Date of Degree
Quorum sensing, the ability to measure the local cell density, is required for animal cells to achieve proper cell growth, differentiation and development. However, because of the genetic intractability of mammalian cells, we study quorum sensing in the genetically pliable unicellular eukaryote Dictyostelium discoideum. D. discoideum cells live as individual cells under vegetative conditions. Starvation sets off development to a multicellular organism. But cells won't develop unless enough cells are under starvation conditions. Approximately 105 cells are needed to form a fruiting body, the final stage of the development. When starved, cells are able to calculate the concentration of surrounding starving cells by simultaneously secreting and sensing a glycoprotein named conditioned medium factor (CMF). A high density of starving cells results in a high level of CMF, which allows cell aggregation and development. Here, we describe the role of a putative phospholipase D (PLD) homologue, PldB, in quorum sensing pathways. PldB is present during early development when quorum sensing is occurring as well as in later development. pldB- cells can aggregate at low cell density. This phenotype is cell autonomous. Cells overexpressing pldB are unable to aggregate or form fruiting bodies even at high cell density. These observations imply that PldB is involved in quorum sensing. CMF decreases cAMP-stimulated GTPase activity in wild-type cells, but not in pldB- cells, confirming that PldB mediates quorum sensing in the CMF signal pathway. pldB- cells develop faster than wild-type cells, indicating that pldB is involved in the timing of development. Analysis of early developmental gene expression shows that cAMP receptor cAR1, required in aggregation, is expressed at higher levels earlier in pldB- cells than in wild-type cells, which may explain the rapid development phenotype of pldB- cells. However, pldB- cells have normal chemotactic rate while pldBOE cells do not chemotax. Cell fractionation and Western blot analysis reveal that PldB protein is localized to cytoplasmic membrane as well as vesicles. PldB takes effect in quorum sensing through its phosphatidylcholine hydrolysis product, phosphatidic acid, since exogenous phosphatidic acid inhibits cell aggregation at low cell density, mimicking the phenotype of overexpressing pldB. Rapamycin promotes cell aggregation at lower cell densities for wild-type, pldB- and pldBOE cells, implying that TOR may have a role in quorum sensing downstream of PldB. The knowledge of PLD in quorum sensing will help us further understand this phenomenon in mammals, which may lead to therapeutic strategies for tissue/organ repair and cancer treatment.
Chen, Yi, "PldB, a Putative Phospholipase D Homologue, Mediates Quorum Sensing in Dictyostelium discoideum Development" (2007). CUNY Academic Works.