Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.
Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.
Pang
K M
KM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Lee
E
E
Knecht
D A
DA
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016764:Cell Polarity; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins