Use of green fluorescent protein-conjugated beta-actin as a novel molecular marker for in vitro tumor cell chemotaxis assay.
To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a beta-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 beta-actin-GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The beta-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, beta-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that beta-actin-GFP could participate in actin polymerization. Although ectopic expression of beta-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.
Hodgson
L
L
Department of Bioengineering, 229 Hallowell Building, and Department of Veterinary Science, 115 Henning Building, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Qiu
W
W
Dong
C
C
Henderson
A J
AJ
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002633:Chemotaxis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D014407:Tumor Cells, Cultured
D000199:Actins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins