Research ArticleCELL BIOLOGY

Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway

See allHide authors and affiliations

Science Advances  08 Jan 2021:
Vol. 7, no. 2, eaba7803
DOI: 10.1126/sciadv.aba7803
  • Fig. 1 Generation and characterization of TfR-eRUSH gene-edited cells.

    (A) Scheme illustrating the insertion of the linker-SBP-EGFP coding sequence in the chromosomal region containing the stop codon (red) of the TFRC gene (transferrin receptor type 1, referred to as TfR). (B) PCR amplification from genomic DNA using primers flanking the TfR stop codon region confirmed the insertion of the SBP-EGFP sequence on both alleles. (C) Flow cytometry analysis indicates the total amount of TfR expressed in wild-type (WT) and TfR-eRUSH cells. MFI is represented ± SD (10,000 cells per condition, n = 3 independent experiments performed in duplicate). Student’s t test (***P < 0.001). (D) Representative live-cell imaging of TfR-eRUSH cells showing protein distribution after biotin addition. Note that TfR-eRUSH is at the PM starting from 23 min after biotin addition (blue arrowheads). (E) Flow cytometry analysis representing the amount of Tf-A647 bound at the surface of TfR-eRUSH cells. Note the increase of Tf fluorescence starting from 20 min after biotin addition. MFI is represented ± SD (5000 cells per condition, n = 3 individual experiment performed in duplicate). (F) Representative confocal immunofluorescence images detecting the arrival of TfR-eRUSH at the PM. TfR-eRUSH (green), Tf-A647 (magenta, top), or anti-TfR antibody (TfR-Ab, bottom) was detected at the PM starting from 20 min after biotin addition. Scale bars, 10 μm.

  • Fig. 2 Proteomics analysis of neosynthesized TfR-containing membranes.

    (A to C) LC-MS/MS proteomics analysis of immunoprecipitated TfR-eRUSH following biotin addition. (A) STRING analysis shows the interaction map of proteins that were enriched at T15 compared to T0. Color codes highlight clusters of proteins of related functions. (B) GO of the proteins enriched at least 1.5 times with a significant P value (<0.05) at T15 compared to T0 (T0-T15) was investigated using the GSEA online software. Relevant GO pathways and their corresponding FDR values are reported for each differential analysis. (C) The table reports the fold enrichment and P values of the Rab proteins significantly enriched at T15 compared to T0 (see table S2). (D) Representative confocal images from a single z-stack indicate the distribution of TfR-eRUSH treated for 12 min with biotin relative to the endogenous Rab5, Rab6, Rab7, and Rab18 proteins and the exogenously expressed Ruby3-Rab10. Note that TfR-eRUSH co-distributes in vesicles containing Rab7, Rab6, or Ruby3-Rab10 (zoomed panels, white arrowheads). Scale bars, 10 μm. Zoomed regions from white squares are represented with scale bars of 1 μm.

  • Fig. 3 Identification of Rab7 as an intermediate compartment of neosynthesized TfR trafficking.

    (A) Live-cell imaging shows localization of TfR-eRUSH in Ruby3-Rab7A–transfected cells. Images were extracted from a single plane at 7, 9, and 12 min after biotin addition. Note that TfR-eRUSH (green) co-distributes with Ruby3-Rab7A (magenta)–positive vesicles (white arrows). Scale bars, 5 μm. (B) TfR-eRUSH cells expressing Ruby3-Rab7A were imaged after apilimod treatment. Images were extracted as a single plane at 42 min after biotin addition. Scale bar, 10 μm. The zoomed region from the white square highlights that TfR-eRUSH (green) localizes at the limiting membrane of Ruby3-Rab7A vesicles (magenta, white arrows). Scale bar, 5 μm. (C) Representative images from a single z-stack indicate the localization of TfR-eRUSH relative to the endogenous Rab5, Rab6, and Rab7. Note that TfR-eRUSH co-distributes with Rab6 or Rab7 (yellow arrows). Scale bars, 5 μm. (D) The graph represents the percentage of colocalization between TfR-eRUSH (±SEM) and endogenous Rab5, Rab6, or Rab7 signal. Data represent n = 30 cells (Rab5), n = 31 cells (Rab6), and n = 27 cells (Rab7) per condition from three independent experiments, and Tukey test was run for significance (*P < 0.05 and ***P < 0.001).

