Figure 7.
WDR81 acts through WDR91 to affect endosomal subdomain formation and recycling of membrane receptors. (A) Representative images from a visual trafficking assay of Flag-β2AR–expressing Ctrl HeLa cells, KO-81 HeLa cells, and KO-81 cells transfected with empty, Myc-WDR81–, or Myc-WDR91–expressing vectors. 24 h after transfection, the 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 45 min (Post washout 45 min). Bars, 5 μm. (B) Quantification of β2AR-EEA1 co-localization for the indicated treatments as shown in A. The y-axis shows the value of Pearson’s correlation coefficient. n = 48 cells (Ctrl, DMSO); n = 48 cells (KO-81, DMSO); n = 40 cells (Ctrl, Agonist); n = 53 cells (KO-81, Agonist); n = 44 cells (Ctrl, Post washout 45 min); n = 46 cells (KO-81, empty, Post washout 45 min); n = 44 cells (KO-81 + WDR81, Post washout 45 min); n = 50 cells (KO-81 + WDR91, Post washout 45 min). (C) Co-immunostaining of endogenous CI-MPR in Ctrl and KO-81 HeLa cells transfected with empty, Myc-WDR81–, or Myc-WDR91–expressing vectors. Zoom images show magnified frames in the merged images. 24 h after transfection, the cells were fixed and stained with CI-MPR and EEA1 antibodies. Bars, 5 µm. (D) Quantification of the co-localization of CI-MPR with EEA1 as shown in C. n = 49 cells (Ctrl); n = 47 cells (KO-81, empty); n = 46 cells (KO-81 + WDR81); n = 49 cells (KO-81 + WDR91). Co-localization was quantified according to Pearson’s correlation coefficient. (E) Representative images of endosomal mCh-PH2C1 in KO-81 cells without or with ectopic expression of GFP-WDR81 and GFP-WDR91. Bars, 1 μm. (F) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane as shown in E. n = 12 endosomes (KO-81); n = 13 endosomes (KO-81 + GFP-WDR81); n = 11 endosomes (KO-81 + GFP-WDR91). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). (G) Representative images of endosomal mCh-PH2C1 in KO-91 cells without or with expression of GFP-WDR81, and GFP-WDR81 together with BFP-WDR91. Bars, 1 μm. (H) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane as shown in G. n = 10 endosomes (KO-91); n = 11 endosomes (KO-91 + GFP-WDR81); n = 11 endosomes (KO-91 + GFP-WDR81 + BFP-WDR91). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). For all quantifications, error bars represent SEM. Data are from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. ***, P < 0.001. NS, P > 0.05.

WDR81 acts through WDR91 to affect endosomal subdomain formation and recycling of membrane receptors. (A) Representative images from a visual trafficking assay of Flag-β2AR–expressing Ctrl HeLa cells, KO-81 HeLa cells, and KO-81 cells transfected with empty, Myc-WDR81–, or Myc-WDR91–expressing vectors. 24 h after transfection, the 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 45 min (Post washout 45 min). Bars, 5 μm. (B) Quantification of β2AR-EEA1 co-localization for the indicated treatments as shown in A. The y-axis shows the value of Pearson’s correlation coefficient. n = 48 cells (Ctrl, DMSO); n = 48 cells (KO-81, DMSO); n = 40 cells (Ctrl, Agonist); n = 53 cells (KO-81, Agonist); n = 44 cells (Ctrl, Post washout 45 min); n = 46 cells (KO-81, empty, Post washout 45 min); n = 44 cells (KO-81 + WDR81, Post washout 45 min); n = 50 cells (KO-81 + WDR91, Post washout 45 min). (C) Co-immunostaining of endogenous CI-MPR in Ctrl and KO-81 HeLa cells transfected with empty, Myc-WDR81–, or Myc-WDR91–expressing vectors. Zoom images show magnified frames in the merged images. 24 h after transfection, the cells were fixed and stained with CI-MPR and EEA1 antibodies. Bars, 5 µm. (D) Quantification of the co-localization of CI-MPR with EEA1 as shown in C. n = 49 cells (Ctrl); n = 47 cells (KO-81, empty); n = 46 cells (KO-81 + WDR81); n = 49 cells (KO-81 + WDR91). Co-localization was quantified according to Pearson’s correlation coefficient. (E) Representative images of endosomal mCh-PH2C1 in KO-81 cells without or with ectopic expression of GFP-WDR81 and GFP-WDR91. Bars, 1 μm. (F) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane as shown in E. n = 12 endosomes (KO-81); n = 13 endosomes (KO-81 + GFP-WDR81); n = 11 endosomes (KO-81 + GFP-WDR91). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). (G) Representative images of endosomal mCh-PH2C1 in KO-91 cells without or with expression of GFP-WDR81, and GFP-WDR81 together with BFP-WDR91. Bars, 1 μm. (H) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane as shown in G. n = 10 endosomes (KO-91); n = 11 endosomes (KO-91 + GFP-WDR81); n = 11 endosomes (KO-91 + GFP-WDR81 + BFP-WDR91). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). For all quantifications, error bars represent SEM. Data are from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. ***, P < 0.001. NS, P > 0.05.

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