Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein–coronin fusion protein

AbstractBackground: The highly motile cells of Dictyostelium discoideum rapidly remodel their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a cytoplasmic actin-associated protein that accumulates at the cortical sites of moving cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins and is known to interact with actin–myosin complexes. In coronin null mutants, cell locomotion is slowed down and cytokinesis is impaired.Results We have visualized the redistribution of coronin by fluorescence imaging of motile cells that have been transfected with an expression plasmid containing the coding sequence of coronin fused to the sequence encoding the green fluorescent protein (GFP). This coronin–GFP fusion protein transiently accumulates in the front regions of growth-phase cells, reflecting the changing positions of leading edges and the competition between them. During the aggregation stage, local accumulation of coronin–GFP is biased by chemotactic orientation of the cells in gradients of cAMP. The impairment of cell motility in coronin null mutants shows that coronin has an important function at the front region of the cells. The mutant cells are distinguished by the formation of extended particle-free zones at their front regions, from where pseudopods often break out as blebs. Cytochalasin A reduces the size of these zones, indicating that actin filaments prevent entry of the particles.Conclusion These data demonstrate that coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of a nascent leading edge, where it participates in the reorganization of the cytoskeleton. Monitoring the dynamics of protein assembly using GFP fusion proteins and fluorescence microscopy promises to be a generally applicable method for studying the dynamics of cytoskeletal proteins in moving and dividing cells

Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein

AbstractBackground: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a ‘leading edge’ of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum.Results: Purified GFP–actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP–actin made up 10–20% of the total actin. Microfilaments containing GFP–actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP–actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP–actin and phalloidin fluorescence, but there were distinct sites in which GFP–actin was more prominent.Conclusions: Double labeling with GFP–actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP–actin are networks or bundles of filamentous actin. Other clusters of GFP–actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins