Actin Organizes WASH Domains Figure 10. Interpretation for the role of actin on WASH dynamics on and off endosomes. To explain how actin depolymerization increases the steady state amount of WASH on endosomes and decreases the exchange of endosomal WASH for cytosolic WASH, we propose that actin branched networks promote the detachment of WASH from endosomes. For example, endosomal WASH may bind to the active Arp2/3 complex at branched junctions of the actin network, and detach from the endosome as the branched junction moves backward because of filament elongation. doi:10.1371/journal.pone.0039774.g010 Germany) were used in the internalization medium in the presence of Tf. Full length human Vps35 was amplified by PCR using a template provided by L. Johannes (Institut Curie, Paris) and cloned into a modified pCDNA5/FRT/V5-His (Invitrogen) vector, in which the CMV promoter was replaced by a EF1a/ HTLV chimera promoter and which tags the ORF by a N- terminal eGFP [7]. Stable 3T3 transfectants obtained by homologous recombination at the FRT site were obtained as previously described [7]. SDS-PAGE and Western Blot Whole cell lysates were prepared by resuspending cells directly in SDS-PAGE loading buffer. Viscosity of samples after boiling was reduced using Benzonase (Sigma). SDS-PAGE was performed using NuPAGE 4–12% Bis-Tris gels (Invitrogen) transferred onto nitrocellulose membranes according to the manufacturer’s in- structions. Western blots were revealed using HRP coupled antibodies, Supersignal kit (Pierce) and a Fuji LAS-3000 (Fujifilm). Immunocytochemistry and image acquisition For standard imaging of fixed samples, 3T3 cells were plated for 2 h onto glass coverslips coated with 50 mg/ml fibronectin (Sigma) before each experiment, fixed in PBS-3% paraformaldehyde, permeabilized in PBS containing 0.05% saponin, then processed for indirect immunofluorescence using standard techniques. Samples were then mounted in ProLongH Gold antifade reagent (Invitrogen). To preserve the structure of enlarged endosomes, cells co- expressing mCherry-Rab5Q79L and GFP-WASH or GFP-Vps35 were plated for 2 h onto fibronectin-coated surfaces (glass plates, from Iwaki, or m-slides, from IBIDI, Martinsried, Germany), fixed in PBS-3% paraformaldehyde for 10 min at 37uC, processed for immunofluorescence as described above, then washed and imaged in PBS. For time-lapse microscopy, cells were plated for 2 h onto fibronectin-coated glass (Iwaki) or plastic (m-Dish, IBIDI) plates and imaging was performed in DMEM medium supplemented with 10 mM Hepes (Invitrogen). PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e39774