Ameboid cells contain an actin cytoskeleton that must be continuously altered during locomotion. Such dynamics are even more dramatic during chemotactic stimulation when profound reorganizations of the actin cytoskeleton must be accomplished rapidly and repeatedly to establish the correct polarity of locomotion. This chapter describes assays that can be used to measure the nucleation activity, polymerization, and cross-linking of actin in cells. Since these assays were developed and/or adapted for use with Dictyostelium discoideum at a stage when it is a rapidly locomoting ameba, and, in particular, during chemotactic stimulation, these assays should be applicable to less dynamic cells and tissues. In order to measure the amount of F-actin in cells at a specific time during locomotion or following agonist stimulation, cells are rapidly fixed and then stained with one of the commercially available fluorescent phalloidin probes. Speed of fixation is the key to using this technique to assay fluctuations in the content of F-actin that occur rapidly in cells (on a seconds time scale) during locomotion or agonist stimulation. Changes in F-actin content occurring in less than 3 sec, as demonstrated by other techniques, can be detected with this fixative while fixation in formaldehyde alone in the absence of Triton can take 15 seconds or more and cause extensive cell surface ruffling.
ASJC Scopus subject areas
- Molecular Biology