Fig. 7

Perturbation of Hax1-IQGAP1 interaction inhibits cell migration. A, Coomassie Blue-stained SDS-PAGE gel was used to assess the quality of purified recombinant TAT-GST-GFP-His₆ and TAT-RGCT-GFP-His₆ proteins. Escherichia coli BL21(DE3) expressed TAT-RGCT-GFP peptide and TAT-GST-GFP (spike-in control) were purified with nickel-nitrilotriacetic acid affinity chromatography and desalted into DMEM. The respective TAT peptides are marked by arrows. B, Directional migration in MCF7 cells treated with TAT-GST-GFP or TAT-RGCT-GFP peptide were evaluated by in vitro scratch wound assays. Aliquots of TAT-GST-GFP or TAT-RGCT-GFP (2.5 μM) were added to confluent monolayers of MCF7 cells for 1 h and were scratched, followed by visualization with phase-contrast microscopy at the indicated time points. C, The kinetics of in vitro wound healing in B are quantified. Note that treatment of TAT-RGCT-GFP peptides leads to a significant delay of in vitro wound healing compared with the control peptides (n = 3, p < 0.05, Student’s t test). D, Real-time imaging of movements of individual MCF7 cells treated with TAT-GST-GFP or TAT-RGCT-GFP peptide. The migration paths of randomly picked transfected cells for each group are presented here as scatter plots (n = 20). E, Relative migration speeds in F are shown as box and whisker plots. Statistical analysis with Student’s t test showed that treatment of TAT-RGCT-GFP peptides leads to a significant decrease in speed compared with the control peptides (p < 0.01, Student’s t test). F, Representative immunofluorescence images of MCF7 cells treated with TAT-GST-GFP or TAT-RGCT-GFP peptide were stained for Vinculin (magenta) and IQGAP1 (red). Aliquots of TAT-GST-GFP or TAT-RGCT-GFP (2.5 μM) were added to cultured MCF7 cells for 1 h followed by fixation, immunocytochemistry and imaged by super-resolution N-SIM microscopy. Scale bar, 10 μm. G, Co-localization of IQGAP1 and FA (vinculin) was determined by Pearson correlation coefficient (n = 20 cells for each). Note that IQGAP1 co-localization with vinculin at the rim of the leading edge is significantly decreased in MCF7 cells treated with TAT-RGCT-GFP peptides compared with the control peptides (p < 0.01). H, Protein abundance in isolated FAs or WCL (20 μg) from MCF7 cells treated with TAT-GST-GFP or TAT-RGCT-GFP peptide (2.5 μM) was determined by immunoblot with different antibodies as indicated. I, Proposed working model accounting for the Hax1 function in directional cell migration. In short, the Hax1-IQGAP1 complex serves as a novel link to orchestrate directional cell migration via affecting focal adhesion dynamics