Figure 7.
Transferrin does not accumulate in NBEA-positive tubules decorated with TGN markers RAB6A or TGN46. (A) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB4A were imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Transferrin accumulates in NBEA-positive tubules representing the tubular segment of RAB4A-positive sorting endosomes (arrows). Scale bar 10 µm in the upper row, 1 µm in other panels. (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB11A imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. NBEA-positive tubules that accumulate transferrin between 2.5 and 6 min after its addition to the media are either negative (open arrowhead) or only weakly positive for RAB11A (arrow). Starting from 9 min 30 s after addition of transferrin, tubules that are positive for RAB11A and transferrin, but negative for NBEA could be detected (solid arrowhead). Scale bar 10 µm in the upper row, 1 µm in other panels. (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB6A were imaged live with 1-min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. RAB6A-positive NBEA-decorated tubules (arrow) do not accumulate transferrin (arrowhead). Scale bar: 10 µm in the upper row, 1 µm in other panels. (D) Schematic summary of data from Fig. 7, A–C. (E) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were treated for 14 min with 10 µg/ml of AlexaFluor647-transferrin in complete media, fixed, stained with an anti-TGN46 antibody, and imaged by confocal microscopy. LRBA-decorated tubules, positive for the TGN marker TGN46 (arrow) do not accumulate the endocytic recycling cargo transferrin (arrowhead). Scale bars 10 µm (left merged) or 1 µm (right merged).

Transferrin does not accumulate in NBEA-positive tubules decorated with TGN markers RAB6A or TGN46. (A) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB4A were imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Transferrin accumulates in NBEA-positive tubules representing the tubular segment of RAB4A-positive sorting endosomes (arrows). Scale bar 10 µm in the upper row, 1 µm in other panels. (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB11A imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. NBEA-positive tubules that accumulate transferrin between 2.5 and 6 min after its addition to the media are either negative (open arrowhead) or only weakly positive for RAB11A (arrow). Starting from 9 min 30 s after addition of transferrin, tubules that are positive for RAB11A and transferrin, but negative for NBEA could be detected (solid arrowhead). Scale bar 10 µm in the upper row, 1 µm in other panels. (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB6A were imaged live with 1-min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. RAB6A-positive NBEA-decorated tubules (arrow) do not accumulate transferrin (arrowhead). Scale bar: 10 µm in the upper row, 1 µm in other panels. (D) Schematic summary of data from Fig. 7, A–C. (E) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were treated for 14 min with 10 µg/ml of AlexaFluor647-transferrin in complete media, fixed, stained with an anti-TGN46 antibody, and imaged by confocal microscopy. LRBA-decorated tubules, positive for the TGN marker TGN46 (arrow) do not accumulate the endocytic recycling cargo transferrin (arrowhead). Scale bars 10 µm (left merged) or 1 µm (right merged).

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