microtubules are responsible for long-range movements between the perinuclear and peripheral region. Therefore, we compared endosomal motility in the presence of LatB and nocodazole (a microtubule depolymerizing drug). In control cells, long-range directional movements toward the cell center were observed (Fig. 4A and movie S7). In contrast, we hardly detected any endosomal movements in nocodazole treated cells, suggesting that endosomal movements largely depend on microtubules (Fig. 4A and movie S8). However, in LatB-treated cells, EGF-containing endosomes moved rapidly in random directions and fused with each other (Fig. 4A and movie S9). Endosomes moved toward the cell center in the control cells, but in LatB-treated cells few movements toward the perinuclear region were observed despite frequent random movements (Fig. 4B). The tracking analysis clearly indicated that actin polymerization was required for directional movement toward the perinuclear region. Next, to observe more directly the endosomal movements induced by actin polymerization, we used both LatB and nocodazole, followed by removal of only LatB to induce actin polymerization. When cells were treated with both LatB and nocodazole, enlarged EEs containing several clusters of EGF were observed (Fig. 4C). However, after removal of LatB, the EGF clusters spread quickly and had both tubular and vacuolar domains. As time progressed, EGF-containing vacuolar domains dispersed further and Tfn gradually disappeared. To quantify this dispersion, we measured the area of endosomes (Fig. 4D). The EE area was significantly increased at 5 min after the LatB washout. These results indicate that actin polymerization is required for both inhibition of homotypic fusion of endosomes through microtubule-independent movements and transport from EEs. Cortactin regulates actin dynamics at EEs We attempted to identify a specific regulator of actin dynamics at EEs since Arp2/3 and its regulatory elements have been suggested to play an essential role in EEs [18,20] and its activator cortactin [37]. We also verified localization of Arp2/3 and cortactin at endosomes (Fig. 5A) and determined whether cortactin was required for endosomal sorting and recovery from LatB treatment. Cortactin was depleted by introducing cortactin- specific siRNAs (Fig. 5B), and the effects on both the recycling and degradation pathways were investigated. As shown in Fig. 5C, Tfn remained with EGF at 30 min after internalization in cortactin siRNA cells. Even at 60 min after internalization, Tfn signals were clearly recognized in cortactin siRNA cells and were partially colocalized with EGF (Fig. 5C and summarized in Fig. 5D). Similar results were obtained by another cortactin siRNA (not shown). These data suggest that segregation at EEs was impaired in cortactin-siRNA cells. Cortactin-siRNA cells were subsequently treated with LatB and their recovery after treatment was observed. As described above, at 10 min after LatB washout, both dispersion and segregation of endosomes was observed in control cells (Fig. 5C, upper panels). On the other hand, clusters of endosomes were still observed at 30 min after washout in cortactin-siRNA cells. These results indicate that cortactin is required for actin assembly in EEs and for subsequent segregation. Discussion The role of actin in endosomes remains unclear, and several not mutually exclusive scenarios can be evoked. These include regulated endosome anchoring onto the actin network at the cell periphery, remodeling of the actin network by endocytic vesicles along their trajectory, endosome motility along existing actin Endosomal Trafficking by Actin Filaments filaments, and possible rocketing via de novo F-actin formation. Alternatively, actin may play an active role in membrane remodeling during endosome biogenesis. In this study, we revealed that actin is required for segregation in EEs, and it induces movement of each endosome toward the cell center by preventing their fusion. Further, we identified cortactin as a key regulator of actin in EEs. We propose that both actin and cortactin are involved in transport from EEs and that these function in two distinct steps (a hypothetical model is illustrated in Fig. 6). Actin filaments are involved in efficient segregation in EEs The recruitment of actin filaments to EEs has been observed previously [30,38]. Here, we demonstrated that inhibition of actin dynamics led to the formation of enlarged EEs and impaired transport from EEs. Simultaneous application of LatB and LY significantly inhibited Tfn recycling compared with individual LatB and LY applications. These results suggest that LatB and LY act in distinct pathways, and that actin might be involved in the EE-to-RE pathway, which is independent of PI3K. In fact, LatB treatment resulted in reduction in tubule formation from EEs. This leads to the major question, what is the role of actin in transport from EEs? Recently, it has been shown that actin dynamics induce scission of membrane tubules [39]. Other researchers have also suggested that actin dynamics play a role in membrane scission [21,40]. These experiments focused on the internalization steps at PM, but this actin-induced scission may also apply to EEs. SNX4 has been shown to be a candidate factor driving membrane tubulation in the EE-to-RE pathway [41], and may contribute to membrane tubulation and scission together with actin dynamics. Another study also reported that myosin VI (a minus end-directed actin motor) and its interacting protein lemur tyrosine kinase 2 siRNAs led to swollen, enlarged EEs and reduced EHD3- containing tubule formation [19]. These results suggest that actin motor proteins also participate in the EE-to-RE pathway. Indeed, at the trans-Golgi network, GOLPH3 bridges phosphatidylinositol and actomyosin to promote efficient tubulation and vesicle formation [42]. We propose that actin filaments contribute to efficient fission by cooperating with factors driving membrane tubulation, such as SNX4 (Fig. 6A). Actin is required for perinuclear localization of endosomes Actin polymerization has been shown to be involved in homotypic fusion of endosomes as well as yeast vacuoles. In Dictyostelium, inhibition of actin polymerization induced LEs to form clusters and blocked endosomal transport and movement, suggesting that the actin coat surrounding LEs prevents endo- somes from clustering, docking, and fusing with each other. [43] On the other hand, fusion between phagosomes and LEs and between LEs themselves is affected by the inhibition of actin polymerization in vitro, indicating that the actin filaments assembled on LEs or phagosomes may facilitate endosomal fusion. These findings suggest that the actin filaments may regulate endosomal fusion at several distinct steps during intracellular transport. We demonstrated that EEs fused with each other forming enlarged EEs after inhibition of actin dynamics. On the other hand, induction of actin-polymerization by LatB washout induced dissociation of EEs and translocation of each vacuolar domain. Furthermore, we observed that the dissociation of EEs by actin polymerization was independent of microtubules. Thus, actin filaments may provide a track for actin-based motor proteins to PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19942