5 min with ligand-free medium. Cells were then incubated with DMSO (as a control) or LatB-containing medium. At 30 min after internalization of Tfn or EGF, few Tfn signals were observed and EGF signals moved to the cell center. Both ligands were not colocalized with EEA1, an EE marker. These data indicate that Tfn was recycled and that EGF reached LEs/lysosomes (Fig. 1B, upper panels). On the other hand, in LatB-treated cells, Tfn signals were still visible and colocalized with EGF in enlarged endosomes. These enlarged endosomes had distinct vacuole domains containing EGF and also colocalized with the early endosomal marker, EEA1 (Fig. 1B, lower panels). These results suggest that intra-endosomal sorting between the degradative and recycling molecules is independent of actin, but that segregation between the pathways depends on actin polymerization. This phenotype was apparently observed only when LatB was added 5 min after ligand internal- ization. Following addition of LatB at 15 min after ligand internalization, the number of vacuole clusters appeared to decrease. Moreover, addition at 30 min after ligand internalization did not induce any vacuole clustering (data not shown). Next, we investigated the effect of jasplakinolide (jasp), a drug that stabilizes actin filaments and promotes actin polymerization [32]. As shown in Fig. 1C, the size and appearance of endosomes were very similar to that observed with LatB-treated cells. Although we couldn’t discriminate whether these enlarged endosomes are composed from Endosomal Trafficking by Actin Filaments fusion or cluster of endosomes, due to limitation of resolution, these results suggest that actin dynamics including polymerization and depolymerization are required for transport from EEs. The ratio of Tfn colocalization with EGF (Fig. 1D) in control cells showed that peak colocalization occurred at 5 min after ligand internalization and rapidly decreased until 30 min; no colocalization was observed at 60 min. In contrast, LatB treatment significantly inhibited the reduction of colocalization; however, colocalization decreased after washout of LatB, as in control cells (Fig. 1D). We further quantified the rate of recycling (Fig. 1E) and degradation (Fig. 1F) under LatB treatment using a biochemical assay. Biotin- labeled Tfn or EGF was internalized, and the intracellular contents were quantified by enzymatic activity using avidin-HRP (see Materials and Methods). In contrast to immunofluorescence data (fig. 1B), there was no significant difference between control and LatB-treated cells at 30 min after internalization. This was probably because microscopic observation largely depends on their size and concentration. At 120 min, LatB treatment significantly reduced Tfn recycling (,40%) compared with control cells (,20%, p,0.01, Fig. 1E). Similarly, EGF degradation was also delayed by LatB treatment (Fig. 1F). These results indicate that actin dynamics play a role in both recycling and degradation. The recycling pathway has two independent routes; one is a direct pathway from EEs to PM and the second is an indirect Figure 1. Actin localized in EEs and was required for transport from EEs. HeLa cells internalized Alexa555-Tfn for 5 min and were then fixed and stained with Alexa488-phalloidin to enable visualization of the F-actin filaments (A). Cells internalized Alexa488-EGF and Alexa555-EGF (B, left panels and C) or Alexa555-Tfn (B, center panels) and Alexa555-EGF (B, right panels) for 5 min. DMSO (B, upper panels), LatB (B, lower panels), or jasp (C) was then added to the medium and cells were incubated for 25 min. Cells were fixed and stained using anti-EEA1 (B, center and right panels; green). Signals where Tfn colocalized with EGF are summarized in D. Intracellular Tfn (E) and EGF (F) were measured using ELISA assay as described in Materials and Methods using biotin-conjugated ligands and are presented as percentages against 5 min. Error bars represent the SEM from three independent experiments. doi:10.1371/journal.pone.0019942.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19942