D. discoideum cells release vesicular trails during cell migration. (A [i]) 3D reconstruction of the surface of two cells migrating toward an aggregation center. The reconstruction was generated by segmentation of PMs captured in an FIB-SEM image stack that spanned the cells. The leading cell is colored yellow, the following cell is colored blue, and the direction of migration is indicated by black arrows. (A [ii]) A single slice from the reconstructed FIB-SEM image volume of ACA-YFP/aca⁻ cells, orthogonal to the scanning electron microscope imaging plane and parallel to the surface of the coverslip in Ai. Cells were stained with anti-GFP conjugated to colloidal gold. The slice shown is ∼20–60 nm from the surface of the coverslip. (A [iii and iv]) Insets depicting areas of vesicle release between cells (green dotted rectangle) and behind the following cell (red dotted rectangle). For clarity, slices at slightly different heights from the coverslip surface are shown in the inset. (A [v and vi]) Magnified view of the inset (blue) showing an MVB just after fusion with and release from the PM. (A [v]) A stack of slices extracted from the FIB-SEM image volume shown in blue dotted inset. These slices were used to segment out and reconstruct this event in 3D, depicted in A (vi). Red arrows are used as reference points marking the groove containing the released vesicle (shown as cyan, indicated by gold arrow). The cytosol is marked in blue, and the extracellular space is colorless. (Inset) An oblique image plane from the FIB-SEM image volume (indicated by the dotted line in A [vi]) showing the release of an intraluminal body from the PM. (B) A single FIB-SEM slice from Video 2 showing MVB fusion with PM. The extracellular space is overlaid in green, the fusing MVBs in pink, and released vesicles in blue.