  • Fig. 4 TfR-eRUSH transiting through Rab7 vesicles localizes at the PM.

    (A) TfR-eRUSH distribution relative to Lamp1 and Rab7+ vesicles. Note that different types of vesicles are observed: TfR-eRUSH vesicles (green) labeled with Rab7 (magenta) and Lamp1 (cyan) (white arrowheads); TfR-eRUSH vesicles labeled with Rab7 but no Lamp1 (yellow arrowheads). Scale bar, 2 μm. (B) Live-cell imaging of TfR-eRUSH cells expressing Ruby3-Rab7A in the presence of LysoTracker. At 10 min after biotin addition, note that TfR-eRUSH-Ruby3-Rab7A+ vesicles do not contain LysoTracker (white arrows, zoomed panels) and are distinct from Ruby3-Rab7A+ (magenta) vesicles containing only LysoTracker (cyan, yellow arrows). Scale bars, 2 μm (right) and 10 μm (left). (C) Confocal imaging showing TfR-eRUSH and Rab7 localization in the presence of DQ-BSA. Scale bar, 10 μm. At 16 min after biotin addition, TfR-eRUSH vesicles (green) labeled with Rab7 (cyan) do not contain DQ-BSA (square 1, white arrows), while DQ-BSA (magenta) is found only in Rab7+ vesicles (cyan) (square 2, white arrows). Scale bars, 2 μm. (D) Quantification of TfR-eRUSH colocalization with Rab7A or DQ-BSA. Data represent the percentage of colocalization with TfR-eRUSH (±SEM) with n = 32 cells (DQ-BSA) and n = 28 cells (Rab7) from n = 3 independent experiments. Student’s t test (***P < 0.001). (E) TfR-eRUSH protein is not degraded upon biotin addition, in the presence of cycloheximide and Baf A1. Actin (loading control), LMW (low molecular weight) marker. Note that the LC3-II bands are more intense in all conditions treated with Baf A1, showing a control for protein degradation. (F) Confocal imaging visualizing TfR-eRUSH-Rab7+ vesicle localization relative to LC3. At 15 min after biotin addition, note that TfR-eRUSH (green)-Rab+ (magenta) vesicles do not co-distribute with LC3 (white arrows), although LC3 (cyan) vesicles are labeled with Rab7 (yellow arrows). Scale bars, 2 and 10 μm (top).

  • Fig. 5 Rab7A is involved in transport of TfR-eRUSH to the PM.

    (A) Flow cytometry assay measuring the level of TfR-eRUSH at the PM in cells treated with pooled siRNA sequences targeting 12 different Rab mRNAs and a nontargeting siRNA control. The screen was performed in duplicate in n = 2 (±SD) independent experiments (2000 cells per condition). Significance was evaluated using ratiometric paired t test (*P < 0.05; ***P < 0.0001). (B) Flow cytometry assay measuring the level of TfR-eRUSH at the PM in cells treated with single sequence-specific siRNA targeting Rab7, RILP, or an irrelevant RNA. Each dot represents an independent experiment performed in quadruplicate (10,000 cells analyzed for each condition). The black bars represent the mean of the three experiments. Ratiometric paired t test (*P < 0.05). (C) Flow cytometry assay measuring the level of TfR-eRUSH at the PM in TfR-eRUSH cells expressing EGFP, GFP-Rab7A, or GFP-Rab7A T22N. Data shown are n = 3 independent experiments (±SD) (5000 cells per condition), and ratiometric paired t test was used for significance (**P < 0.001). ns, nonsignificant. (D) TfR-eRUSH cells transfected with Ruby3-Rab7A were imaged by TIRF microscopy. A representative image extracted from movie S4 is shown at 726 s. Scale bar, 10 μm. Note that TfR-eRUSH (green) is carried to the PM by Ruby3-Rab7 (magenta) vesicle (cropped regions). Scale bar, 1 μm. (E) TfR-eRUSH cells transfected with Ruby3-Rab6A were imaged by TIRF microscopy 24 hours after transfection. A representative image extracted from movie S5 is shown at 860 s. Scale bar, 10 μm. Ruby3-Rab6A (magenta) vesicle carried TfR-eRUSH (green) at the PM (cropped regions). Scale bar, 1 μm.

Supplementary Materials

Stay Connected to Science Advances

Navigate This Article