Endosomal Trafficking by Actin Filaments pathway to PM via REs [9]. The direct pathway uses vesicle transport, which requires PI3-kinase activity and is inhibited by LY294002 (LY), a specific inhibitor of PI3-kinase [7]. To determine which pathway is dependent on actin dynamics, we used LY in combination with LatB. As shown in Figure S1A, Tfn was recycled or dissociated from EGF in control or LY-treated cells. EGF-containing endosomes were relocated to the perinucle- ar region, suggesting that EGF was transported to LEs/lysosomes. On the other hand, in LatB- or LatB/LY-treated cells, Tfn remained colocalized with EGF even at 30 min after internaliza- tion and these EEs were enlarged. This colocalization persisted until at least 60 min after internalization (data not shown). The quantitative analysis showed that intracellular Tfn was significant- ly increased in LatB/LY-treated cells (,82%) compared with control cells (,45%), LY- (,62%), and LatB-treated cells (,66%, Fig. S1B). These effects of LatB/LY on Tfn recycling suggest that the LatB-sensitive pathway differs from the LY-sensitive pathway and may be an EE-to-RE pathway. Disruption of actin dynamics induces enlarged endosomes As disruption of actin dynamics induced the formation of abnormal enlarged endosomes, we compared endosomal structure between control-, LatB-, and jasp-treated cells using 3D reconstruction (Fig. 2A). In control cells, endosomes containing both EGF and Tfn were observed until 15 min after internaliza- tion (Fig. 2B and movie S1), but not at 30 min after internalization (see Fig. 1B). On the other hand, in LatB- or jasp-treated cells, endosomes containing several vacuolar domains as well as tubular domains were seen even at 30 min after internalization (Fig. 2B, movie S2 and S3). The vacuolar domain-containing endosomes were not detected in control cells. Figure 2. Inhibition of actin dynamics induced homotypic fusion of EEs. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of DMSO (left), LatB (5 mg/ml, center), or jasp (right). Images were taken at 15 min (DMSO) or 25 min (LatB and jasp) (A). The structure of endosomes containing both EGF and Tfn was visualized by 3D reconstruction with a rotation angle of 30u (B). Live imaging of EEs. Cells were treated as in A, and images were taken at 10 min (DMSO) or 25 min (LatB) after the addition of each drug. After 30 min, LatB-treated cells were washed and images were taken at 5 min (LatB w/o). Representative images are shown. The arrow indicates a fission event. doi:10.1371/journal.pone.0019942.g002 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19942