Figure 4.
Brief inhibition of PI(3)P synthesis upon cLTP blocks the recruitment of SNX17 and SNX27 to dendritic spines. (A) Diagram of experiment. DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 or mScarlet-SNX27 along with a filler to visualize dendritic morphology. 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the intensity of SNX17 or SNX27 at dendritic spines and in the dendritic shaft was quantified. (B) Example confocal images of DIV17 neurons expressing mNeonGreen-SNX17 and mCherry (filler) under the indicated conditions. mNeonGreen-SNX17 intensity is presented in the fire LUT color scheme to facilitate the visualization of dendritic spines. Scale bar, 2.5 µm. (C) Example confocal images of DIV17 neurons expressing mScarlet-SNX27 and eGFP (filler) under the indicated conditions. mScarlet-SNX27 intensity is presented in the fire LUT color scheme to facilitate the visualization of dendritic spines. Scale bar, 2.5 µm. (D) The mean intensity of mNeonGreen-SNX17 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mNeonGreen-SNX17 mean intensity in the dendritic shaft. Baseline: 0.657 ± 0.026, N = 30 neurons; cLTP+DMSO: 0.823 ± 0.036, N = 30 neurons; cLTP+VPS34-IN1: 0.673 ± 0.026, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (E) The mean intensity of mScarlet-SNX27 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mScarlet-SNX27 mean intensity in the dendritic shaft. Baseline: 0.759 ± 0.035, N = 30 neurons; cLTP+DMSO: 0.976 ± 0.060, N = 30 neurons; cLTP+VPS34-IN1: 0.729 ± 0.049, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. DIV, days in vitro.

Brief inhibition of PI(3)P synthesis upon cLTP blocks the recruitment of SNX17 and SNX27 to dendritic spines. (A) Diagram of experiment. DIV16 hippocampal neurons were transfected with mNeonGreen-SNX17 or mScarlet-SNX27 along with a filler to visualize dendritic morphology. 24 h later, cells were treated with or without cLTP stimulation. cLTP-treated cells were washed and incubated in HBS containing either DMSO or 1 μM VPS34-IN1 for 10 min. Cells were fixed, and the intensity of SNX17 or SNX27 at dendritic spines and in the dendritic shaft was quantified. (B) Example confocal images of DIV17 neurons expressing mNeonGreen-SNX17 and mCherry (filler) under the indicated conditions. mNeonGreen-SNX17 intensity is presented in the fire LUT color scheme to facilitate the visualization of dendritic spines. Scale bar, 2.5 µm. (C) Example confocal images of DIV17 neurons expressing mScarlet-SNX27 and eGFP (filler) under the indicated conditions. mScarlet-SNX27 intensity is presented in the fire LUT color scheme to facilitate the visualization of dendritic spines. Scale bar, 2.5 µm. (D) The mean intensity of mNeonGreen-SNX17 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mNeonGreen-SNX17 mean intensity in the dendritic shaft. Baseline: 0.657 ± 0.026, N = 30 neurons; cLTP+DMSO: 0.823 ± 0.036, N = 30 neurons; cLTP+VPS34-IN1: 0.673 ± 0.026, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (E) The mean intensity of mScarlet-SNX27 was measured in the dendritic spines present in the first 30 μm of secondary dendrites and normalized to mScarlet-SNX27 mean intensity in the dendritic shaft. Baseline: 0.759 ± 0.035, N = 30 neurons; cLTP+DMSO: 0.976 ± 0.060, N = 30 neurons; cLTP+VPS34-IN1: 0.729 ± 0.049, N = 30 neurons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. DIV, days in vitro.

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