BLTP2-positive tubular internal membranes are Rab10-dependent tubular recycling endosomes continuous with the PM. (A) Endogenous BLTP2 localizes at the tip of mCherry-Rab10–positive tubular endosomes in HeLaM cells. (B) BLTP2^Halo- and mCherry-Rab10–positive tubular endosomes in HeLaM cells are connected with the PM as revealed by labeling with the membrane-impermeant PM dye CellBrite. (C) mCherry-Rab10–positive tubular endosomes in HeLaM cells are also labeled with the PM marker GFP-CAAX. (D and E) Absence of GFP-CAAX–positive tubules in HeLaM cells expressing dominant-negative Rab10 (mCh-Rab10 T23N). Fluorescence image in D and quantification in E. Two-tailed t test. Mean ± SEM. n = 3 independent experiments. 48 cells for WT and 57 cells for the T23N mutant. (F and G) Expression of dominant-negative Rab10 (mCh-Rab10 T23N) abolished BLTP2^EGFP-positive tubules. Fluorescence images in F and quantification in G. Two-tailed t test. Mean ± SEM. n = 3 independent experiments. 127 cells for WT Rab10 and 121 cells for the T23N mutant. (H) Tubular structures positive for endogenous Rab10 immunoreactivity disappear after nocodazole treatment for 2 h but are restored after washing out the drug. n = 3 independent experiments. Pretreatment: 269 Rab10 tubules from 28 cells; treated: zero tubules observed in the 46 cells examined; washout: 353 Rab10 tubules from 26 cells. (I) Live imaging of a HeLaM cells after nocodazole washout shows the recovery of a Rab10-positive tubular endosome and the recruitment of BLTP2^EGFP after the tubule reaches the PM and fuses with it. (J) Schematic drawing of BLTP2 recruitment and localization at a Rab10-positive tubular endosome that is continuous with the PM.
Figure 2.

BLTP2-positive tubular internal membranes are Rab10-dependent tubular recycling endosomes continuous with the PM. (A) Endogenous BLTP2 localizes at the tip of mCherry-Rab10–positive tubular endosomes in HeLaM cells. (B) BLTP2^Halo- and mCherry-Rab10–positive tubular endosomes in HeLaM cells are connected with the PM as revealed by labeling with the membrane-impermeant PM dye CellBrite. (C) mCherry-Rab10–positive tubular endosomes in HeLaM cells are also labeled with the PM marker GFP-CAAX. (D and E) Absence of GFP-CAAX–positive tubules in HeLaM cells expressing dominant-negative Rab10 (mCh-Rab10 T23N). Fluorescence image in D and quantification in E. Two-tailed t test. Mean ± SEM. n = 3 independent experiments. 48 cells for WT and 57 cells for the T23N mutant. (F and G) Expression of dominant-negative Rab10 (mCh-Rab10 T23N) abolished BLTP2^EGFP-positive tubules. Fluorescence images in F and quantification in G. Two-tailed t test. Mean ± SEM. n = 3 independent experiments. 127 cells for WT Rab10 and 121 cells for the T23N mutant. (H) Tubular structures positive for endogenous Rab10 immunoreactivity disappear after nocodazole treatment for 2 h but are restored after washing out the drug. n = 3 independent experiments. Pretreatment: 269 Rab10 tubules from 28 cells; treated: zero tubules observed in the 46 cells examined; washout: 353 Rab10 tubules from 26 cells. (I) Live imaging of a HeLaM cells after nocodazole washout shows the recovery of a Rab10-positive tubular endosome and the recruitment of BLTP2^EGFP after the tubule reaches the PM and fuses with it. (J) Schematic drawing of BLTP2 recruitment and localization at a Rab10-positive tubular endosome that is continuous with the PM.

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