A EUKARYOTIC TISSUE DENSITY SENSOR

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This proposal concerns eucaryotic mass effectors: extracellular molecules that allow individual cells within a tissue to sense the mass of the entire tissue. Such molecules would be centrally involved in the regulation of growth during development wound healing and tissue regeneration. Disruption of the masssensing mechanism could lead to tumor formation. As a model system, we will use the conditioned medium factor (CMF) secreted by developing Dictyostelium discoideum cells. In submerged monolayer culture, Dictyostelium cells differentiate at high cell densities but not at low densities; cells at low densities will however differentiate in medium in which a high density of cells was previously starved (a conditioned medium). An explanation this phenomenon is that during development, Dictyostelium cells need to be able to sense whether they are far from an aggregation center and thus need to continue expressing aggregation specific genes, or whether they are at or near an aggregation center and thus need to be continue expressing aggregation specific genes, or whether they are at or near an aggregation center and thus can begin expressing differentiation- specific genes. Since developing Dictyostelium cells do not divide, CMF is a mass effector rather than a mitogen or growth factor. In higher eucaryotes, one could envision similar mechanisms with different effectors and receptors so that liver cell could sense the number of other liver cells, pancreas the number of other pancreas cells, etc. Fractionation of the conditioned medium shows that the activity that allows low density cells to differentiate copurifies with a 70 kD protein as well as with a ~5 kD molecule; interestingly, the two molecules both have the same activity and do not need to be combined to allow differentiation. The 5 kD CMF's and whether they are related and/or if one is derived from the other. The gene encoding the 70 kD CMF will be isolated and sequenced for comparison with other known proteins. If the 5 kD CMF is a polypeptide, its gene will similarly be characterized. The physiological role of CMF will be examined by characterizing the temporal regulation of its secretion, which cell types secrete it, possible factors that might regulate its secretion and the extent of its interaction with the Dictyostelium cAMP mediated chemotaxis mechanism. The long term goal is to understand on a molecular basis the entire transduction mechanism whereby cells sense whether they are in the presence of a large mass of other cells.