Immunofluorescence HeLa cells cultured on coverslips were starved with serum-free medium containing 0.1% BSA/DMEM for 1 h. For Tfn and EGF binding, the medium was replaced with serum-free DMEM containing Alexa488- or Alexa555-conjugated Tfn or Alexa555- conjugated EGF (Molecular Probes) and cells were incubated for 1 h on ice. Cells were then washed with cold phosphate-buffered saline and incubated with 10% FBS/DMEM at 37uC. LatB (5 mg/ ml), Jasp (1 mM) or LY294002 (100 mM) were added 5 min after Tfn and EGF internalization and cells were incubated for the time periods described. The treated cells were fixed with 3.7% formaldehyde for 15 min at RT and immunofluorescence was performed as described previously [49]. Imaging analysis Colocalization of Tfn and EGF fluorescence at EEs was calculated using MetaMorph software (Universal Imaging Corpo- ration). The threshold of fluorescence was determined by a 15 min-based DMSO control. Colocalization was calculated by measuring the ratio of Tfn to EGF signals. We quantified 10 cell images and averaged these (excluding two extreme values). For 3D reconstruction, Z-series imaging of the samples was performed in 0.1-mm increments. These images were reconstructed with MetaMorph software. For area measurements, the endosome cluster area was enclosed with a circle and measured with Image J software (National Institutes of Health). Live imaging and tracking analysis HeLa cells plated on a 35-mm diameter dish with a glass base were treated with DMSO or LatB as in the immunofluorescence experiments. Time-lapse images were taken using a confocal microscope and were acquired every 2 s. These images were reconstituted with MetaMorph software. The tracking analysis samples were taken using video microscopy, and the tracking analysis was performed with Image J software (National Institutes of Health). ELISA assay HeLa cells were plated on a 24-well plate, grown in serum-free medium for 1 h, and incubated with biotin-human transferrin or biotin-human EGF for 1 h on ice to facilitate binding to the cell surface. The ligands were internalized at 37uC, and cells were incubated for the times indicated with the appropriate reagents. To measure Tfn recycling and EGF degradation, ELISA assays were performed as described [50]. A peroxidase coloring kit (SUMILON) was used for the enzymatic reaction and light absorption was determined with a microplate reader (MTP 300; Hitachi) in order to quantify intracellular Tfn and EGF. Supporting Information Figure S1 Actin polymerization participates in EE-to- RE transport. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min before the addition of DMSO, LY294002 (LY), and LatB or both LY and LatB. Cells were then incubated for further 30 min, fixed, and observed (A). Intracellular Tfn was measured as in Fig. 1E (B). Error bars represent the SEM from three independent experiments performed in duplicate. (TIF) Movie S1 3D reconstitution of early endosomes con- taining both Tfn and EGF. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of DMSO. Images were taken at 15 min (DMSO). The Endosomal Trafficking by Actin Filaments structure of endosomes containing both EGF and Tfn was visualized by 3D reconstruction with a rotation angle of 2u. Frame rate, 7 frames/sec (181 frames). (AVI) Movie S2 3D reconstitution of early endosomes under LatB treatment. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of LatB. Images were taken at 25 min. The structure of endosomes containing both EGF and Tfn was visualized by 3D reconstruc- tion with a rotation angle of 2u. Frame rate, 7 frames/sec (181 frames). (AVI) Movie S3 3D reconstitution of early endosomes under Jasplakinolide treatment. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of Jasp. Images were taken at 25 min. The structure of endosomes containing both EGF and Tfn was visualized by 3D reconstruction with a rotation angle of 2u. Frame rate, 7 frames/ sec (181 frames). (AVI) Movie S4 Tubular endosomes was segregated from early endosomes. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of DMSO, and images were taken at 10 min after the addition of DMSO. Images were collected every 2 sec for 210 sec. Frame rate, 7 frames/sec (105 frames). (AVI) Movie S5 Large early endosomes were formed by clustering of several early endosomes under LatB treatment. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of LatB, and images were taken at 25 min after the addition of the drug. Images were collected every 2 sec for 2 min. Frame rate, 7 frames/sec (60 frames). (AVI) Movie S6 Actin polymerization induced by removal of LatB made extension and fission of tubular endosomes. HeLa cells internalized both Alexa488-Tfn and Alexa555-EGF for 5 min at 37uC before the addition of LatB. After 30 min, LatB- treated cells were washed and images were taken at 5 min. Images were collected every 2 sec for 108 sec. Frame rate, 7 frames/sec (54 frames). (AVI) Movie S7 Directional movements of EGF-containing endosomes. HeLa cells internalized Alexa555-EGF for 5 min at 37uC, and DMSO was added subsequently. Live cell images were taken at 15 min after internalization using confocal microscopy. Images were collected every 2 sec for 3 min, and a pattern of colors and dots was assigned to each particle. Frame rate, 7 frames/sec (90 frames). (AVI) Movie S8 Microtubules were required for movements of EGF-containing endosomes. HeLa cells internalized Alexa555-EGF for 5 min at 37uC. Nocodazole was added before the addition of the ligands and cells were incubated for 1 h at 37uC. Live cell images were taken at 15 min after internalization using confocal microscopy. Images were collected every 2 sec for 3 min, and a pattern of colors and dots was assigned to each particle. Frame rate, 7 frames/sec (90 frames). (AVI) PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19942