Figure 8.
WDR91 coordinates with the FAM21-containing WASH complex to organize the actin network for formation of endosomal retrieval subdomains. (A) Co-localization of phalloidin-stained F-actin with Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Quantifications of linear pixel fluorescence of F-actin (green) and Rab7 (magenta) along the arrows across the endosomes in the zoom images are shown on the right. Bars, 5 µm. (B) Co-immunostaining of Rab7 with endogenous ARPC2 (top), and FAM21 (bottom) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Quantifications of linear pixel fluorescence of Rab7 (magenta), ARPC2 (green), or FAM21 (green) along the arrows across the endosomes in the zoom images are shown on the right. Bars, 5 µm. (C) Co-IP of Flag-FAM21 with GFP-WDR91. IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (D) Co-IP of endogenous FAM21 with WDR91 in control HeLa cells. IP was performed with WDR91 antibody and precipitated proteins were detected with antibodies against the indicated proteins. (E) Co-localization of GFP-FAM21 with mCh-PH2C1 in Ctrl, KO-91 cells, and KO-91 cells re-expressing BFP-WDR91. Bars, 2 μm. Quantification of linear pixel fluorescence of GFP-FAM21 (green), mCh-PH2C1 (magenta), and BFP-WDR91 (blue) along the arrows across the endosomes in the merge images are shown on the right. (F) Immunoblotting of endogenous FAM21, VPS35, and SNX27 in KO-91 cells treated with negative control siRNA (siNC), VPS35 siRNA (siVPS35), and SNX27 siRNA (siSNX27). (G) Co-immunostaining of endogenous FAM21 with Rab7 in KO-91 cells treated with siNC, siVPS35, and siSNX27. Bars, 5 μm. Quantification of endogenous FAM21 intensity on endosomes is shown on the right. n = 50 cells (siNC); n = 50 cells (siVPS35); n = 48 cells (siSNX27). Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001. (H) Immunoblotting of endogenous KIAA1033 in Ctrl (top) and KO-91 (bottom) cells treated with siNC and KIAA1033 siRNA (siKIAA1033). (I) Immunostaining of endogenous VPS35 (top) and SNX27 (bottom) in Ctrl HeLa cells treated with siNC and siKIAA1033. Zoom images show magnified frames in the left column in each group. Membrane tubules are indicated with arrowheads. Bars, 5 μm. (J) Immunostaining of endogenous VPS35 (top) and SNX27 (bottom) in KO-91 cells treated with siNC and siKIAA1033. Zoom images show magnified frames in the left column in each group. Bars, 5 μm. Source data are available for this figure: SourceData F8.

WDR91 coordinates with the FAM21-containing WASH complex to organize the actin network for formation of endosomal retrieval subdomains. (A) Co-localization of phalloidin-stained F-actin with Rab7 in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Quantifications of linear pixel fluorescence of F-actin (green) and Rab7 (magenta) along the arrows across the endosomes in the zoom images are shown on the right. Bars, 5 µm. (B) Co-immunostaining of Rab7 with endogenous ARPC2 (top), and FAM21 (bottom) in Ctrl and KO-91 cells. Zoom images show magnified frames in the merged images. Quantifications of linear pixel fluorescence of Rab7 (magenta), ARPC2 (green), or FAM21 (green) along the arrows across the endosomes in the zoom images are shown on the right. Bars, 5 µm. (C) Co-IP of Flag-FAM21 with GFP-WDR91. IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (D) Co-IP of endogenous FAM21 with WDR91 in control HeLa cells. IP was performed with WDR91 antibody and precipitated proteins were detected with antibodies against the indicated proteins. (E) Co-localization of GFP-FAM21 with mCh-PH2C1 in Ctrl, KO-91 cells, and KO-91 cells re-expressing BFP-WDR91. Bars, 2 μm. Quantification of linear pixel fluorescence of GFP-FAM21 (green), mCh-PH2C1 (magenta), and BFP-WDR91 (blue) along the arrows across the endosomes in the merge images are shown on the right. (F) Immunoblotting of endogenous FAM21, VPS35, and SNX27 in KO-91 cells treated with negative control siRNA (siNC), VPS35 siRNA (siVPS35), and SNX27 siRNA (siSNX27). (G) Co-immunostaining of endogenous FAM21 with Rab7 in KO-91 cells treated with siNC, siVPS35, and siSNX27. Bars, 5 μm. Quantification of endogenous FAM21 intensity on endosomes is shown on the right. n = 50 cells (siNC); n = 50 cells (siVPS35); n = 48 cells (siSNX27). Statistical analyses were performed with the Kruskal–Wallis test. ***, P < 0.001. (H) Immunoblotting of endogenous KIAA1033 in Ctrl (top) and KO-91 (bottom) cells treated with siNC and KIAA1033 siRNA (siKIAA1033). (I) Immunostaining of endogenous VPS35 (top) and SNX27 (bottom) in Ctrl HeLa cells treated with siNC and siKIAA1033. Zoom images show magnified frames in the left column in each group. Membrane tubules are indicated with arrowheads. Bars, 5 μm. (J) Immunostaining of endogenous VPS35 (top) and SNX27 (bottom) in KO-91 cells treated with siNC and siKIAA1033. Zoom images show magnified frames in the left column in each group. Bars, 5 μm. Source data are available for this figure: SourceData F8.

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