Figure 8.
The influx of secretory cargo redistributes LRBA and NBEA from recycling to secretory compartments and sequester NBEAL1 and LRBA to different subdomains of secretory tubules. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-mCherry-RUSH were treated for 12 min with 10 µg/ml of AlexaFluor647-Transferrin and 50 µM of biotin to release transport of CTLA4-mCherry-RUSH from the ER, followed by live imaging before and after the treatment. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Scale bar 10 µm in the upper and middle panel and 1 µm in the lower panel. (B) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-RUSH-mCherry were imaged before and 12 min after the addition of 50 µM of biotin to the culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and transiently transfected with CTLA4-RUSH construct imaged before and 12 min after the addition of 50 µM of biotin to culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (D) Schematic summary of data from Fig. 8, A–C. (E) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and transiently transfected with CTLA4-RUSH-mCherry were imaged 12 min after the addition of 50 µM of biotin. NBEAL1 decorates the tip of growing Golgi-connected secretory tubules positive for CTLA4-RUSH-mCherry (arrows) that form transport carriers upon tubular fission. Scale bars 1 µm. (F) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and mScarlet-LRBA and transiently transfected with CTLA4-RUSH-mTagBFP2 were imaged live 12 min after the addition of 50 µM of biotin. NBEAL1 and LRBA segregate into different subdomains of Golgi-connected secretory tubules (arrows). mTagBFP2 signal was verified by visual observation, but not imaged, due to the toxicity of the blue-channel imaging setup. Scale bar 10 µm (left merged) or 1 µm (right merged). (G) Schematic summary of data from Fig. 8, E and F.

The influx of secretory cargo redistributes LRBA and NBEA from recycling to secretory compartments and sequester NBEAL1 and LRBA to different subdomains of secretory tubules. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-mCherry-RUSH were treated for 12 min with 10 µg/ml of AlexaFluor647-Transferrin and 50 µM of biotin to release transport of CTLA4-mCherry-RUSH from the ER, followed by live imaging before and after the treatment. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Scale bar 10 µm in the upper and middle panel and 1 µm in the lower panel. (B) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-RUSH-mCherry were imaged before and 12 min after the addition of 50 µM of biotin to the culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and transiently transfected with CTLA4-RUSH construct imaged before and 12 min after the addition of 50 µM of biotin to culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (D) Schematic summary of data from Fig. 8, A–C. (E) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and transiently transfected with CTLA4-RUSH-mCherry were imaged 12 min after the addition of 50 µM of biotin. NBEAL1 decorates the tip of growing Golgi-connected secretory tubules positive for CTLA4-RUSH-mCherry (arrows) that form transport carriers upon tubular fission. Scale bars 1 µm. (F) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and mScarlet-LRBA and transiently transfected with CTLA4-RUSH-mTagBFP2 were imaged live 12 min after the addition of 50 µM of biotin. NBEAL1 and LRBA segregate into different subdomains of Golgi-connected secretory tubules (arrows). mTagBFP2 signal was verified by visual observation, but not imaged, due to the toxicity of the blue-channel imaging setup. Scale bar 10 µm (left merged) or 1 µm (right merged). (G) Schematic summary of data from Fig. 8, E and F.

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