The Developmental Role of Membrane in the Cellular Slime Mold, Dictyostelium Discoideum

Citation

Tuchman, Jessica (1974) The Developmental Role of Membrane in the Cellular Slime Mold, Dictyostelium Discoideum. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8272-S965. https://resolver.caltech.edu/CaltechTHESIS:03062018-093229873

Abstract

This thesis concerns the role of the cell surface membrane in cell-cell interactions. Specifically, the purpose of these experiments was to discover whether the aggregation of the cellular slime mold Dictyostelium discoideum is accompanied by, and perhaps dependent on, specific differentiation of the cell surface membrane, and if so, whether such components could be isolated in an active state. To this end, partially purified cell surface membranes were prepared from both vegetative (0 hour) and developing (14 hour)cells. The membranes were characterized by sucrose gradient centrifugation, SDS polyacrylamide gel electrophoresis, and electron microscopy. It was found that membranes from both 0 and 14 hour cells possessed the ability to inhibit the developmentally controlled aggregation of slime mold cells when mixed with these cells and plated under normal laboratory condition. HeLa cell membranes, even at the highest obtainable concentrations, were inert in this respect. Aggregation phase membranes were able to prevent cell aggregation at significantly lower concentrations than was required for vegetative membranes, and were also markedly more resistant to heat degradation. It appears that aggregation phase membranes block aggregation by preventing the attainment of aggregation competence in the developing cells, whereas vegetative membranes appear to act through a direct competition for available aggregation antigen receptor sites on the cell surface.

The effect of the differentiated membranes on several develop­mentally controlled enzymes was tested. Membrane treatment leads to the induction of some developmentally controlled enzymes and the re­pression or excretion of ethers. In one case, alkaline phosphatase, enzyme induction occurs 12 hours earlier than in nonnal cells, and the enzyme reaches approximately double its normal activity. The distribution of effected and uneffected enzymes bears no resemblance to the normal sequence of enzyme induction. The only characteristic with which the membrane effect can be linked, is the intracellular localization of the effected enzyme. The results indicate that there are some difficulties in the generally accepted view of the slime mold developmental program, and point out the crucial role played by the formation and maintenance of cell-cell contacts during normal development.

A study of the changes in protein synthesis during slime mold development was also undertaken. Total cell protein was displayed on SDS polyacrylamide gels after a 2 hour pulse label. It was found that during the first few hours of development, a single major band accounts for more than 20 percent of the total protein on the gel. Actin was purified from vegetative cells by a known procedure, and was found to correspond to the major band in several respects. The discovery of a single protein being synthesized in such quantity, and its identification as actin, provide a powerful tool for the isola­tion of a specific messenger RNA molecule, and for an intensive study of all the factors involved in regulating protein synthesis in a eucaryotic organism.

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