Figure 4. AVIL colocalizes with and interacts with PLCE1 and the ARP2/3 complex.

(A) Human podocytes transfected with a GFP-tagged, full-length (FL) mouse Avil construct were immunostained with mouse anti-paxillin (red), mouse anti-ARP3 (red), and rabbit anti-PLCE1 (red) antibodies, respectively. Note the colocalization of AVIL and ARP3 at podocyte lamellipodia (empty white arrowheads) and the colocalization of AVIL and PLCE1 at lamellipodia (solid white arrowheads). AVIL also overlapped with paxillin at FAs. Scale bars: 10 μm. Inset scale bars: 25 μm. (B) AVIL interacted with PLCE1 upon co-overexpression in HEK293T cells. GFP-tagged AVIL was co-overexpressed with Flag-tagged PLCE1 in HEK293T cells. Co-IP using Flag showed that GFP-tagged AVIL interacted with Flag-tagged PLCE1. (C and D) Upon co-overexpression in HEK293T cells, Myc-tagged ACTR2 (ARP2 complex) (C) and ACTR3 (ARP3 complex) (D) interacted with GFP-tagged AVIL. (E) Under EGF stimulation (100 ng/ml) in human cultured podocytes, PLCE1 (green) translocated to cell membrane ruffles, where it colocalized with ARP3 (red, solid white arrowheads). Cells depleted of AVIL by shRNA failed to recruit PLCE1 to the ARP-marked lamellipodia (empty white arrowheads) upon EGF stimulation. Scale bars: 10 μm. Inset scale bars: 25 μm. (F) Human podocytes were transfected with scrambled siRNA, siRNA-9, or siRNA-11 for AVIL. EGF stimulation increased the concentration of DAG in scrambled siRNA–treated control cells (black circles). siRNA-mediated knockdown of AVIL decreased active DAG levels (pink circles). The overexpression of WT Avil rescued this effect (green circles), while the 3 mutant clones (Arg135Gln, Leu425Met, and Phe656Valfs*7) from patients with SRNS did not (red circles). Individual data points were derived from 3 independent experiments and are displayed as the mean and SD. Statistical analysis was performed using a 2-tailed, 1-way ANOVA [F (7,16) = 92.09, P < 0.0001]. ***P < 0.0001, by Sidak’s multiple comparisons test.