Use of Green Fluorescent Protein-Conjugated -Actin as a Novel Molecular Marker for in Vitro Tumor Cell Chemotaxis Ass
详细信息 查看全文
- 作者：Louis Hodgson ; Wei Qiu ; Cheng Dong ; and Andrew J. Henderson
- 刊名：Biotechnology Progress
- 出版年：2000
- 出版时间：December 2000
- 年：2000
- 卷：16
- 期：6
- 页码：1106 - 1114
- 全文大小：627K
- 年卷期：v.16,no.6(December 2000)
- ISSN：1520-6033
文摘
To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryoticexpression vector expressing a 
-actin-GFP fusion protein was generated. Theexpression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed -actin-GFP fusion protein, which colocalized with endogenous cellular actin as determinedby histoimmunofluorescence staining. The -actin-GFP was also observed to bereorganized in response to treatments with the chemoattractant type IV collagen. Cellsextended pseudopodial protrusions and altered the morphology of their corticalstructure in response to type IV collagen stimulation. More importantly, -actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicatingthat -actin-GFP could participate in actin polymerization. Although ectopic expression of -actin-GFP lead to minor side effects on cell proliferation, these studiessuggest that this strategy provides an alternative to the invasive techniques currentlyused to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues.
-actin-GFP fusion protein was generated. Theexpression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed -actin-GFP fusion protein, which colocalized with endogenous cellular actin as determinedby histoimmunofluorescence staining. The -actin-GFP was also observed to bereorganized in response to treatments with the chemoattractant type IV collagen. Cellsextended pseudopodial protrusions and altered the morphology of their corticalstructure in response to type IV collagen stimulation. More importantly, -actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicatingthat -actin-GFP could participate in actin polymerization. Although ectopic expression of -actin-GFP lead to minor side effects on cell proliferation, these studiessuggest that this strategy provides an alternative to the invasive techniques currentlyused to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues.