Developmental mechanisms of the cellular slime mold, Dictyostelium discoideum
Bishop, John David
Gomer, Richard H.
Doctor of Philosophy
Our laboratory has shown that an extracellular signalling protein, Conditioned Medium Factor (CMF), acts as a cell-type specific cell-density sensing factor that is required for cells of Dictyostelium discoideum to undergo both differentiation and morphogenesis during development. The work pursued in this thesis was initiated both to determine how CMF controls development and to discover additional components of the developmental pathway. Chapter one describes investigations into the CMF signal transduction pathway. In the first section, it is demonstrated that CMF null cells fail to produce viable spores and furthermore that application of exogenous cAMP pulses rescues the inability of the CMF null cells to develop. It is further demonstrated that in CMF antisense cells CMF is required for cAMP-induced cGMP production. In the second section it is demonstrated that CMF does not affect the phosphorylation state of the cAMP receptor cAR1, nor does CMF affect the ability of cAR1 to adopt either the low affinity AL form or the super slow disassociating BSS form. However, CMF does slightly increase the ability of high levels of cAMP to induce a loss of ligand binding to cAR1. These data are contrary to previous reports, and reasons for these discrepancies are discussed. In the third section, it is demonstrated that the cAR1 phosphorylation sites are not required for the CMF effect on cAMP stimulated GTPase activity, cAMP-stimulated calcium influx, or low cell density aggregation. Finally, in the fourth section, it is shown that although CMF binds a G-protein coupled receptor that is linked to Galpha1, and the cAMP and CMF cell surface binding sites are very tightly linked, the sites must be two separate proteins because the linkage between the binding sites is disrupted in Gbeta-mutants. Furthermore, heterologously expressed cAR1 binds cAMP but not CMF. Chapter two describes the search for novel developmental mutants. The initiation of a mutagenesis screen is described, as are the cloning and partial characterization of five mutants. These mutants are caused by disruptions of genes encoding a novel deubiquitinating enzyme, an extracellular signal regulated kinase, a novel uridine diphosphoglucose pyrophosphorylase isozyme, and two completely novel proteins.
Molecular biology; Cell biology; Biochemistry