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UBC Theses and Dissertations

Endocytosis and membrane lipid composition in wild-type and mutant Paramecium tetraurelia Pollock, Carol

Abstract

Eighty-two temperature-sensitive mutants of Paramecium tetraurelia that involve some aspect of endocytosis or vacuolar processing were isolated following nitrosoguanidine treatment. Two of these, the recessive mutations fvc (food vacuole clumping) and dml (defective membrane) , were examined in some detail. At the restrictive temperature, 34.5°C, the food vacuoles of fvc cells clumped in one area of the cell, usually the posterior. Fewer food vacuoles accumulated in fvc cells than in wild-type cells after 20 minutes in a food vacuole marker, such as blue watercolor (BP). The fvc mutation was originally recovered as a double mutation and was linked by 34 ± 3 (standard deviation) map units to the tnd (temperature-sensitive, trichocyst non-discharge) locus. The mutant, dml, had morphologically abnormal vacuoles which, in the light microscope, appeared as a mass of fused or disrupted vacuoles. An ultrastructural study revealed that the structural integrity of the food vacuoles was lost. Abnormal mitochondria and vesicular structures were also observed, as well as cells in which large portions of the cytoplasm were missing, presumably having been digested by the hydrolytic enzymes that were released from the food vacuole. A gas-liquid chromatographic analysis of wild-type and mutant cells indicated that the major fatty acids were: palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), docosatetraenoic (22:4), tetracosadienoic (24:2), and tetracosatetraenoic (24:4). With an increase in temperature from 27°C to 34.5°C the percent composition, in wild-type whole cell lipids, of 16:0 increased and that of 24:4 decreased. However, in dml cells, the opposite occurred, i.e. the percent composition of 16:0 decreased and 24:4 increased. In wild-type cells these changes were reflected mainly in the fatty acids of the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) with only slight changes occurring in the phosphonolipid, 2-aminoethylphosfhonolipid (AEPL). In dml cells, the fatty acids of PC, PE and AEPL varied with temperature in a manner similar to the pattern observed with whole dml cells. The unsaturation index (U.I. = number of double bonds per 100 acyl groups) decreased in wild-type cells, in whole cell fatty acids and particularly in PE, with an increase in temperature. However, the U.I. in dml whole cell fatty acids and phospholipids increased with an increase in temperature. A survey of several chemical agents indicated that dibucaine, sodium dodecyl sulfate and colchicine inhibited endocytosis. The mutant, fvc, was more sensitive to sodium dodecyl sulfate and colchicine than wild-type celis. .Dimethyl sulfoxide produced partial phenocopies of the dml mutant in wild-type cells. After treatment with 6-12% dimethyl sulfoxide the food vacuoles fused and separation of the nascent food vacuole from the gullet was inhibited., dmj. cells were more sensitive to dimethyl sulfoxide than were wild-type cells. The results indicated that the mutant, fvc was abnormal in some aspect of intracellular transport which may involve microtubules or microfilaments. The mutant, dml, did not undergo the same changes in fatty acid composition as wild-type cells in response to an increase in temperature. The vacuolar membranes in this mutant degenerated and material which was normally sequestered in the vacuoles was released, resulting in cellular destruction.

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