Endosomal Trafficking by Actin Filaments Figure 6. Hypothetical model of the role of actin filaments in EEs. EEs are transported on microtubules from the cell periphery to the perinuclear region. EEs are segregated into degradative vacuoles or recycling tubular endosomes. Actin filaments may regulate transport from EEs at two distinct steps. (A) In the EE-to-RE pathway, actin filaments may produce tension facilitating segregation of tubular endosomes. (B) In the EE-to-LE/ lysosome pathway, actin filaments may prevent homotypic fusion of EEs, enabling each endosome to be transported from the cell periphery to the perinuclear region. doi:10.1371/journal.pone.0019942.g006 has been identified as a filamentous, actin-binding protein and is a major substrate for Src kinase [48]. In addition to linking actin organization and signal transduction, cortactin has emerged as a key protein involved in the coordination of membrane dynamics and cytoskeleton remodeling. Recent evidence shows that protein kinase Cd and calmodulin regulate receptor recycling from EEs through Arp2/3 and cortactin [26]. Our results show that cortactin may also be a key regulator of actin dynamics at EEs as cortactin siRNA impaired transport from EEs. A recent report has also revealed that WASH (an NPF) is required for endosome fission [18]. WASH localizes on Tfn-containing EEs and is required for efficient recycling from these. Cortactin may be a downstream target of WASH at EEs as WASH siRNA resulted in the loss of cortactin recruitment onto EEs. Determining the network of actin regulators and tubulation driving factors would provide precise knowledge of the sorting machinery involved in intracellular membrane transport. Materials and Methods Cells, reagents, antibodies and construct HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37uC under 5% CO2. LatB was purchased from Biomol, jasp from Calbiochem, nocodazole, dynasore from Sigma. Rabbit monoclo- nal anti-EEA1 was purchased from Cell signaling technologies. Mouse monoclonal anti-EEA1 was purchased from BD Trans- duction Laboratories, and mouse monoclonal anti-LAMP1 and rabbit polyclonal anti-cortactin were purchased from Santa Cruz. Rabbit polyclonal anti-p34-Arc was purchased from Upstate Biotechnology. Alexa555-conjugated Tfn, Alexa555-conjugated EGF, Alexa488-conjugated goat anti-mouse IgG, anti-rabbit IgG, Alexa488-conjugated phalloidin, and Lysotracker DND-26 were purchased from Invitrogen. GFP-Rab11 was a generous gift from M. Fukuda (Tohoku Univ., Japan). RNA interference and plasmid transfection Cortactin siRNAs (siRNA CTTN-1: 59-CCGAAUGGAUAA- GUCAGCUtt-39, siRNA CTTN-2: 59-GGUUUCGGCGG- CAAAUACGtt-39), N-WASP siRNAs (siRNA WASL-1: 59- GCAGAUCGGAACUGUAGUGtt-39, siRNA WASL-2: 59- GGGUAUCCAACUAAAAUCUtt-39, siRNA WASL-3: 59- GGUAAUAUUAUGGUAACUCtt-39), and negative control siRNA were purchased from Ambion. HeLa cells plated in 35- mm diameter dishes were transfected with each duplex siRNA (100 pmol) using Lipofectamin2000 (Invitrogen), according to the manufacturer’s instructions. The cells were used in the different experiments after 72 h of incubation. To observe recycling endosomes, GFP-rab11 was transfected by Effectene (QIAGEN) and analyzed at 24 h after transfection. PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19942