Fig. 7. BFA induces transport of both Tf and IgA to the same alkaline apical endosomes. (A-H) Cells were labeled to steady state and imaged alive in the presence of FR-Tf and Cy5-IgA. An apical optical section of control cells shows that FR-Tf (A) is missing from the AREs in which Cy5-IgA concentrates (arrowheads in A,B). In a lower focal plane of the same cells, the two probes closely colocalize in uniformly acidic endosomes (arrows in E and F). In cells treated with BFA for 15 minutes, and then incubated for 25 minutes in probes and BFA, FR-Tf is delivered to apical, alkaline compartments containing Cy5-IgA (arrows in C and D). A separate field of cells, incubated for 15 minutes with BFA, then with probes and BFA for 45 minutes (G,H) shows the two are closely colocal in medial acidic (orange) compartments as well as in alkaline tubular endosomes (green, at the top of the panel). (I) A field of untreated cells imaged in the presence of FR-IgA shows that IgA is located in both acidic endosomes as well as in relatively alkaline AREs located at the cell apices. These two distinct compartments are apparent also in images of cells incubated with BFA for 15 minutes, then with BFA and FR-IgA for another 20 (J) or 25 (K) minutes, showing IgA present in both medial acidic endosomes and relatively alkaline apical endosomes. Note that because of differences in cell height, single optical sections collect apical planes of some cells and medial planes of others. (L) Sorting endosomes remain distinct after protracted exposure of cells to BFA. 3-D images of living, polarized PTR cells were collected in the presence of basolateral Alexa488-Tf and diI-LDL following a 15 minute pretreatment with BFA and an 85 minute incubation with both BFA and fluorescent ligands. Whereas Tf is largely distributed into extended tubules, LDL, found in both sorting endosomes containing Tf and in late endosomes lacking Tf, is restricted to vesicular compartments. Panel L is 29 µm in height.