A green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium.
We transformed Dictyostelium discoideum cells by a vector for expression of a chimerical fusion protein consisting of Aequorea Victoria green fluorescent protein (GFP) and D. discoideum actin at its amino- and carboxy-terminal, respectively. The amount of expressed GFP-actin was about 3% of total actin molecules in the transformed cells. The expression of GFP-actin in D. discoideum completely inhibited cytokinesis in suspension culture. The expression decreased the rate of random cell locomotion to about a half of that of control cells. The expression also caused the cells to round up. These phenotypic observations suggested that GFP-actin acts as a dominant negative form of actin in the cells. The rounding up by expression of GFP-actin was suppressed by genetical elimination of myosin II heavy chain. This result suggested that myosin II is necessary for the rounding up of GFP-actin expressing cells. GFP-actin constructed cortical actin filament architectures together with intrinsic actin in the cells. Purified GFP-actin polymerized and de-polymerized repetitively according to the solution conditions in vitro. The critical concentration of GFP-actin for polymerization is the same as that of actin. The GFP-actin filaments was able to bind to coverglass surfaces coated with myosin head fragments. However, the GFP-actin filaments did not slide at all on the coverglass by addition of ATP. This indicates that the GFP-actin filaments form rigor complex with myosin II in vitro even in the presence of ATP. The formation of rigor complex may cause the cells to round up.
Aizawa
H
H
Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Japan.
Sameshima
M
M
Yahara
I
I
D000199:Actins / Q000502:physiology*; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D004023:Dictyostelium / Q000166:cytology* / Q000502:physiology; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology*
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins