The Eps8/IRSp53/VASP Network Differentially Controls Actin Capping and Bundling in Filopodia Formation
Figure 6
Eps8:IRSp53 induces filopodia formation in VASP-deficient MVD7 cells after RNAi-mediated removal of CP.
a. RNAi-mediated downregulation of CP in MVD7 cells over-expressing IRSp53 increases filopodia formation. Control (WT scr) or CP (CP KD) RNAi-treated MVD7 cells transfected with Flag–IRSp53 were fixed and stained with rhodamine–phalloidine or anti-flag to detect F-actin (red) or IRSp53 (blue), respectively. Right panels, magnifications corresponding to the white dashed squares of the pictures on the left (the different channels are indicated). DI are digitalized images obtained with Adobe Photoshop filters starting from the actin channel to highlight cells protrusions [12]. Bar is 10 µm (4 µm for the magnifications). b. The expression of endogenous CP in cells interfered for CP (CP KD) or treated with scrambled oligo (WT scr) was analyzed by immunoblotting with the indicated abs. CP reduction (85%) was determined using the software ImageJ, by analyzing the intensity of the signals for CP in control cells (WT scr) or cells interfered for CP (CP KD), normalizing over vinculin signal. c. Change in RFI and FII (i.e., Eps8:IRSp53:Fa normalized by its wild type value, see main text) in WT and CP knockout MVD7 cells. Empty rectangles represent experimental results, filled rectangles simulations of equations in Table 1 in Text S1 and parameters in Table 2 and Table 3 in Text S1. d. Complexes formed in HeLa cells by Eps8, IRSp53 and Abi1 in CP Kd and WT plotted as percentage of total protein concentration in the wild type. Simulations as in c.