Figure S5.
SNX27 is recruited to dendritic spines upon cLTP, which is dependent on the CaMKII pathway. (A) Representative confocal images from DIV17 hippocampal neurons expressing mScarlet-SNX27 and eGFP (filler) under conditions of cLTP or HBS control (mock). Images of live neurons were captured before cLTP (baseline); during cLTP; and at 0, 5, 10, 15, 20, 25, and 30 min after the cLTP stimulus. Representative images of baseline, t = 10, and t = 30 are shown. Intensity presented in the fire LUT color scheme. Scale bar, 2.5 µm. (B) The endpoint of the experiment (30 min) was used to measure the cLTP-dependent increase in dendritic spine width. To calculate the increase in spine size, the final spine width was normalized to the baseline width for each spine. Mock: 1.024 ± 0.025, N = 98 spines across 15 cells; cLTP: 1.498 ± 0.056, N = 108 spines across 15 cells. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) The mean intensity of mScarlet-SNX27 was measured in the spines that remained in the same plane at each time point following cLTP (or mock) and normalized to the baseline for each individual spine. Mock: N = 98 spines across 15 cells (baseline: 1,000, cLTP: 0.989 ± 0.023, 0: 1.037 ± 0.023, 5: 1.037 ± 0.025, 10: 0.991 ± 0.028, 15: 1.032 ± 0.027, 20: 1.022 ± 0.031, 25: 1.015 ± 0.029, 30: 1.017 ± 0.028); cLTP: N = 108 spines across 15 cells (baseline: 1,000, cLTP: 1.117 ± 0.034, 0: 1.252 ± 0.057, 5: 1.347 ± 0.070, 10: 1.506 ± 0.073, 15: 1.523 ± 0.065, 20: 1.566 ± 0.066, 25: 1.651 ± 0.073, 30: 1.673 ± 0.064). Two-way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance, **P < 0.01, ****P < 0.001. Error bars are SEM. (D) The number of puncta containing mScarlet-SNX27 in 30 µm of dendritic spines at the indicated time points was quantified for the cLTP and mock conditions and normalized to the baseline for each dendrite. Mock: N = 15 dendrites (baseline: 1,000, 10: 1.020 ± 0.025, 30: 1.079 ± 0.028); cLTP: N = 15 dendrites (baseline: 1,000, 10: 1.312 ± 0.049, 30: 1.544 ± 0.074). Three independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance, ****P <0.001. Error bars are SEM. (E) DIV16 hippocampal neurons were co-transfected with mScarlet-SNX27 and either an empty pRK5 vector or pRK5-HA-CaMKII-T286D. 24 h later, neurons were fixed, permeabilized, and incubated with an anti-HA antibody. 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. Ctrl: 0.775 ± 0.035, N = 30 neurons; CaMKII-T286D: 0.909 ± 0.041, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, *P < 0.05. Error bars are SEM. (F) Same as E, but the number of puncta containing mScarlet-SNX27 in 30 µm of dendritic spines was quantified. Ctrl: 0.257 ± 0.016, N = 30 neurons; CaMKII-T286D: 0.365 ± 0.018, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

SNX27 is recruited to dendritic spines upon cLTP, which is dependent on the CaMKII pathway. (A) Representative confocal images from DIV17 hippocampal neurons expressing mScarlet-SNX27 and eGFP (filler) under conditions of cLTP or HBS control (mock). Images of live neurons were captured before cLTP (baseline); during cLTP; and at 0, 5, 10, 15, 20, 25, and 30 min after the cLTP stimulus. