Figure 7.
Figure 7. WDR91 is required for endosome conversion in mouse neurons. (A) Endosome conversion is compromised in neuronal cells of Wdr91−/− mice. Immunostaining of EEA1, mCh-Rab7, and Map2 in cultured primary hippocampal neurons isolated from Wdr91+/+ and Wdr91−/− mice (left). Neurons were transfected with vectors expressing mCh-Rab7 at the fourth day after isolation and subjected to immunostaining 3 d after transfection. Quantification of endosomes that were positive for EEA1 and negative for Rab7 (EEA1+;Rab7−), positive for both EEA1 and Rab7 (EEA1+;Rab7+), and positive for Rab7 and negative for EEA1 (EEA1−;Rab7+) in cultured primary hippocampal neurons (right). ≥10 neurons were scored. Data representing means ± SEM are from three independent experiments. (B) Rescuing effects of Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) on EEA1-positive endosomes in the primary hippocampal neurons isolated from Wdr91−/− mice. Neurons isolated from Wdr91+/+ or Wdr91−/− mice were transfected with vectors expressing the indicated proteins and then subjected to immunostaining of EEA1 (red) and GFP or Flag (green) 3 d after transfection. Insets show magnified images of the boxes in the merged images. Quantification of neurons with giant endosomes after transfection is shown at the bottom right. ≥25 neurons were scored. Data representing means ± SEM are from three independent experiments. (C) Representative images of primary hippocampal neurons isolated from Wdr91+/+ or Wdr91−/− mice. Neurons with the indicated genotype were transfected with a GFP-expressing vector and individual vectors expressing the indicated WDR91 proteins. (D) Quantification of the cumulative length of the dendrites of hippocampal neurons shown in C. ≥35 neurons were scored. (E) Quantification by Sholl analysis of the dendritic branches of hippocampal neurons shown in C. ≥30 neurons were scored. Data represent means ± SEM in all quantifications. ***, P < 0.001. Bars, 20 µm.

WDR91 is required for endosome conversion in mouse neurons. (A) Endosome conversion is compromised in neuronal cells of Wdr91−/− mice. Immunostaining of EEA1, mCh-Rab7, and Map2 in cultured primary hippocampal neurons isolated from Wdr91+/+ and Wdr91−/− mice (left). Neurons were transfected with vectors expressing mCh-Rab7 at the fourth day after isolation and subjected to immunostaining 3 d after transfection. Quantification of endosomes that were positive for EEA1 and negative for Rab7 (EEA1+;Rab7−), positive for both EEA1 and Rab7 (EEA1+;Rab7+), and positive for Rab7 and negative for EEA1 (EEA1−;Rab7+) in cultured primary hippocampal neurons (right). ≥10 neurons were scored. Data representing means ± SEM are from three independent experiments. (B) Rescuing effects of Flag-WDR91, Flag-WDR91(3A), and Flag-WDR91(Δ5) on EEA1-positive endosomes in the primary hippocampal neurons isolated from Wdr91−/− mice. Neurons isolated from Wdr91+/+ or Wdr91−/− mice were transfected with vectors expressing the indicated proteins and then subjected to immunostaining of EEA1 (red) and GFP or Flag (green) 3 d after transfection. Insets show magnified images of the boxes in the merged images. Quantification of neurons with giant endosomes after transfection is shown at the bottom right. ≥25 neurons were scored. Data representing means ± SEM are from three independent experiments. (C) Representative images of primary hippocampal neurons isolated from Wdr91+/+ or Wdr91−/− mice. Neurons with the indicated genotype were transfected with a GFP-expressing vector and individual vectors expressing the indicated WDR91 proteins. (D) Quantification of the cumulative length of the dendrites of hippocampal neurons shown in C. ≥35 neurons were scored. (E) Quantification by Sholl analysis of the dendritic branches of hippocampal neurons shown in C. ≥30 neurons were scored. Data represent means ± SEM in all quantifications. ***, P < 0.001. Bars, 20 µm.

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