Expression of Ecad-GFP and transport in PGCs. HeLa cells were transiently transfected with Ecad-GFP (A–J) or VSVG-GFP (K and L). Ecad-GFP is concentrated in the perinuclear region 3 h after transfection (A) and is then visible at the PM in cell-cell contacts by 24 h (B). Shortly after transfection, newly synthesized Ecad-GFP (C) and the Golgi marker GM130 (D) colocalize in a merged image (E) in fixed HeLa cells. Ecad-GFP is visible in the Golgi region and in PGCs exiting the Golgi in a live HeLa cell (F). Regions from the image in F are magnified, digitally filtered and shown in relief to demonstrate two regions of interest containing Ecad-GFP–positive tubular PGCs (dashed box shown in G; dotted box shown in H). Arrows show tubular PGCs (5–20 μm; G and H), as they exit the Golgi region. Individual PGCs imaged after exit from the Golgi carrying either Ecad-GFP (I and J) or VSVG-GFP (K and L), from different transfected cells, include tubulovesicular PGCs (∼1 μm length) with concentrations of cargo at the trailing edge (I and K) or spherical carriers (250–300 nm diameter; J and L). Scale bars, (A–H) 10 μm; (I–L) 200 nm.

Ecad-GFP–positive PGC trajectories and direct Golgi-to-PM transport. Ecad-GFP was imaged in live HeLa cells to follow the trajectories of PGCs. A 60-s movie sequence (4 images/s) was recorded and then sequential images back-subtracted to produce a single image, showing the paths of seven Ecad-GFP–positive PGCs, with beginnings and ends of paths designated by yellow and red dots, respectively (A). A 90-s back-subtracted sequence, converted to an intensity-based color palette, highlights the concentration of curvilinear PGC tracks in the center of the cell (B). Imaging of a live HeLa cell at a focal plane level with the Golgi complex, shows the trajectory of a single spherical PGC (red trace) moving toward the cell periphery over a period of 40 s (C–E; Supplementary Movie 1). This is further enlarged in a back-subtracted image (D). Individual frames from this movie sequence (4 frames/s) show this 300-nm, spherical PGC (circled in red) as it exits the Golgi (frame 1) and finally disappears from the focal plane (after frame 111; E). To assess the fates of such PGCs in other live cells, imaging was performed at a focal plane level with the basal cell surface (F–H). Fusion events between Ecad-GFP-PGCs and the PM (F) are depicted in a region of interest (white box), converted to a pixel intensity-based color display palette (Supplementary Movie 2). Over 120 s, five fusion events were recorded, designated by white circles (G). Fully loaded PGCs arrived in the region, remained stationary, and then fused with the PM. In H, two stationary carriers (dashed box from G) are shown at 0.267-s intervals, fusing in succession. Analysis of the carrier fluorescence intensity distribution of the circled in H, is graphed I. The progressive flattening of the fluorescence distribution curve, indicates lateral dispersal of the PGC contents during fusion (Schmoranzer et al., 2000; Toomre et al., 2000). Scale bars, (A–C and F) 10 μm; (D, E, and G) 1 μm; (H) 250 nm.

Ecad-GFP-PGCs fuse most frequently with intermediate compartments before PM delivery. Imaging in live HeLa cells shows the recorded track of an Ecad-GFP-PGC (red line; 56-s duration, 0.267 s/frame Supplementary Movie 3; A). The same trajectory is shown enlarged in a back-subtracted image (B) cropped from the dashed box in A. At the terminus of this trajectory (box from B) the PGC fuses with an intermediate, Ecad-GFP–positive structure, demonstrated by three sequential stages (C). Fluorescence intensities in the PGC and intermediate structure, objects β and α, respectively, in C, merge to give an equivalent total fluorescence in the final fused object, π in C, as graphed (D). Scale bars, (A and B) 2 μm; (C) 1 μm.

