Next, we observed the formation of enlarged endosomes in LatB-treated cells using live cell imaging. In control cells, Tfn- containing tubules extended from endosomes and fission was observed frequently (Fig. 2C, upper panel and movie S4). However, in LatB-treated cells, endosomes rapidly fused with each other, resulting in enlarged endosomes with few short tubules (Fig. 2C, middle panel and movie S5). After washout of LatB, Tfn- containing tubular structures immediately segregated from endosomes and clusters of vacuolar domains dissociated from each other (Fig. 2C, bottom panel and movie S6). At 15 min after washout, these clusters were dissociated, and at 60 min after washout, EGF-containing endosomes localized around the peri- nuclear region and finally disappeared (data not shown). These data clearly indicate that disruption of the actin filaments induced aggregation of EEs, resulting in the formation of enlarged EEs. On the other hand, actin polymerization made the vacuolar domains pull apart and severed the tubules containing recycling molecules. Disruption of actin filaments is not required for transition to the LE and RE stage We demonstrated that LatB treatment induced abnormal enlargement of EEs, judging from colocalization with EEA1. However, there was a possibility that LatB treatment blocked the Endosomal Trafficking by Actin Filaments transition from EEs to LEs and/or REs because EEs have a mosaic structure [33]. EEs move from the cell periphery to perinuclear region in a microtubule-dependent manner and mature to LEs; this process is accompanied by both recruitment of an LE marker LAMP1 and intraluminar acidification [2,4,34]. Therefore, we investigated the effect of actin polymerization on endosomal maturation. In control cells, the EGF signals were colocalized with Lamp1 at 30, 60, and 120 min after internalization (Fig. 3A). Interestingly, the same results were obtained in LatB-treated cells, indicating that EEs containing EGF were partially converted to LEs. The same results were obtained using lysotracker, an acidic sensor (Fig. 3B). On the other hand, Rab11, a marker of REs, was not colocalized with EGF (Fig. 3C), suggesting that transferrin did not reach recycling endosomes. When we analyzed whether early and late endosomes fuse together in a heterotypic manner by localization of these specific markers, they were not co-localized but adjacently localized (Fig. 3D). These results indicate that the transition from EE to LE did not depend on actin dynamics, although the degradative/ recycling components remain the same organelle. Actin contributes to early endosomal motility Actin filaments have been reported to be responsible for short- range movement of peripheral endosomes [35,36]. In contrast, Figure 3. Actin depolymerization blocked transition from EE to LE. HeLa cells internalized Alexa555-EGF for 5 min at 37uC and DMSO or LatB was then added. After 30, 60, or 120 min, cells were fixed and stained with anti-Lamp1 (A) or incubated with Lysotracker (B) for 15 min or 30 min. GFP-rab11 transfected HeLa cells were internalized Alexa555-transferrin, treated with LatB for 30 min and fixed (C). Cells, which were treated with LatB for 30 min, fixed and stained with anti-EEA1 (red) and anti-Lamp1 (green. D). Samples were observed using a confocal microscope. doi:10.1371/journal.pone.0019942.g003 PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19942