Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells.
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.


CONCLUSIONS
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.
Nakahara
Hirokazu
H
The First Department of Oral & Maxillofacial Surgery, Osaka University Graduate School of Dentistry, Suita 565-0871, Japan. nakahara@dent.osaka-u.ac.jp
Otani
Tomohiro
T
Sasaki
Takuya
T
Miura
Yasuhiro
Y
Takai
Yoshimi
Y
Kogo
Mikihiko
M
D000199:Actins / Q000032:analysis; D045744:Cell Line, Tumor; D021961:Cell Membrane Structures / Q000737:chemistry / Q000648:ultrastructure; D048429:Cell Size; D005353:Fibronectins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D009361:Neoplasm Invasiveness; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000502:physiology*
D000199:Actins; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors; D019869:Phosphatidylinositol 3-Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins