Figure 6: MTSS1 interacts with Rho-cofilin signalling pathways.

(a) B16 melanoma cell transiently expressing EGFP-MTSS1 (green) together with mCherry-VASP (red). MTSS1 localized in the cytoplasm along with VASP (yellow, overlay of EGFP-MTSS1 and mCherry-VASP), and along the cell periphery of the protruding (white arrow) but not the retracting (yellow arrow) front. Inset, the protruding front of another melanoma cell depicting EGFP-MTSS1 localization in filopodial extensions identified by mCherry-VASP at their tips (cyan arrow). A representative picture is shown from a series of slides. Scale bar, 5 μm. (b) Migration of empty-vector and ectopic MTSS1-expressing HMEL cells through fibronectin-coated culture inserts for 20 h after RNA interference with cofilin expression for 48 h. Bars represent the mean of five random microscopic fields ±s.d. from three replicates. Data were compared with control-treated cells (Ct.siRNA) using the two-tailed Student’s t-test, *P≤0.05, ***P≤0.001, NS: not significant. (c) Left panel, immunoblotting for phospho(Ser3)-cofilin and cofilin levels in empty-vector and ectopic MTSS1-expressing HMEL cells in the presence of fibronectin (10 μg ml⁻¹). Middle panel, pull-down fractions from a Rac activity assay immunoblotted for active (Rac-GTP) and total Rac. Right panel, pull-down fractions from a RhoA activity assay immunoblotted for active (Rho-GTP) and total Rho. Bottom panel, corresponding densitometric plots. (d) Upper panel, pull-down fractions from a Rho activity assay with ectopic MTSS1-expressing HMEL cells in the presence of excess GTPγS or GDP and immunoblotted for MTSS1 and RhoA. Lower panel, densitometric quantification of the immunoblots.