Figure 5.

LRBA- and NBEA- positive tubules accumulate transferrin starting from 2 min after its addition to the culture media. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and AP1M1-mScarlet were imaged live 10 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Scale bar 10 µm (main figure) or 1 µm (insert magnifications). (B) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were fixed 2 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin and imaged by confocal microscopy. Scale bars 10 µm (left merged) or 1 µm (right merged). (C) Examples of colocalization of stably transfected 3xFlag-EGFP-LRBA and AlexaFluor647-Transferrin, 2 min after addition of transferrin to the culture media. Scale bars 1 µm. (D) HeLa cells stably transfected with 3xFlag-EGFP-NBEA were fixed 3 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin and imaged by confocal microscopy. Scale bars 10 µm (left panel) or 1 µm (zoomed-in panels). (E) Quantification of the mean signal intensity ratio of LRBA or transferrin on LRBA/transferrin positive tubule vs. nearby LRBA/transferrin positive vesicle. The mean signal intensity of green (endogenous LRBA) or magenta (endocytosed transferrin, 3 min) channels within a 4 × 4 pixels rectangular box over the tubular or vesicular part of the transferrin-positive compartment was used for calculations. n = 20. (F) Schematic summary of data from Fig. 5, A–E. (G) Quantification of the dynamics of colocalization of LRBA or NBEAL2 positive structures and transferrin. HeLa cells stably transfected with 3xFlag-EGFP-LRBA or -NBEAL2 were fixed and imaged at the indicated time points after the addition of 10 µg/ml of AlexaFluor647-Transferrin to the culture media. EGFP or AlexaFluor647 positive structures were segmented and each dot represent the percentage of EGFP-positive objects partially or completely overlapping with AlexaFluor647 positive objects per cell. n ≥ 50 per condition.