Figure 6.
WDR44 tubules are in close contact with the ER via binding to VAPA/B. (A) Confocal images of HeLa cells incubated with 10 kD Alexa Fluor 546–dextran (5 mg/ml, 30 min) and stained with α-WDR44. There was no colocalization. (B) Confocal images of HeLa cells stained with α-WDR44 and α-ERGIC53, α-GM130, α-TGN46, or α-LAMP2. For visualization of recycling endosomes, cells were incubated with Alexa Fluor 546–Transferrin (10 µg/ml, 1 h); for the ER, cells were transfected with Calnexin-GFP. WDR44 tubules only colocalized with Calnexin-GFP. (C) Confocal images of transfected HeLa cells stained with α-Calreticulin, which colocalized with GFP–WDR44 ΔC patches. (D and F) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. The efficiency of binding of myc-VAPA (D) and myc-WDR44 (F) was quantified using binding to GFP-WDR44 (D) or to GFP-VAPA (F) as reference. n = 3 or 4. (D) myc-VAPA was coimmunoprecipitated by GFP-WDR44, but not by mutants lacking the first 14 aa of the protein. (E) Schematic representation of human VAPA and of the mutants used. VAPA and VAPB have a cytoplasmic Major Sperm Protein (MSP) domain, a coiled coil (CC) region, and a transmembrane (TM) tail for anchoring in the ER. (F) myc-WDR44 was coimmunoprecipitated by GFP-tagged VAPA/B, but not by VAPA ΔMSP or VAPA DD. (G and H) Confocal images of transfected HeLa cells showing colocalization of RFP-WDR44 tubules with GFP-VAPA (G) and with endogenous VAPB (H). (I) Confocal images of live HeLa cells. Snapshots were taken every 5 s and are shown at relevant time points. RFP-WDR44 and Calnexin-GFP tubules were closely associated throughout time. Scale bars: 5 µm. (J) STED images of myc-WDR44–transfected HeLa cells stained with α-myc and α-Calnexin, and fluorescence intensity profiles along the line drawn, showing that the myc-WDR44 (red line) and Calnexin (cyan line) peaks were superimposed on tubule 1 but shifted by ∼80 nm on tubules 2 and 3. (K) Confocal stack of transfected HeLa cells. GFP-WDR44 ΔFFAT was associated with tubules. (L) Confocal images of transfected HeLa cells. GFP-WDR44 ΔFFAT tubules did not colocalize with RFP-VAPA. (M) Percentage of shRNA-expressing HeLa cells with endogenous (endo.) WDR44 tubules. n = 5–8. (N) Confocal images of transfected HeLa cells showing colocalization of GRAF2-RFP tubules with GFP-VAPA. (O) Confocal images of transfected HeLa cells stained with α-myc and α-Calnexin. When myc-WDR44 was coexpressed, GRAF2-GFP–positive tubules colocalized with an endogenous ER marker. (D, F, and M) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (A–C, G, H, J–L, N, and O) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

WDR44 tubules are in close contact with the ER via binding to VAPA/B. (A) Confocal images of HeLa cells incubated with 10 kD Alexa Fluor 546–dextran (5 mg/ml, 30 min) and stained with α-WDR44. There was no colocalization. (B) Confocal images of HeLa cells stained with α-WDR44 and α-ERGIC53, α-GM130, α-TGN46, or α-LAMP2. For visualization of recycling endosomes, cells were incubated with Alexa Fluor 546–Transferrin (10 µg/ml, 1 h); for the ER, cells were transfected with Calnexin-GFP. WDR44 tubules only colocalized with Calnexin-GFP. (C) Confocal images of transfected HeLa cells stained with α-Calreticulin, which colocalized with GFP–WDR44 ΔC patches. (D and F) Immunoprecipitation (IP) of transfected 293T cells with α-GFP. The efficiency of binding of myc-VAPA (D) and myc-WDR44 (F) was quantified using binding to GFP-WDR44 (D) or to GFP-VAPA (F) as reference. n = 3 or 4. (D) myc-VAPA was coimmunoprecipitated by GFP-WDR44, but not by mutants lacking the first 14 aa of the protein. (E) Schematic representation of human VAPA and of the mutants used. VAPA and VAPB have a cytoplasmic Major Sperm Protein (MSP) domain, a coiled coil (CC) region, and a transmembrane (TM) tail for anchoring in the ER. (F) myc-WDR44 was coimmunoprecipitated by GFP-tagged VAPA/B, but not by VAPA ΔMSP or VAPA DD. (G and H) Confocal images of transfected HeLa cells showing colocalization of RFP-WDR44 tubules with GFP-VAPA (G) and with endogenous VAPB (H). (I) Confocal images of live HeLa cells. Snapshots were taken every 5 s and are shown at relevant time points. RFP-WDR44 and Calnexin-GFP tubules were closely associated throughout time. Scale bars: 5 µm. (J) STED images of myc-WDR44–transfected HeLa cells stained with α-myc and α-Calnexin, and fluorescence intensity profiles along the line drawn, showing that the myc-WDR44 (red line) and Calnexin (cyan line) peaks were superimposed on tubule 1 but shifted by ∼80 nm on tubules 2 and 3. (K) Confocal stack of transfected HeLa cells. GFP-WDR44 ΔFFAT was associated with tubules. (L) Confocal images of transfected HeLa cells. GFP-WDR44 ΔFFAT tubules did not colocalize with RFP-VAPA. (M) Percentage of shRNA-expressing HeLa cells with endogenous (endo.) WDR44 tubules. n = 5–8. (N) Confocal images of transfected HeLa cells showing colocalization of GRAF2-RFP tubules with GFP-VAPA. (O) Confocal images of transfected HeLa cells stained with α-myc and α-Calnexin. When myc-WDR44 was coexpressed, GRAF2-GFP–positive tubules colocalized with an endogenous ER marker. (D, F, and M) Data are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. (A–C, G, H, J–L, N, and O) Insets show magnifications of the boxed areas. Scale bars: 10 µm; scale bars of insets: 2 µm.

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