Fig. 1: OFD1 functions as a class II NPF to promote centrosomal actin branching. | Nature Communications

Fig. 1: OFD1 functions as a class II NPF to promote centrosomal actin branching.

From: An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesis

Fig. 1

a SDS-PAGE (Coomassie blue stained) and Immunoblot analysis of Flag pull-down samples, OFD1-Flag pulled down the purified 7-subunit Arp2/3 complex. b Immunoblot analysis of co-immunoprecipitation (IP) of OFD1-Flag with seven individual subunits of the Arp2/3 complex. c SDS-PAGE (Coomassie blue staining) analysis of F-actin pelleting assay. BSA and α-Actinin were used as negative and positive controls, respectively, S (Supernatant), P (Pellet). d OFD1 synergistic effect with class I NPF on actin polymerization. Polymerization of 0.875 μM 20% pyrene-labeled actin monomers was carried out in the presence of 20 nM Arp2/3 complex, 5 nM GST-VCA and 1000, 500 or 250 nM OFD1. e Representative imaging of ARP2 (green) and OFD1 (red) in RPE1 cells transiently transfected with control or OFD1 siRNA for 72 h and after fixation (Left Panel). 3D reconstruction of the zoom images (step size: 150 nm) were performed using Imaris Viewer software. ARP2 fluorescence integrated over a 1-μm-diameter circle around the centrosome for si-Control or si-OFD1 condition, 51 si-Control cells and 36 si-OFD1 cells examined over three independent experiments, P = 4.9 × 10−11, two-tailed unpaired student’s t-test (Right Panel). f Representative imaging of endogenous OFD1 (green) and F-actin (phalloidin, red, Gamma-adjusted (0.5)) in HeLa cells transiently transfected with control or OFD1 siRNA for 72 h and after fixation with PFA-PEM (Left Panel). 3D reconstruction of the zoom images (step size: 140 nm) were performed using Imaris Viewer software. F-actin fluorescence integrated over a 3-μm-diameter circle around the centrosome for si-Control or si-OFD1 condition, 35 si-Control cells and 43 si-OFD1 cells examined over three independent experiments, P = 7.05 × 10−6, two-tailed unpaired student’s t-test (Right Panel). g Sequence alignments of OFD1 and other NPFs. Conserved tryptophan residues are shown in red. h Peptide competition assay. Upper Panel, schematic representation of the structural domains of OFD1 peptides. Lower Panel, immunoblot analysis of pull-down assay of OFD1-Flag with the purified 7-subunit Arp2/3 complex (100 nM) with titration of OFD1p-WT peptides or OFD1p-W1012A (WA) peptides. The numbers under the gel lanes represent the ratio of ARP2 band intensity to Flag band intensity, which were normalized relative to the line 2 sample. i Actin polymerization upon OFD1 peptide treatment. Polymerization of 0.875 μM 20% pyrene-labeled actin monomers was carried out in the presence of 10 nM Arp2/3 complex, 5 nM GST-VCA, 500 nM OFD1 with 200 or 100 μM CPP-OFD1p-WT peptides, or with 200 or 100 μM CPP-OFD1p-W1012A(WA) peptides.

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