Figure 3

Tir binds cortactin and promotes its activation of Arp2/3-mediated actin polymerization. (A) Cortactin binds Tir through the N-terminal and the SH3 domains in vitro. Recombinant Tir was incubated with WT cortactin (FL) and with the three mutants expressed as GST fusion proteins: the SH3 domain mutant (W525K), the N-terminal (NH2) domain and the SH3 domain (SH3). GST alone served as a negative control. The pull-downs were subjected to SDS-PAGE and blotted with anti-Tir monoclonal antibody. In the last line, one-fifth of the total amount of Tir that was used as input per pull-down sample (upper left panel). Coomassie staining of proteins is also shown (lower panels). Half of the GST-proteins and on-fifth of Tir that was used in the pull-downs was stained with Coomassie blue. (B) Cortactin binds Tir independently of phosphorylation in vitro. Recombinant Tir was incubated with WT cortactin (FL) and with the three mutants expressed as GST fusion proteins: Arp2/3 domain mutant (W22A), Erk-phosphorylation-mimicking mutant (SD) and Src-phosphorylation- mimicking mutant (3D). GST alone served as a negative control (right upper panel). Coomassie staining of proteins is also shown (lower panels). Half of the GST-proteins and one-fifth of Tir that were used in the pull-downs were stained with Coomassie blue (shown in A). (C) Immunofluorescence images of Tir-coupled beads incubated with actin, Arp2/3 and cortactin/mutants. Carboxilate beads (diameter 1 μm) uncoupled (left panels) or coupled to Tir protein (right panels) were incubated with a solution of 500 nM WT cortactin or cortactin mutants proteins for 1 hour. Then, actin and Arp were added and incubated at RT to allow actin polymerization. After 1 hour TRITC-phalloidin was added, and the samples were observed immediately on a microscope. Pictures were taken at 600× magnification. Scale bar 40 μm.