Structural Proteins in Cellular Slime Molds

Dictyostelium discoideum is a free-living amoeba that lives on decaying organic matter in the soil or in the forest. It crawls about by locomotion, divides by cytokinesis, and eats bacteria in a process termed phagocytosis. When food is scarce, the cells come together by chemotaxis to create a multicellular organism that differentiates to form a mature fruiting body composed of a stalk extending up from the substrate with a sphere of spore cells at its tip.

The processes of phagocytosis, cytokinesis, and locomotion are mediated by actin and myosin in the cells. The structure and organization of the actin and myosin in the cells are regulated by actin-binding proteins. These actin-binding proteins help to organize and regulate the actin and myosin so that the proper structures are assembled at the correct site in the cytoplasm of the cell at the time when they are needed.

Four undergraduate students work in our laboratory to investigate a specific actin-binding protein, the 30,000-dalton actin-bundling protein of Dictyostelium discoideum, and its contributions to cell structure and movement. The DNA encoding the protein is being employed to create strains of the Dictyostelium that either lack the protein entirely (gene knockout; Chris's project), or that make too much of it (overexpression; Stephen's project). These reverse genetic experiments directly test the requirement of the 30,000-dalton protein for cell movements. Rob is trying to find the regions of the 30,000-dalton protein required to bind actin filaments. A combination of protein biochemistry and molecular genetics is needed for his experiments. Kristen is interested in how the 30,000-dalton protein can become localized at the proper place in the cell at the time when it is needed. She is using an epitope tag approach to identify signals in the amino acid sequence of the 30,000-dalton protein. These signals may target the protein to be localized to the phagocytic cup, the cleavage furrow, or regions of cell-to-cell contact in developing cells. An antibody that reacts with the epitope tag is used to determine the intracellular localization of known fragments of the protein. This antibody is also used to determine whether short pieces of the sequence of the intact protein are sufficient to act as signals to direct its localization in moving cells.

Experiments such as these are being performed in laboratories all over the world on dozens of cytoskeletal and contractile proteins that are highly conserved from a simple amoeba or a yeast to human cells. One day, we hope to understand how all of these proteins work together to control the shape of the cell and to produce the beautiful, highly coordinated movements that mediate essential process in the life of a cell.