Endosomal Trafficking by Actin Filaments treatment (B). HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of LatB (5 mg/ml). Fifteen minutes after the 37uC incubation, nocodazole (10 uM) was added to the mixture and this was incubated further in the presence of LatB and nocodazole. At 60 min after the addition of nocodazole, the medium was replaced with one containing only nocodazole. Cells were then further incubated for 5, 15, or 30 min (D). The EE cluster area was measured as described in Materials and Methods (E). Error bars represent the SEM from three independent experiments. doi:10.1371/journal.pone.0019942.g004 prevent aggregation or homotypic fusion (Fig. 6B). As the actin- dependent movement was limited among endosomes, the observation of endosomal movement under nocodazole treatment would be difficult (Fig. 4A). Actin polymerization is not required for the EE-to-LE transition We found that inhibition of actin dynamics impaired EGF degradation. Although EGF-containing endosomes were EEA1- positive, they also colocalized with LAMP1, an LE marker, and exhibited intra-endosomal acidification. These results suggest that actin dynamics may be required downstream of endosomal acidification. EEs and other endocytic compartments are acidified by V-ATPase [44,45]. V-ATPase interacts with ARNO and Arf6 in EEs and regulates the protein degradative pathway [46]. As Arf6 has been implicated in the rearrangement of the actin cytoskeleton [47], LatB might inhibit actin rearrangement downstream of Arf6. Cortactin regulates actin polymerization at EEs We have shown that the Arp2/3 complex, which initiates assembly of new filaments from the sides of pre-existing filaments to generate a network of branched filament arrays, exists in EEs. In general, the Arp2/3 complex is stimulated by binding to the side of actin filaments as well as to a nucleation-promoting factor (NPF). NPFs include N-WASP, WASP, and cortactin. Cortactin Figure 5. Cortactin is required for endosome biogenesis. HeLa cells internalized Alexa555-Tfn for 5 min at 37uC and were then fixed and stained with anti-cortactin (A, upper panels) or anti-p34-Arc (A, lower panels). For depletion of cortactin, negative control or cortactin-specific siRNAs were transfected into cells and these were then incubated for 72 h. Cells were lysed and processed for Western blotting using anti-cortactin or anti- actin (B). SiRNA-transfected cells internalized both Alexa488-Tfn and Alexa555-EGF for 30 or 60 min (C) or Alexa555-EGF for 5 min and LatB for 30 min. After washout of LatB, cells were incubated for 10 or 30 min and then fixed and stained using Alexa488-phalloidin to enable visualization of the F-actin filaments (D). The rate of EGF colocalized with Tfn was calculated and summarized in E. doi:10.1371/journal.pone.0019942.g005 PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19942