Figure 6: Phosphorylation of ACF7 on tyrosine 259 promotes focal adhesion dynamics and cell motility in vitro.

(a) Lysates are collected from WT, ACF7 KO and ACF7 KO cells re-expressing ACF7 or ACF7-Y259F mutant. Lysates were analysed by SDS–PAGE and immunoblotting with different antibodies as indicated. (b) Immunofluorescence for focal adhesions (vinculin, red) and exogenous ACF7 (green) shows localization of WT but not Y259F mutant of ACF7 at focal adhesions. Boxed areas are magnified as insets. Scale bar, 20 μm. (c) Confluent monolayers of keratinocytes with different genotypes were subjected to in vitro scratch-wound assays. Phase contrast images of the site were taken at hours (h) indicated after scratch-wounding. (d) Quantification of the kinetics of in vitro wound closure. Error bar represents s.d. One-way ANOVA indicates that the difference between WT versus KO, or KO versus KO+ACF7 or KO+ACF7 versus KO+ACF7-YF is statistically significant (P<0.05). (e) Box and whisker plots of cell velocities (left panel) and focal adhesion disassembly (right panel) in different keratinocyte cell lines as indicated. One-way ANOVA indicates that the difference between WT versus KO, or KO versus KO+ACF7, or KO+ACF7 versus KO+ACF7-YF, or KO+ACF7 versus KO+ACF7-SD or KO+ACF7 versus KO+ACF7-YFSD is statistically significant (P<0.05). (f) Behaviour of microtubule targeting to focal adhesions was monitored by confocal videomicroscopy and manually traced. Targeting frequencies to individual focal adhesions were quantified and depicted by bar graphs. Error bar represents s.d. One-way ANOVA indicates that the difference between WT versus KO, or KO versus KO+ACF7, or KO+ACF7 versus KO+ACF7-YF is statistically significant (P<0.05). ANOVA, analysis of variance.