Figure 2.

Cortactin is required for adhesion-dependent cell edge protrusion. (A) Domain structure of RFP-tagged cortactin mutants used in this study. Cortactin has an NTA domain required for Arp2/3 binding followed by 6.5 cortactin repeats (shaded). A Pro-rich region (white box) contains three Tyr (Y) that can be phosphorylated by Arg and is followed by an SH3 domain. Cortactin 3F contains Tyr to Phe (F) point mutations in the phosphorylatable Tyr (Y421F, Y466F, and Y482F). The C-terminal SH3 domain is deleted in cortactinΔSH3 (aa 1–494). Cortactin W525A contains a mutation in a key Trp in the SH3 domain (W525A) that abrogates SH3 binding to ligands. (B–H) Representative frames from 10-min time-lapse videos of cells plated on 10 µg/ml fibronectin. For kymography analysis, a radial grid of eight lines was placed over the phase images (as shown in B), and kymographs were constructed for each of the indicated lines. WT (n = 24 cells; B), WT expressing empty pSuper (pS) RNAi vector (n = 23 cells; C), cortactin KD (n = 26 cells; D), cortactin KD expressing RNAi resistant FL cortactin-RFP (n = 20 cells; E), cortactin 3F–RFP (n = 20 cells; F), cortactinΔSH3-RFP (n = 17 cells; G), and cortactin WA–RFP (n = 23 cells; H) are shown. (I and J) Quantification of the number of protrusions (I) and retractions (J) per 10-min video. Mean ± SEM. ANOVA between all cell types: protrusions, P < 0.0001; retractions, P < 0.0001. Fisher’s protected least significant difference (PLSD) for all cells versus WT: *, P ≤ 0.0002. Bars, 10 µm.