Figures and data
Endocytic vesicles in VE cells
(A) Schematic diagram of the imaging setup. An E8.5 whole mouse embryo was observed with a laser confocal microscope. (B) Confocal images of VE cells stained with FM 1-43, LysoTracker Red, and anti-EEA1 antibody, or electroporated with GFP-Rab7. The yellow dotted lines indicate the cell boundaries. The yellow arrows show some vesicles that were positive for LysoTracker Red in zone 2. (C) For descriptive purposes, the supranuclear portion of VE cells is divided into 3 zones. Zone 1, zone 2, and zone 3 contain early endosomes (EE), late endosomes (LE), and lysosomes, respectively. (D) Confocal images of VE cells labeled with Alexa 488-transferrin. Images taken 1, 10, 20, and 40 min after pulse labeling are shown. The yellow dotted lines indicate the cell boundaries. (E) Time-course analysis of the Alexa 488-transferrin-containing vesicles in VE cells. n = 4. The scale bars indicate 4 μm (B) and 10 μm (D).
Homotypic fusion between late endosomes
(A) Time-lapse imaging of endocytic vesicles in VE cells. After 5 min of pulse-labeling with Alexa 488-transferrin, homotypic fusion of late endosomes (arrows) was frequently observed in zone 2. (B) By labeling of VE cells with FM1-43, the fusion process of the cell membranes in zone 2 was observed. (C) Histograms showing the size distribution of total late endosomes (black line, n = 265) and the late endosomes that underwent homotypic fusion (gray line, n = 37). (D) Correlation between the size of the late endosomes that underwent homotypic fusion and the time required for completion of fusion. The sizes of the larger of the fused vesicles are plotted. Thirty-seven fusion events were measured. The scale bars indicate 5 μm (A, top) and 1.5 μm (A, bottom; B).
Heterotypic fusion between late endosomes and lysosomes
(A) Time-lapse imaging of VE cells. After 15 min of pulse-labeling with Alexa 488-transferrin, heterotypic fusion of late endosomes was observed: they shrank gradually and disappeared from the focal plane (arrows). (B) Time-lapse imaging of late endosomes in VE cells. Late endosomes and lysosomes were labeled with Alexa 488-transferrin (Tfn, green) and rhodamine-dextran (Dex, red), respectively. The bottom figures show optical sectioning through the dotted line in the upper figure. (C and D) Time course of the fluorescence intensity. In a late endosome undergoing fusion (ROI 1 in [B-C]), the fluorescence intensity of transferrin (green) slightly increased and then quickly decreased after membrane fusion. The fluorescence signal of dextran (red) in an underlying lysosome leaked into the fusing late endosome. The white box in (C) indicates the area shown in (B). The gray line in (D) indicates the diameter of the transferrin-positive late endosomes. The blue dotted lines in (D) indicate the moment the fusion pore was formed in ROI 1. ROI 2 is a negative control in a lysosome that did not undergo fusion. (E) Electron microscopic image showing pore formation between a late endosome (LE) and a lysosome (Ly). The inset shows a magnified picture of the boxed region. (F) Correlation between the size of late endosomes that underwent heterotypic fusion and the time required for completion of fusion. Twenty-five fusion events were measured. (G) Histograms showing the size distribution of late endosomes at 5 min (gray line, n = 256) and 15 min (black line, n = 242) after labeling with Alexa 488-transferrin. The average size of the vesicles was larger at 15 min than at 5 min (**P<0.01, unpaired t-test). The scale bars indicate 5 μm (A, top), 1.5 μm (A, bottom), 2 μm (B), 3 μm (C), and 1 μm (E).
Mathematical modeling analysis of membrane deformation after connection of 2 vesicles
(A) Definition of shape parameters. (B) Free-energy landscape for small vesicles. The gray arrow (D) indicates a typical orbit of membrane deformation from the initial condition (w, a) = (0.0,0.3). (C) Free-energy landscape for large vesicles. The solid gray arrow (E) indicates the typical orbit of membrane deformation, whereas the dashed gray arrow (D’) indicates the typical orbit of membrane deformation under fluctuated conditions. The inset is a blowup of the region w = 0∼0.15, a = 0.25∼0.35. (D–E) Time series of shape variation on the typical orbits indicated in (B) and (C), respectively. Note that the exact time scale cannot be estimated in this analysis. (F) Probability of the explosive fusions in the Flarge landscape (the orbit of the dashed gray arrow in [C]) for various vesicle sizes, osmotic pressure differences, and fluctuation energies, estimated by Monte-Carlo simulations. 
