Figure 6.
WDR91 enriches and restricts active Rab7 and SNX-retromer components for formation of the endosomal retrieval subdomain. (A) Schematic depiction of an active Rab7-detecting sensor derived from the PH2-C1 domain of PLEKHM1. (B) Co-IP of GFP-Rab7, GFP-Rab7(Q67L), and Rab7(T22N) with Myc-PH2C1. IPs were performed with GFP-trap beads and immunoblotted using Myc and GFP antibodies. (C) Co-localization of mCherry-PH2C1 (mCh-PH2C1) with GFP-Rab7, GFP-Rab7(Q67L), and GFP-Rab7(T22N) in HeLa cells. Bars, 5 μm. (D) Representative images of endosomal mCh-PH2C1 in Ctrl cells, KO-91 cells, and KO-91 cells expressing GFP-WDR91, GFP-RILP, or GFP-FYCO1. Bars, 1 μm. (E) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane (as indicated in the upper right) as shown in D. n = 12 endosomes (Ctrl); n = 12 endosomes (KO-91); n = 12 endosomes (KO-91 + GFP-WDR91); n = 12 endosomes (KO-91 + GFP-RILP); n = 12 endosomes (KO91 + GFP-FYCO1). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). (F) FRAP analysis of mCh-PH2C1 in Ctrl, KO-91 cells, and KO-91 cells re-expressing WDR91. The frame in each individual image indicates the photo-bleached area. Bars, 1 μm. (G) Quantification of fluorescence recovery of mCh-PH2C1 (left), and recovery half-time (t1/2; right). n = 18 endosomes (Ctrl); n = 16 endosomes (KO-91); n = 16 endosomes (KO-91 + WDR91). Data represent mean ± SEM. Statistical analyses were performed with the Kruskal–Wallis test. **, P < 0.01; ***, P < 0.001. (H and I) Co-localization of SNX27-GFP, VPS35-GFP, SNX6-GFP with mCh-PH2C1 in Ctrl (H) and KO-91(I) HeLa cells. Quantifications of linear pixel fluorescence of GFP-tagged proteins (green) and mCh-PH2C1 (magenta) along the arrows across the endosomes in the merge images are shown on the right. Bars, 2 μm. (J) Co-localization of SNX27-GFP, VPS35-GFP, SNX6-GFP with mCh-PH2C1 in KO-91 cells re-expressing BFP-WDR91. Quantifications of linear pixel fluorescence of GFP-tagged proteins (green), mCh-PH2C1 (magenta), and BFP-WDR91 (blue) along the arrows across the endosomes in the merge images are shown on the right. Bars, 2 μm. (K and L) Time-lapse chasing of SNX27-GFP–labeled (K) or VPS35-GFP–labeled (L) tubulation events (indicated by arrowheads in different colors) from mCh-WDR91–enriched endosomal domains. Bars, 1 μm. Source data are available for this figure: SourceData F6.

WDR91 enriches and restricts active Rab7 and SNX-retromer components for formation of the endosomal retrieval subdomain. (A) Schematic depiction of an active Rab7-detecting sensor derived from the PH2-C1 domain of PLEKHM1. (B) Co-IP of GFP-Rab7, GFP-Rab7(Q67L), and Rab7(T22N) with Myc-PH2C1. IPs were performed with GFP-trap beads and immunoblotted using Myc and GFP antibodies. (C) Co-localization of mCherry-PH2C1 (mCh-PH2C1) with GFP-Rab7, GFP-Rab7(Q67L), and GFP-Rab7(T22N) in HeLa cells. Bars, 5 μm. (D) Representative images of endosomal mCh-PH2C1 in Ctrl cells, KO-91 cells, and KO-91 cells expressing GFP-WDR91, GFP-RILP, or GFP-FYCO1. Bars, 1 μm. (E) Measurement of mCh-PH2C1 fluorescence intensity in a clockwise direction along the endosomal membrane (as indicated in the upper right) as shown in D. n = 12 endosomes (Ctrl); n = 12 endosomes (KO-91); n = 12 endosomes (KO-91 + GFP-WDR91); n = 12 endosomes (KO-91 + GFP-RILP); n = 12 endosomes (KO91 + GFP-FYCO1). The profiles of endosomes were obtained and normalized to achieve the average endosomal mCh-PH2C1 fluorescence intensity (mean ± SEM). (F) FRAP analysis of mCh-PH2C1 in Ctrl, KO-91 cells, and KO-91 cells re-expressing WDR91. The frame in each individual image indicates the photo-bleached area. Bars, 1 μm. (G) Quantification of fluorescence recovery of mCh-PH2C1 (left), and recovery half-time (t1/2; right). n = 18 endosomes (Ctrl); n = 16 endosomes (KO-91); n = 16 endosomes (KO-91 + WDR91). Data represent mean ± SEM. Statistical analyses were performed with the Kruskal–Wallis test. **, P < 0.01; ***, P < 0.001. (H and I) Co-localization of SNX27-GFP, VPS35-GFP, SNX6-GFP with mCh-PH2C1 in Ctrl (H) and KO-91(I) HeLa cells. Quantifications of linear pixel fluorescence of GFP-tagged proteins (green) and mCh-PH2C1 (magenta) along the arrows across the endosomes in the merge images are shown on the right. Bars, 2 μm. (J) Co-localization of SNX27-GFP, VPS35-GFP, SNX6-GFP with mCh-PH2C1 in KO-91 cells re-expressing BFP-WDR91. Quantifications of linear pixel fluorescence of GFP-tagged proteins (green), mCh-PH2C1 (magenta), and BFP-WDR91 (blue) along the arrows across the endosomes in the merge images are shown on the right. Bars, 2 μm. (K and L) Time-lapse chasing of SNX27-GFP–labeled (K) or VPS35-GFP–labeled (L) tubulation events (indicated by arrowheads in different colors) from mCh-WDR91–enriched endosomal domains. Bars, 1 μm. Source data are available for this figure: SourceData F6.

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