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 expressi on of a chimerical fusion protein consisting of Aequorea Victoria gree n 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 expres sion 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 a lso caused the cells to round up. These phenotypic observations sugges ted that GFP-actin acts as a dominant negative form of actin in the ce lls. The rounding up by expression of GFP-actin was suppressed by gene tical elimination of myosin II heavy chain. This result suggested that myosin LI is necessary for the rounding up of GFP-actin expressing ce lls. GFP-actin constructed cortical actin filament architectures toget her with intrinsic actin in the cells. Purified GFP-actin polymerized and de-polymerized repetitively according to the solution conditions i n vitro. The critical concentration of GFP-actin for polymerization is the same as that of actin. The GFP-actin filaments was able to bind t o 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 comple x with myosin II in vitro even in the presence of ATP. The formation o f rigor complex may cause the cells to round up.