Actin filaments are associated with late endosomes in VE cells
(A–C) Confocal images of E8.5 mouse embryos stained with Alexa 488-phalloidin and anti-mouse IgG. Mouse IgG was incorporated into late endosomes and used as a marker for endosomes. Punctate cytoplasmic staining of phalloidin (green) was associated with the IgG-containing endocytic vesicles (red) in zone 2 (B–B’’) and at lower frequencies with the lysosomes in zone 3 (arrows, C–C’’). (D) Optical sectioning of VE cells along the dotted line in the upper panel. Actin filaments, extending from the apical surface into the cytoplasm (white arrowheads), were closely associated with the endocytic vesicles (red). (E and F) Three-dimensional surface rendering of actin filaments and a late endosome. The side view (E) and basal view (F) are shown. (G) Alexa 488-phalloidin staining of mRFP-fascin-1-expressing VE cells. Actin punctates were highly colocalized with fascin-1, whereas fine actin filaments extending radially in the x-y plane from endosomes were negative for mRFP-fascin-1 (white arrow heads). The fluorescence intensity plot along the white arrow in the middle panel shows colocalization of fascin-1 and phalloidin in the large spot. (H) Deconvolution images of late endosomes and actin. In addition to strong phalloidin-positive spots (white arrow heads), which were observable by use of conventional confocal microscopy, actin filaments extending from the endosomal surface (orange arrow heads) were visualized in the x-y axis image. In the x-z axis image, a late endosome was surrounded by actin filaments, which extended from the apical cell surface to the basal side in the cytoplasm. The scale bars indicate 3 μm (A–D), 5 μm (G), and 1 μm (E–F, H).
Dynamic regulation of actin filaments surrounding late endosomes
(A) Representative time-lapse images of GFP-actin spots on a late endosome. VE cells were electroporated with GFP-actin at E7.5, and time-lapse analysis was performed on the next day. The arrow indicates the fusion of actin spots. The arrowhead indicates the appearance of a new actin spot. (B) The turnover rate of actin was analyzed by use of a FRAP assay. The area (4 μm x 4 μm) covering a single late endosome in VE cells expressing GFP-actin was bleached, and the fluorescence recovery was observed by use of time-lapse microscopy. The yellow boxes indicate the laser-irradiated area. The time laser irradiation was ended is indicated as 0. (C-D) Graphs showing the fluorescence recovery of GFP-actin surrounding late endosomes in the apical region of zone 2 (light blue in [C], n = 4) and the basal region of zone 2 (dark blue in [C], n = 3) or at the cell surface of VE cells (D, n = 8). Fluorescence intensity was normalized to prebleaching intensities. (E) GFP-actin localization during vesicle movement. Endocytic vesicles (red) visualized by use of rhodamine-dextran in zone 2 are shown. When a vesicle (arrow) moved in a certain direction (upward in this figure), the actin patch (arrowhead) was observed at the rear end of the movement direction. The dotted line indicates the initial position of the vesicle. The scale bars indicate 1 μm (A) and 3 μm (B, E).
Actin dynamics regulate homotypic fusion in VE cells
(A) Confocal images of endocytic vesicles in zone 2. E8.5 mouse embryos pretreated with 0.1 μM cytochalasin D (CD) or 0.1 μM jasplakinolide (Jasp) were labeled with Alexa 488-transferrin for 5 min. Time-lapse images were taken for 400 s from 5 min after labeling. Arrows indicate homotypic fusion. (B) Frequency of homotypic fusion calculated from the number of fusions that occurred in 400 s. Data are represented as means ± SEMs of 4–9 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (**P<0.01; ***P<0.001). (C) Quantitation of types of homotypic fusion. Fusion types are classified into normal explosive fusion, slow explosive fusion (more than 60 s), and bridge fusion. Cytochalasin D treatment slowed down the fusion process in a dose-dependent manner and induced bridge fusion at 1 μM. Independent experiment numbers are shown on the top of the bars. (D) Quantitation of the time for completion of homotypic fusion. Treatment with 0.1 μM or 1 μM cytochalasin D or with 20 μM CK666, but not with 0.1 μM jasplakinolide, resulted in a significant increase in the fusion time. Data are represented as means ± SEMs of 4–30 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (**P<0.01; ***P<0.001). (E) Representative images of homotypic fusion of late endosomes in VE cells treated with 1 μM cytochalasin D. Endosomes were visualized by incubation with Alexa 488-transferrin. The arrows indicate the vesicles undergoing homotypic fusion in the bridge fusion mode. (F) Time-lapse imaging of GFP-actin localization during homotypic fusion of late endosomes. Localization of GFP-actin (green) and endocytic vesicles (red), visualized by use of rhodamine-dextran, were observed. When a pair of endosomes (arrows) fused, GFP-actin was newly polymerized at the opposite positions of the docking site and its fluorescence intensity was increased (arrowheads). (G) Distribution of GFP-actin during homotypic fusion of late endosomes. The fluorescence intensities of GFP-actin (dark gray, n = 5) and FM 1-43 (light gray, n = 5) were measured during homotypic fusion. The percentages of their signals in the proximal (docking site, shown as 1), lateral (shown as 2), and distal (shown as 3) regions at the predocking, docking, rounding, and postfusion stages are shown. Data are represented as means ± SEMs (*P<0.05, unpaired t-test). (H) The time course of changes in the fluorescence intensity of GFP-actin around a pair of late endosomes during homotypic fusion. Time 0 s indicates the moment the fusion pore of two late endosomes was formed. The graph indicates the average of 5 independent experiments. The scale bars indicate 10 μm (A), 2 μm (E), and 3 μm (F).
Actin stabilization induces heterotypic fusion of late endosomes with lysosomes
(A) Confocal images of endocytic vesicles in zone 2. E8.5 mouse embryos pretreated with 0.1 μM cytochalasin D (CD) or with 0.01 μM jasplakinolide (Jasp) were labeled with Alexa488-transferrin for 5 min. Time-lapse images were taken for 400 s from 15 min after labeling. The arrows indicate heterotypic fusion with lysosomes. (B) Frequency of heterotypic fusion calculated from the number of fusions that occurred in 400 s from 5 min after labeling. Data are represented as means ± SEMs of 4–7 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (**P<0.01, ***P<0.001). (C) Quantitation of heterotypic fusion frequency at 15 min after labeling of VE cells pretreated with cytochalasin D (CD), jasplakinolide (Jasp), or CK666. Data are represented as means ± SEMs of 4–8 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (*P<0.05, **P<0.01). (D) Effects of myosins on heterotypic fusion. Frequency of heterotypic fusion was calculated at 15 min after labeling. Data are represented as means ± SEMs of 5–8 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (*P<0.05, ***P<0.001). (E) Colocalization of EGFP-myosin IIA or EGFP-myosin Va with phalloidin in zone 2 of VE cells. The insets show the magnified images of the boxed area. Following electroporation of the EGFP-fused myosin expression plasmids and 24-h culture, whole embryos were stained with Alexa546-phalloidin. EGFP-myosin IIA signals overlapped with phalloidin-positive spots on late endosomes, whereas most of the EGFP-myosin Va signals were juxtaposed with phalloidin-positive spots on late endosomes (see the schemas and fluorescence intensity plots). The scale bars indicate 10 μm (A) and 3 μm (D).
Transition from homotypic to heterotypic fusion
(A) Time course of frequencies of homotypic (blue line) and heterotypic fusion (red line). The graph indicates the means of 6 independent experiments. (B) Distribution of cofilin in zone 2 of VE cells. After whole embryos were electroporated with YFP-cofilin and cultured for 1 d, they were stained with Alexa 546-phalloidin. The lower graph plotting the fluorescence intensity along the white arrow in the upper image indicates colocalization of actin filaments and cofilin. (C) Optical sectioning images (right) of VE cells along the dotted line in the x-y axial panel (left). Embryos, which were electroporated with YFP-cofilin and cultured for 1 d, were stained with Alexa 546-phalloidin. The actin filaments, extending from the apical to the basal region, were highly colocalized with cofilin. Note that the white arrows and blue arrowheads indicate an actin filament that surrounds late endosomes and the cell membrane, respectively. (D) Quantification of the fluorescence intensity of phalloidin and cofilin along the apical-basal axis. The intensity of spots colocalized with phalloidin and cofilin around late endosomes observed in the x-y images was quantified along the z-axis. Fluorescence intensity was normalized to the intensities in the most apical plane (8 late endosomes from 3 VE cells). Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test and represented as means ± SEMs (**P<0.01, ***P<0.001). (E) FRAP assay to evaluate the dynamics of actin at the apical region of zone 2 in VE cell pretreated with the S3 peptide. After treatment with the S3 or RV peptide overnight, the area (4 μm x 4 μm) covering a single late endosome in VE cells expressing EGFP-actin was bleached and the fluorescence recovery was observed by time-lapse microscopy. Three independent experiments were performed. (F–G) Frequency of homotypic (F) and heterotypic fusion (G) in embryos pretreated with 15 μg/ml S3 or RV peptides. Frequencies of homotypic and heterotypic fusions were calculated according to the numbers of fusions in 400 s from 5 min (F) and 15 min (G) after labeling with Alexa 488-transferrin. Data are represented as means ± SEMs of 3–4 independent experiments; individual data are shown by circular dots. Data were analyzed by use of one-way ANOVA with the Tukey multiple comparison test (*P<0.05). The scale bars indicate 8 μm (B) and 2 μm (C).
Summary of late endosome fusion in VE cells
In VE cells, late endosomes (LE) show two different types of fusion with different targets. In homotypic fusion, once a membrane pore is formed between two late endosomes, the pore quickly expands and a single fused vesicle is formed. In contrast, in heterotypic fusion, the pore does not expand; instead, the late endosome gradually shrinks and disappears over time as the result of a transfer of its vesicle content into lysosomes. Two different types of actin are associated with the late endosomal membrane in VE cells: the actin filaments that extend along the apical-to-basal axis and the filaments that are radially polymerized from the membrane. Cofilin, an actin-binding protein that severs and depolymerizes actin filaments activates actin turnover. In the apical region, late endosomes receive squeezing forces via active actin modification by cofilin and Arp2/3, which leads to homotypic fusion in an explosive mode. In contrast, in the basal region, actin is more static and late endosomes receive sliding forces via myosins.
