain factors of the mRNA decay pathway andtherefore, are likely to be sites in which mRNAs targeted for degradation arelocalised. In contrast, EGP-bodies lack decay components and contain onlytranslation initiation factors and RNA binding proteins. Thus EGP-bodies have beensuggested to be storage repositories for mRNAs that need to be rapidly translatedfollowing glucose readdition. In this study we utilised the m-TAG system to investigate the localisation ofendogenous MS2-tagged mRNAs with P-bodies and EGP-bodies. A triplefluorescent labelled system developed show that a class of unregulated mRNAslocalised into P-bodies following glucose starvation. It was also observed that thesespecific abundant classes of mRNAs can be found in aggregates prior to any cellularstress and upon glucose starvation these aggregates coalesce into larger granules thatcolocalise with P-body components. This coalescence of mRNA aggregatesfollowing glucose starvation does not rely upon the recruitment of mRNA decayfactors and appears to precede this event. Indeed mRNAs in mutants deficient in Pbodyformation still develop large aggregates following glucose stress. In unstressedcells it appears that the mRNA granules are implicated in high-level translation ofthese specific abundant mRNAs. Following the inhibition of translation initiation inresponse to stress, these granules nucleate P-body formation via aggregation and therecruitment of mRNA decay factors.