Imaging Actin Cytoskeleton Dynamics in Dictyostelium Chemotaxis

This chapter will focus on responses that the chemoattractant cyclic AMP elicits in the motility system of Dictyostelium . These cells can be permanently transfected to express cytoskeleton-associated proteins tagged with fluorescent proteins. Multiple proteins that are distinguishable by the excitation and emission spectra of their tags can be simultaneously expressed. This makes it possible to relate the spatial and temporal patterns of their chemoattractant-induced translocation to each other in one cell by a single recording. Since actin polymerization in live cells progresses with velocities of about 3 μm/s, high image frequencies and short acquisition times in the millisecond range are required. Techniques of total internal reflection fluorescence (TIRF) and spinning-disc confocal microscopy provide appropriate temporal and spatial resolution for the analysis of actin dynamics.