A Green Fluorescent Protein-actin Fusion Protein Dominantly Inhibits Cytokinesis, Cell Spreading, and Locomotion in Dictyostelium

Cell Structure and Function

Masazumi Sameshima¹  Ichiro Yahara¹  Hiroyuki Aizawa¹ 

[1] Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science

关键词: actin;    green fluorescent protein;    dominant negative;    cytokinesis;    cell movement;   

DOI  :  10.1247/csf.22.335

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(34)Cited-By(28)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 aminoand 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.

【 授权许可】

Unknown