FIGURE 9.

Kymograph analysis of IRSp53, Dyn1, Mena, and Eps8 during filopodia assembly and disassembly. A, filopodia were followed as described in Fig. 6. The kymographs are dual channel analysis of one filopodia from each cDNA transfection, with lifeact marking F-actin (panels i–v) and GFP (panel vi), IRSp53 (panel vii), Dyn1 (panel viii), Mena (panel ix), Eps8 (panel x). B, schematic of the tip complex is shown with the circle representing the diameter of the filopodia. IRSp53 dimers with the I-BAR forming a zeppelin shape (gray) (50) with two SH3 domains per dimer attracting protein partners to the barbed end of actin filaments. Cdc42 (filled circles) brings IRSp53 to membrane sites and activates the protein exposing I-BAR and SH3 domains. Oligomerization of IRSp53 I-BAR domain in vitro induces the protrusion of membranes in the shape of a cylinder with a specified diameter (51). The IRSp53 SH3 domains attract partners to membrane sites associated with short actin filaments. Our previous work suggests the short actin filaments may be generated by IRSp53-N-Wasp·Toca-1 complex (19). The kymograph analysis shows that IRSp53 levels remain relatively constant through the lifetime of a filopodia. In contrast, Dyn1, Mena, and Eps8 arrive in sequence marking initiation, assembly, and disassembly phases (circles and arrows in A). This timeline of protein arrival fits with the role of Dyn1 in extending barbed ends (30), Mena in preventing actin capping (52), and Eps8 in capping actin (53). Thus, we hypothesize that filopodial dynamics are regulated by switching of IRSp53 partners, Dyn1, Mena, and Eps8. Further work will be required to establish how these IRSp53 partners (Dyn, Mena, and Eps8) compete with each other and modulate actin dynamics during filopodia formation. C, key to A and B.