Light microscopy to image and quantify cell movement.

For decades, Dictyostelium discoideum has been an efficacious and attractive model system for the study of cell motility, primarily because cells become highly motile during the transition from growth phase to aggregation competence and because the haploid genome is readily amenable to mutation. These crawling amoebae, as well as other motile cells such as polymorphonuclear neutrophils (PMNs), extend pseudopodia, retract pseudopodia, and translocate across a substratum even in the absence of chemoattractant. This phenomenon, referred to as basic motile behavior, has been investigated in Dictyostelium through analysis of cytoskeletal mutants. Likewise, many chemotactic signal transduction pathways and networks have been inferred from studies of Dictyostelium mutants. However, before concluding from mutational analyses that a particular molecule or protein plays a role in chemotaxis, it is imperative to first precisely define its contribution, if any, to basic motile behavior. Here, we describe two-dimensional and three-dimensional technologies that can be coupled with 2D and 3D Dynamic Image Analysis System (2D and 3D-DIAS) software for the analysis of cell motility, shape changes, pseudopod formation, and localization of tagged molecules during basic motile behavior. In addition, we describe a method to analyze the 3D trajectories of microspheres attached to the surface of crawling Dictyostelium cells. We include information on microscopy, image acquisition techniques, and computer hardware that could be reproduced in a typical laboratory setting for motion analysis using 2D and 3D-DIAS software. Finally, we highlight features available in DIAS that have proven insightful in identifying defects in basic motile behavior exhibited by various cytoskeletal and putative signal transduction mutants.