USE OF A FUSION PROTEIN BETWEEN GFP AND AN ANTI-BINDING DOMAIN TO VISUALIZE TRANSIENT FILAMENTOUS-ACTIN STRUCTURES

Many important processes in eukaryotic cells involve changes in the qu antity, location and the organization of actin filaments [1-3], We hav e been able to visualize these changes in live cells using a fusion pr otein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequ orea victoria and the 25 kDa highly conserved actin-binding domain (AB D) from the amino terminus of the actin cross-linking protein ABP-120 [4], In live cells of the soil amoeba Dictyostelium that were expressi ng GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cell s coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin, On the ventral surface of n on-polarized vegetative cells, a broad ring of F actin periodically as sembled and contracted, whereas in polarized cells there were transien t punctate F actin structures; cells cycled between the polarized and non-polarized morphologies, During the formation of pseudopods, an inc rease in fluorescence intensity coincided with the initial outward def ormation of the membrane,This is consistent with the models of pseudop od extension that predict an increase in the local density of actin fi laments, In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneo usly.