Figure 10.
RUFY1 interaction with the dynein-dynactin complex is required for CI-M6PR retrieval from endosomes to the TGN. (A–C) Confocal images of HeLa cells co-transfected with CD8α-M6PR and GFP-SNX1 (A) or RUFY1-GFP (B) or YFP-Golgi (C). Surface labeling of CD8α-M6PR was performed as described in Fig. 6 K and cells were fixed at the indicated time points, followed by immunostaining with secondary antibodies to anti-CD8 antibodies. (D) Quantification of the percentage of processive and non-processive tracks of CD8α-M6PR endosomes in HeLa cells treated with control or RUFY1 siRNA. Data represents mean ± SD analyzed from live-cell imaging experiments, and each dot represents one cell (****P < 0.0001; *P < 0.05; unpaired two-tailed t test). (E) A histogram displaying the distribution of run lengths obtained from particle tracking of CD8α-M6PR endosomes in HeLa cells treated with either control or RUFY1 siRNA. For both D and E, around 200 tracks were analyzed from 8 cells. (F–H) Representative confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with RUFY1 (WT)-GFP (Rescue) or RUFY1 (ΔCC2)-GFP (Rescue) constructs and immunostained for CI-M6PR and Giantin. Cells expressing the rescue construct are marked with a white boundary. Bars: (main) 10 µm; (insets) 2 µm. (I) Pearson’s colocalization coefficient quantification of CI-M6PR with Giantin in control siRNA and RUFY1 siRNA-treated HeLa cells and in cells transfected with either RUFY1 (WT)-GFP (Rescue) or RUFY1 (ΔCC2)-GFP (Rescue) constructs. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; n.s., not significant; unpaired two-tailed t test). (J) Proposed role of RUFY1 in regulating cargo sorting from endosomes to the TGN: RUFY1’s binding to Arl8b and Rab14 regulates its endosomal localization. RUFY1 recruits the dynein-dynactin complex on these endosomes and mediates CI-M6PR retrieval from the endosome to the TGN. In cells depleted of RUFY1, retrograde motility of CI-M6PR endosomes is reduced, resulting in a defect in sorting of pro-cathepsins to lysosomes. Alternatively, sorting of CI-M6PR into tubular endosomes might be affected, eventually leading to a defect in CI-M6PR retrieval from endosomes to the TGN.

RUFY1 interaction with the dynein-dynactin complex is required for CI-M6PR retrieval from endosomes to the TGN. (A–C) Confocal images of HeLa cells co-transfected with CD8α-M6PR and GFP-SNX1 (A) or RUFY1-GFP (B) or YFP-Golgi (C). Surface labeling of CD8α-M6PR was performed as described in Fig. 6 K and cells were fixed at the indicated time points, followed by immunostaining with secondary antibodies to anti-CD8 antibodies. (D) Quantification of the percentage of processive and non-processive tracks of CD8α-M6PR endosomes in HeLa cells treated with control or RUFY1 siRNA. Data represents mean ± SD analyzed from live-cell imaging experiments, and each dot represents one cell (****P < 0.0001; *P < 0.05; unpaired two-tailed t test). (E) A histogram displaying the distribution of run lengths obtained from particle tracking of CD8α-M6PR endosomes in HeLa cells treated with either control or RUFY1 siRNA. For both D and E, around 200 tracks were analyzed from 8 cells. (F–H) Representative confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with RUFY1 (WT)-GFP (Rescue) or RUFY1 (ΔCC2)-GFP (Rescue) constructs and immunostained for CI-M6PR and Giantin. Cells expressing the rescue construct are marked with a white boundary. Bars: (main) 10 µm; (insets) 2 µm. (I) Pearson’s colocalization coefficient quantification of CI-M6PR with Giantin in control siRNA and RUFY1 siRNA-treated HeLa cells and in cells transfected with either RUFY1 (WT)-GFP (Rescue) or RUFY1 (ΔCC2)-GFP (Rescue) constructs. The values plotted are the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; n.s., not significant; unpaired two-tailed t test). (J) Proposed role of RUFY1 in regulating cargo sorting from endosomes to the TGN: RUFY1’s binding to Arl8b and Rab14 regulates its endosomal localization. RUFY1 recruits the dynein-dynactin complex on these endosomes and mediates CI-M6PR retrieval from the endosome to the TGN. In cells depleted of RUFY1, retrograde motility of CI-M6PR endosomes is reduced, resulting in a defect in sorting of pro-cathepsins to lysosomes. Alternatively, sorting of CI-M6PR into tubular endosomes might be affected, eventually leading to a defect in CI-M6PR retrieval from endosomes to the TGN.

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