To complement studies being conducted by NCI investigators to understand the signaling cascades that occur during chemotaxis in Dictyostelium discoideum, we are determining the high-resolution three-dimensional arrangement of vesicles that are observed by fluorescence light microscopy in migrating Dictyostelium. Cells are introduced into plastic capillary tubes under a cAMP gradient to provide conditions of directed cell migration. The capillary tubes are then quickly frozen at high pressure to preserve the cellular ultrastructure. The frozen blocks are freeze-substituted, embedded in plastic, sectioned to a thickness of 0.3 to 0.5 micrometers, stained and analyzed by automated electron tomography in an energy-filtering 300 kV field-emission transmission electron microscope. Experiments are in progress to reconstruct whole cells to visualize ultrastructural differences between the anterior and posterior regions of the migrating amoebae.
