Comparison of differentiating Dictyostelium discoideum cell types separated by an improved method of density gradient centrifugation

Abstract

During the migrating slug stage of development of the cellular slime mold Dictyostelium discoideum, the precursor cells of the mature spores and stalk are known to differ from each other. In order to explore these differences on a variety of levels, we have improved existing methods for the isopyknic purification of the precursor types. Prespore and prestalk amoebae obtained from mechanically dissociated slugs were separated by centrifugation on preformed Percoll density gradients. The isolated precursor cells are greater than 90-95% pure, show good viability and developmental competence, and can be obtained in relatively large amounts. We have performed initial comparisons of the differentiating amoebae with respect to their location within the slug, protein composition, and developmental capabilities. Density centrifugation reveals heterogeneity among posterior slug cells: The majority of these cells have the expected prespore characteristics, but there is also a minor (lighter) density fraction resembling anterior, prestalk amoebae. Thirty polypeptides synthesized preferentially by prespore or prestalk slug cells were detected by two-dimensional gel electrophoresis and fluorography. Some of these proteins are equally cell-specific when their accumulation is measured by silver staining; others are remarkably non-specific when stained. When the isolated precursor populations were allowed to complete development, they differed in terms of developmental territory size and timing, and in ways which suggest differences in cell cohesion and chemotaxis.