Biosynthetic E-cadherin colocalizes with Rab11wt-GFP in HeLa cells. HeLa cells cotransfected with Ecad-YFP and Rab11wt-GFP were fixed and labeled with an antibody to Golgi marker GM130 (A). Confocal microscopy and spectral unmixing show the individual patterns of each protein and the merged image. Ecad-YFP and Rab11wt-GFP colocalized in punctate structures (arrowheads) seen in the enlarged, merged image. Ecad-YFP also colocalized in some separate structures with GM130 (arrows). In B, HeLa cells coexpressing hEcad (red) and Rab11wt-GFP (green) were incubated at 20°C to block TGN exit and then fixed immediately (no release) or after 15- or 60-min release incubations at 37°C. An antibody used to label hEcad shows perinuclear Golgi accumulation, without PM labeling, and no colocalization with peripheral Rab11wt-GFP puncta, at 20°C. Enlargement of a typical perinuclear fluorescence pattern (dotted box) shows hEcad and Rab11wt-GFP closely adjacent, but not colocalizing. On release, hEcad then colocalizes in scattered punctate structures with Rab11wt-GFP (see solid arrows, 15 min, and hollow arrows, 60 min in merged images). hEcad labeling at the PM is evident 60 min after release. In HeLa cells expressing Rab11wt-YFP (green), hEcad (red), and Rab5QL-GFP (blue; C), hEcad colocalization with Rab11wt-YFP–positive punctae is clearly evident 15 min after 20°C block release (hollow arrows), although no significant colocalization is observed with Rab5QL-GFP–positive enlarged early endosomes (solid arrows). Scale bars, 10 μm.

Colocalization of Ecad-mRFP with Rab11wt-GFP in live HeLa cells. HeLa cells, cotransfected with Rab11wt-GFP and Ecad-mRFP, were imaged live. A single image at the plane of the Golgi complex shows colocalization in some of the larger (1–3 μm), stationary structures (A). Ecad-mRFP is concentrated in the Golgi, in PGCs and in the larger structures colabeled with Rab11wt-GFP. Sequential images (cropped from dashed box in A) show colabeled intermediate compartments that move locally, whereas surrounding PGCs move on longer trajectories through this field (B). PGCs containing Rab11wt-GFP, Ecad-mRFP, or both, can be observed budding from and fusing with the intermediate compartments in the live movie sequence (Supplementary Movie 4). Scale bars, (A) 10 μm; (B) 5 μm.

Localization and surface delivery of hEcad and ΔS1-Ecad in preconfluent HeLa and MDCK cells expressing Rab11 mutants. HeLa (A–C, E, and F) and MDCK (D) cells were fixed 24 h after transfection with combinations of either untagged hEcad or ΔS1-Ecad and either Rab11wt-GFP or Rab11QL-GFP. Cells were imaged by confocal microscopy as ZY/ZX/XY sections (A and E) or by epifluorescence (B–D and F). Immunolabeling of cells expressing either hEcad (A) or ΔS1-Ecad (E) alone, reveals efficient surface delivery of both cargos to the whole surface of HeLa cells. Coexpression of Rab11wt-GFP (B) or Rab11QL-GFP (C) greatly reduced hEcad surface delivery, so that no surface labeling was detectable. In these same cells, hEcad was concentrated in intracellular structures, many of which also contained either Rab11wt-GFP or Rab11QL-GFP. Similar results were observed in preconfluent MDCK cells expressing hEcad and Rab11QL-GFP, with hEcad surface delivery blocked, and strong intracellular accumulation in enlarged Rab11QL-GFP–positive compartments (D). In contrast to hEcad, ΔS1-Ecad was efficiently transported to the cell surface of HeLa cells in the presence of Rab11QL-GFP (F) and was colocalized in intracellular structures with Rab11QL-GFP. Scale bars, 10 μm.

Rab11 mutants mistarget hEcad and transform polarized MDCK cells. Confluent monolayers of MDCK cells were fixed and labeled, and confocal imaging was performed to generate XZ and XY sections. Single expression of hEcad (A) or ΔS1-Ecad (B) shows their basolateral or apical distribution, respectively (red labeling), relative to nuclear staining (DAPI, blue). In cells coexpressing Rab11SN-GFP (green), hEcad (red) is dramatically shifted to the apical surface (C). Coexpression of Rab11SN-GFP (green) does not alter the apical delivery of ΔS1-Ecad (red; D). The expression of Rab11SN-GFP notably transforms the cells initially causing enhanced spreading and invasive protrusion at the basal cell surface, as seen in basal XY sections (C and D). This phenotypic change was even more striking after expression of Rab11QL-GFP as seen in a 3D reconstruction of cells cotransfected with Rab11QL-GFP (green) and hEcad (red; E). 3D rendered animations of confocal sections pertaining to each panel are available in the supplementary material (Supplementary Movies 5–9) Scale bars, 10 μm.