Figure 4.
NBEA-positive tubules are highly dynamic sites of clathrin-coated vesicle formation. (A) HeLa cells stably expressing 3xFlag-EGFP-NBEA together with clathrin coat adaptors AP1M1-mScarlet (1), AP3M1-mScarlet (2), GGA1-mScarlet (3), GGA3-mScarlet (4), mScarlet-EpsinR (5), and mScarlet-clathrin heavy chain (6) were imaged live. Scale bar 10 µm (left merged) or 1 µm (right merged). (B) Quantification of the percentage of AP1M1 dots colocalizing with BDCPs in HeLa cells expressing 3xFlag-EGFP-LRBA or 3xFlag-EGFP-NBEA together with AP1M1-mScarlet (data shown in Fig. 3 H or Fig. 4 A, respectively). n = 15–20 cells and 150–300 AP1M1 dots/cell were quantified. (C) HeLa cells stably expressing 3xFlag-EGFP-NBEAL1 and mScarlet-tagged AP1M1 (1), GGA1 (2), or clathrin heavy chain (3) were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (D) Schematic summary of the localization of LRBA/NBEA, NBEAL1, and NBEAL2-positive tubular compartments with clathrin coat adaptors and clathrin. PM- plasma membrane, EE/SE- early/sorting endosomes, RE- recycling endosomes, GtPM- Golgi to plasma membrane carriers, TGN- trans-Golgi network. (E) LRBA- and AP1M1-positive tubules undergo rapid fission at or close to AP1M1-positive speckles (indicated by arrows). HeLa cells stably transfected with 3xFlag-EGFP-LRBA and mScarlet-AP1M1 were imaged live with representative snap-shot images shown. Scale bars 1 µm. (F) LRBA and AP1M1 partition in a mutually exclusive manner along the length of growing LRBA and AP1M1 positive tubule. HeLa cells stably transfected with 3xFlag-EGFP-LRBA and mScarlet-AP1M1 were imaged live with representative snap-shot images shown. Scale bars 1 µm. (G) Schematic summary of the dynamics of LRBA and AP1M1 positive tubule from Fig. 4, E and F.

NBEA-positive tubules are highly dynamic sites of clathrin-coated vesicle formation. (A) HeLa cells stably expressing 3xFlag-EGFP-NBEA together with clathrin coat adaptors AP1M1-mScarlet (1), AP3M1-mScarlet (2), GGA1-mScarlet (3), GGA3-mScarlet (4), mScarlet-EpsinR (5), and mScarlet-clathrin heavy chain (6) were imaged live. Scale bar 10 µm (left merged) or 1 µm (right merged). (B) Quantification of the percentage of AP1M1 dots colocalizing with BDCPs in HeLa cells expressing 3xFlag-EGFP-LRBA or 3xFlag-EGFP-NBEA together with AP1M1-mScarlet (data shown in Fig. 3 H or Fig. 4 A, respectively). n = 15–20 cells and 150–300 AP1M1 dots/cell were quantified. (C) HeLa cells stably expressing 3xFlag-EGFP-NBEAL1 and mScarlet-tagged AP1M1 (1), GGA1 (2), or clathrin heavy chain (3) were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (D) Schematic summary of the localization of LRBA/NBEA, NBEAL1, and NBEAL2-positive tubular compartments with clathrin coat adaptors and clathrin. PM- plasma membrane, EE/SE- early/sorting endosomes, RE- recycling endosomes, GtPM- Golgi to plasma membrane carriers, TGN- trans-Golgi network. (E) LRBA- and AP1M1-positive tubules undergo rapid fission at or close to AP1M1-positive speckles (indicated by arrows). HeLa cells stably transfected with 3xFlag-EGFP-LRBA and mScarlet-AP1M1 were imaged live with representative snap-shot images shown. Scale bars 1 µm. (F) LRBA and AP1M1 partition in a mutually exclusive manner along the length of growing LRBA and AP1M1 positive tubule. HeLa cells stably transfected with 3xFlag-EGFP-LRBA and mScarlet-AP1M1 were imaged live with representative snap-shot images shown. Scale bars 1 µm. (G) Schematic summary of the dynamics of LRBA and AP1M1 positive tubule from Fig. 4, E and F.

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