Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells
Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells
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DOI：
10.1111/j.1365-2443.2003.00695.x
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发表时间：
2003-12-01
期刊：
影响因子：
2.1
通讯作者：
Kogo, M
中科院分区：
文献类型：
--
作者：
Nakahara, H;Otani, T;Kogo, M
背景：侵袭伪足是侵袭性肿瘤细胞向细胞外基质的膜性突起。在体外纤连蛋白降解/侵袭测定中，这些结构与恶性肿瘤细胞的基质降解和侵袭性位点相关。Rho家族小G蛋白由Rho、Rac和Cdc 42亚家族组成，通过肌动蛋白细胞骨架的重组参与各种细胞功能，如细胞形状改变、粘附和运动。我们研究了Rho家族小G蛋白在侵袭伪足形成中的作用。结果：我们首次证明了RPMI 7951人黑色素瘤细胞的侵袭伪足在垂直方向上延伸到基质基质中，使用激光扫描共聚焦显微镜系统。我们证实，侵入伪足富含肌动蛋白丝（F-actin），并在垂直视图和水平视图上用F-actin染色清晰可见。然后，我们研究了Rho，Rac和Cdc 42在同一细胞系的侵袭性中的作用。在体外纤连蛋白降解/侵袭试验中，Cdc 42的显性活性突变体增强了点样降解，而Rac的显性活性突变体增强了扩散型降解。此外，frabin，GDP/GTP交换蛋白Cdc 42与F-肌动蛋白结合活性，增强点样和扩散型降解。然而，Rho的显性活性突变体不影响纤连蛋白的降解。结论：Cdc 42和Rac通过frabin-Cdc 42/Rac-PI 3 K信号通路协同参与纤维连接蛋白的降解和侵袭。
Background: Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation.Results: We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation.Conclusion: These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.