Use of Green Fluorescent Protein-Conjugated β-Actin as a Novel Molecular Marker for in Vitro Tumor Cell Chemotaxis Assay

摘要

To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a β-actin–GFP fusion protein was generated. The expression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed β-actin–GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The β-actin–GFP was also observed to be reorganized in response to treatments with the chemoattractant type IV collagen. Cells extended pseudopodial protrusions and altered the morphology of their cortical structure in response to type IV collagen stimulation. More importantly, β-actin–GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that β-actin–GFP could participate in actin polymerization. Although ectopic expression of β-actin–GFP lead to minor side effects on cell proliferation, these studies suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues.