Figure 1.
Loss of WDR91 causes retromer-mediated trafficking defects. (A) Schematic representation of pH-GluA2. (B) Time-course recording of pH-GluA2 in mouse primary hippocampal neurons. Neurons were isolated from Wdr91+/+ or Wdr91−/− mice, transfected with pH-GluA2–expressing vector, and subjected to glutamate perfusion for 5 min. Glutamate was removed at the time point 5 min. Neurons were continuously imaged for pH-GluA2 signals. Bars, 5 μm. (C) Time-course tracing of pH-GluA2 fluorescence change in neurons shown in B. n = 9 neurons (Wdr91+/+); n = 7 neurons (Wdr91−/−). (D) Left: Maximum amplitudes of pH-GluA2 fluorescence intensity change induced by glutamate. Right: Average half-time (T1/2) for recycling after glutamate washout. n = 9 neurons (Wdr91+/+); n = 7 neurons (Wdr91−/−). Statistical analyses were performed with the Mann–Whitney U-test. (E) Schematic representation of Flag-β2AR. (F) Representative images from a visual trafficking assay of Ctrl and KO-91 HeLa cells expressing Flag-β2AR. Cells were fixed under the indicated conditions including no treatment (DMSO), isoproterenol perfusion for 30 min (Agonist), and isoproterenol perfusion for 30 min followed by washout and further incubation for the indicated time (Post washout). Bars, 5 μm. (G) Quantification of β2AR-EEA1 co-localization for the indicated treatments as shown in F. The y-axis shows the value of Pearson’s correlation coefficient. n = 56 cells (Ctrl, DMSO); n = 64 cells (KO-91, DMSO); n = 61 cells (Ctrl, Agonist); n = 99 cells (KO-91, Agonist); n = 47 cells (Ctrl, Post washout 30 min); n = 42 cells (KO-91, Post washout 30 min); n = 45 cells (Ctrl, Post washout 45 min); n = 45 cells (KO-91, Post washout 45 min); n = 49 cells (Ctrl, Post washout 60 min); n = 43 cells (KO-91, Post washout 60 min). Statistical comparisons are between Ctrl and KO-91 cells. Statistical analyses were performed with the two-tailed unpaired t test. (H) Co-immunostaining of endogenous CI-MPR with EEA1 or TGN46 in Ctrl and KO-91 HeLa cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (I) Quantification of the co-localization of CI-MPR with endosomal or TGN markers as shown in H. The y-axis shows the value of Pearson’s correlation coefficient. n = 53 cells (Ctrl, CI-MPR-EEA1); n = 65 cells (KO-91, CI-MPR-EEA1); n = 54 cells (Ctrl, CI-MPR-TGN46); n = 46 cells (KO-91, CI-MPR-TGN46). Statistical comparisons are between Ctrl and KO-91 cells. Statistical analyses were performed with the two-tailed unpaired t test. For all quantifications, error bars represent SEM. Data are from three independent experiments. ***, P < 0.001. NS, P > 0.05.

Loss of WDR91 causes retromer-mediated trafficking defects. (A) Schematic representation of pH-GluA2. (B) Time-course recording of pH-GluA2 in mouse primary hippocampal neurons. Neurons were isolated from Wdr91+/+ or Wdr91−/− mice, transfected with pH-GluA2–expressing vector, and subjected to glutamate perfusion for 5 min. Glutamate was removed at the time point 5 min. Neurons were continuously imaged for pH-GluA2 signals. Bars, 5 μm. (C) Time-course tracing of pH-GluA2 fluorescence change in neurons shown in B. n = 9 neurons (Wdr91+/+); n = 7 neurons (Wdr91−/−). (D) Left: Maximum amplitudes of pH-GluA2 fluorescence intensity change induced by glutamate. Right: Average half-time (T1/2) for recycling after glutamate washout. n = 9 neurons (Wdr91+/+); n = 7 neurons (Wdr91−/−). Statistical analyses were performed with the Mann–Whitney U-test. (E) Schematic representation of Flag-β2AR. (F) Representative images from a visual trafficking assay of Ctrl and KO-91 HeLa cells expressing Flag-β2AR. Cells were fixed under the indicated conditions including no treatment (DMSO), isoproterenol perfusion for 30 min (Agonist), and isoproterenol perfusion for 30 min followed by washout and further incubation for the indicated time (Post washout). Bars, 5 μm. (G) Quantification of β2AR-EEA1 co-localization for the indicated treatments as shown in F. The y-axis shows the value of Pearson’s correlation coefficient. n = 56 cells (Ctrl, DMSO); n = 64 cells (KO-91, DMSO); n = 61 cells (Ctrl, Agonist); n = 99 cells (KO-91, Agonist); n = 47 cells (Ctrl, Post washout 30 min); n = 42 cells (KO-91, Post washout 30 min); n = 45 cells (Ctrl, Post washout 45 min); n = 45 cells (KO-91, Post washout 45 min); n = 49 cells (Ctrl, Post washout 60 min); n = 43 cells (KO-91, Post washout 60 min). Statistical comparisons are between Ctrl and KO-91 cells. Statistical analyses were performed with the two-tailed unpaired t test. (H) Co-immunostaining of endogenous CI-MPR with EEA1 or TGN46 in Ctrl and KO-91 HeLa cells. Zoom images show magnified frames in the merged images. Bars, 5 µm. (I) Quantification of the co-localization of CI-MPR with endosomal or TGN markers as shown in H. The y-axis shows the value of Pearson’s correlation coefficient. n = 53 cells (Ctrl, CI-MPR-EEA1); n = 65 cells (KO-91, CI-MPR-EEA1); n = 54 cells (Ctrl, CI-MPR-TGN46); n = 46 cells (KO-91, CI-MPR-TGN46). Statistical comparisons are between Ctrl and KO-91 cells. Statistical analyses were performed with the two-tailed unpaired t test. For all quantifications, error bars represent SEM. Data are from three independent experiments. ***, P < 0.001. NS, P > 0.05.

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