Chemotaxis is a dynamic process that involves directional sensing, cell polarity, and cell motility. Cells continuously rearrange their cytoskeleton and plasma membrane, resulting in an asymmetric cell shape, and periodically extend pseudopods. Actin polymerization in pseudopods at the leading edge of the cell is synchronized with contractile forces generated by myosin motor proteins at the rear. A directional sensing system biases pseudopodia formation towards the source of the chemoattractant and thus orients cell movement along the extracellular chemical gradient. To understand the molecular mechanisms of chemotaxis, a variety of assays have been used to dissect the individual sub-reactions involved in Dictyostelium.
This model organism is an excellent system for the study of cell migration. The molecular mechanisms underlying chemotaxis, such as actin polymerization, intracellular signaling, and cell migration, are highly conserved among eukaryotes. Therefore many powerful experimental tools used to dissect these processes in other organisms are applicable to Dictyostelium. When there are sufficient nutrients Dictyostelium cells proliferate as haploid single amoebae; however, when nutrients are depleted starvation immediately triggers the developmental program for surviving harsh conditions through spore formation. Chemotaxis plays an essential role in Dictyostelium development. During differentiation, ~100,000 cells migrate toward aggregation centers that release the chemoattractant cAMP and form multicellular structures. Differentiating cells secrete cAMP every 6 min and waves of extracellular cAMP reinforce the expression of the cAMP receptors and other signaling molecules that are required to respond to cAMP.
Differentiation normally takes several hours and the chemotactic ability peaks at 5–6 hrs after starvation. Around this time, cells establish an increased cell polarity due to down-regulation of basal cytoskeletal activity and become highly sensitive to chemoattractant stimulation. Thus, the developing amoebae display robust and rapid chemotactic responses.
In this chapter, we will describe how Dictyostelium mutants can be analyzed to determine whether and how they are defective in chemotaxis. Cell movement toward chemoattractants can be examined by direct observation using time-lapse microscopy. Quantification of cell movement and shape provides information on cell polarity, directionality, and rate of cell migration. In addition, the many biochemical reactions involved in chemotaxis can be examined in mutant and wildtype cells. Assays for chemoattractant-receptor interactions, G-protein activation, phosphatidylinositol (3,4,5)-triphosphate (PIP3) production and activation of Tor complex 2 (TorC2) and Ras signaling are described in this chapter. It should be noted that these biochemical reactions are often localized and therefore it is critical to determine where the reactions occur using microscopic approaches in addition to biochemical measurements. These assays will reveal the molecular mechanisms underlying chemotaxis and the function of proteins involved in this process. Identification of genes that are mutated in chemotaxis-defective mutants will help us understand the function of proteins involved in chemotaxis.