Fig. 3
CD93 is recycled via large vesicles moving along F-actin cables. a: Exponentially growing HUVECs were detached from the plate, resuspended in complete growth medium, plated on the substrate, and fixed at late phases of spreading (from 30 to 50 min). Cells were analyzed by immunofluorescence using phalloidin and an anti-CD93 (4E1) antibody. Scale bar, 11 μm. b: Exponentially growing HDBECs were treated as in a. Cells were analyzed by immunofluorescence using phalloidin and two different anti-CD93 antibodies as indicated. Arrowheads indicate CD93+ve vesicles. Images were acquired using a Leica SP8 confocal microscope equipped with Leica White Light Laser, notch filters (488 nm, 568 nm, and 647 nm) and a HC PL APO CS2 63x/1.40 oil objective. Images were taken in 1024 × 1024 format, speed 400 Hz, and pinhole set at 1.0 Airy diameter. Scale bars, 20 μm. c: HUVECs were pretreated for 4 h with 50 μg/mL cycloheximide, then they were detached from the plate, resuspended in complete growth medium, and plated on the substrate in the presence of cycloheximide. Cells were analyzed by immunofluorescence at late phases of spreading using anti-CD93 antibodies. DIC image of stained cells is shown. The dotted line indicates nucleus boundary. Scale bar, 8 μm. d: Periodic z-series of ECs treated as in a were acquired at 50 min after plating to generate a color-coded projection such that the basal cell surface, which adheres to the substrate is red and the apical cell surface is violet. A vertical section (lateral xz, 47 z-sections of 115 nm each) obtained from the same images is shown. A dotted ring containing an asterisk indicates apical bud boundary. In the xz view, a dotted line indicates the cell boundary and an arrowhead the point of view of the image. Scale bar, 8 μm. e: Details of late spreading HUVECs. Cells treated as in a were stained for F-actin and CD93. Overlay of stained cells and white dot colocalization (wdc) images are shown. Arrowheads indicate colocalization between CD93+ve vesicles and actin cables. Manders and Costes quantitative analyses of CD93 colocalization with F-actin and vice versa are shown (n = 8 cells). Scale bars are 4 μm