counterpart in the Tf channel. Aberrant trajectories and recoveries were discarded. Supporting Information Figure S1 LatA treatment induces close to complete depoly- merization of branched actin networks associated with WASH- positive endosomes. (A) 3T3 cells were treated as in Fig. 2A, and pre-extracted before fixation, as described in Derivery et al (2009). Cells were then processed for immunofluorescence using WASH and cortactin antibodies, and observed by epifluorescence microscopy (single planes). Scale bar: 10 mm. (B) corresponds to the insets shown in (A). The fluorescence intensities of WASH and cortactin were measured along linescans drawn on the endosomes (white lines). Under LatA treatment, WASH signal increases, whereas cortactin signal decreases, indicating the disappearance of the endosomal branched actin network. (TIF) Figure S2 CytoD treatment increases endosomal WASH similarly to LatA. (A) 3T3 cells were loaded with fluorescent Tf until equilibrium, then treated with 10 mM nocodazole in the continuous presence of Tf for 1 h, then treated with 1 mM CytoD or carrier in the presence of nocodazole and Tf for 30 min. Cells were then processed for immunofluorescence and observed as in Fig. 2. Scale bar: 10 mm. (B–C) Image stacks (25 cells, 3324 endosomes for control; 28 cells, 5203 endosomes for Cyto D) were processed and presented as in Fig. 2D–E, after normalization of endosomes volumes (*: p,0.001 compared with control within the same volume bin, ANOVA2 followed by a Tukey pairwise comparisons test). (B) The intensity of GFP-WASH domains increases when actin is depolymerized. (C) The ‘apparent concentration’ of GFP-WASH in domains increases upon actin depolymerization and does not depend on endosome volume. (TIF) Figure S3 Actin depolymerization increases the amount of GFP- WASH associated with endosomes. (A) Stable 3T3 cells expressing GFP or GFP-WASH were analyzed by Western Blot using indicated antibodies. GFP-WASH overexpression is limited. GFP- WASH replaces endogenous WASH in the stable cell line. (B) Cells expressing GFP-WASH were treated as in Fig. 2A, then fixed and imaged by spinning disk confocal microscopy (single planes). Scale bar: 10 mm. (C–D) Image stacks (14 cells, 1639 endosomes for control; 21 cells, 3056 endosomes for LatA) were processed and presented as in Fig. 2D–E (*: p,0.001 compared with control within the same volume bin, ANOVA2 followed by a Tukey pairwise comparisons test). (C) The intensity of GFP-WASH domains increases when actin is depolymerized. (D) The ‘apparent concentration’ of GFP-WASH in domains increases upon actin depolymerization and does not depend on endosome volume. (TIF) Figure S4 FAM21 colocalizes with WASH, whether or not actin is depolymerized. (A) Stable 3T3 cells expressing GFP-WASH were treated as in Fig. 6A, then processed for immunofluorescence using FAM21 antibodies and observed by epifluorescence microscopy followed by deconvolution (single planes). Scale bar: 10 mm. (B) corresponds to the insets displayed in (A). Scale bar: 1 mm. FAM21, another subunit of the WASH complex, References 1. Pollard TD (2007) Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 36: 451–477. 2. Rottner K, Hanisch J, Campellone KG (2010) WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond. Trends Cell Biol 20: 650–661. Actin Organizes WASH Domains colocalizes with WASH and forms crescents with WASH upon actin depolymerization. (TIF) Movie S1 FRAP analysis of GFP-WASH dynamics. Several WASH domains were bleached in a nocodazole-treated cell. The bleached regions are indicated by circles in the 6 frames before bleaching. The insets show WASH domains (green) and the Tf- positive endosomes (red). Frame rate: 20 images per second. Scale bar: 10 mm. (MOV) Movie S2 Actin depolymerization inhibits WASH recovery on endosomes after photobleaching. This movie corresponds to Fig. 3. Frame rate: 20 images per second. (MOV) Movie S3 Depletion of the Arp2/3 complex induces the formation of enlarged endosomes harboring a crescent-shaped WASH domain. Figure 5C is extracted from this movie. Frame rate: 1 image per second, 2 planes with an increment of 0.5 mm z- projected per time point. Scale bar: 10 mm. (MOV) Movie S4 Actin depolymerization by LatrunculinA induces the formation of enlarged endosomes harboring a crescent-shaped WASH domain. 3T3 cells stably expressing GFP-WASH were treated as in Fig. 2A and imaged by live spinning disk confocal microscopy. During the 1-min course of this movie, large endosomes displaying large and static crescent-shaped WASH domain at their surface are observed. Frame rate: 1 image per second, 2 planes with an increment of 0.3 mm z-projected per time point. Scale bar: 10 mm (1 mm in insert). (MOV) Movie S5 3D reconstructions of WASH domains on Rab5Q79L-enlarged endosomes in the presence or not of LatrunculinA. Figure 6B is extracted from this movie. (MOV) Movie S6 Direct observation of WASH domain coalescence upon actin depolymerization. Two examples are shown. Figure 8 is extracted from the left movie. Scale bar: 2 mm. (MOV) Acknowledgments We thank Marcos Gonzalez-Gaitan, Nir Gov, Sylvain Loube´ry, Fabrice Cordelie`res, Marie-Noe¨lle Soler, Marc Bailly-Bechet, Guillaume Romet- Lemonne and Dafne Chirivino for their advice on image acquisition and analysis, Philippe Chavrier, Ludger Johannes and Dominique Lallemand for kind gifts of reagents, and Ludger Johannes and Patricia Bassereau for critical reading the manuscript. We acknowledge IBiSA Imaging Facilities of Institut Curie and IMAGIF. EH belongs to the CellTiss Consortium. Author Contributions Conceived and designed the experiments: ED EH AG. Performed the experiments: ED EH. Analyzed the data: ED EH AG. Contributed reagents/materials/analysis tools: VH. Wrote the paper: AG ED EH. Developed the procedure for quantitative image analysis and wrote the custom MATLAB code: ED. 3. Derivery E, Gautreau A (2010) Generation of branched actin networks: assembly and regulation of the N-WASP and WAVE molecular machines. BioEssays 32: 119–131. PLoS ONE | www.plosone.org 15 June 2012 | Volume 7 | Issue 6 | e39774