The responses of Chlamydomonas reinhardtii CW15+ (a wall-less mutant) to freezing stress have been examined: Tests following freezing of bulk (0.5ml) samples reveal the existence of an optimum cooling rate f o r preservation of viability, close to l*C min-1. Direct observation of cells during freezing and thawing on a cryo light microscope have allowed different forms of injury to be classified. At suboptimal cooling rates, reduction in surface area during shrinkage can be achieved by severe distortion or by the formation of layered stacks of membrane. Lethal injury is not evident until thawing takes place. The manifestation of injury depends upon the severity of the freezing treatment. The first symptom of injury is membrane blebbing at the cell surface, leading to swelling of the entire cell, followed by collapse. The membrane involved in the swelling might originate i n the mitochondrion and chloroplast envelope. Regions of fusion between membrane of these organelles and the plasma membrane have been observed during rewarming by electron microscopy. Fluorescent markers of the mitochondrial membrane have been detected i n the membrane involved in blebbing. Further damage during slow cooling contributes to rapid lysis on re-expansion. If the freezing treatment is still more severe, osmotic unresponsiveness will result. At supraoptimal cooling rates loss inviability is associated with intra cellular freezing. These responses have been examined in the light of available data regarding the freeze-thaw responses of higher plant protoplasts . The first symptom of damage seen in 'C reinhardtii ie. that of blebbing and massive swelling, is not common to both systems. Thus general rules concerning the responses of plant cells to freezing stresses cannot be made by using either of these protoplasts as a model system, although each may be useful for study of particular aspects of higher plant and algal cell freezing as separate investigations.

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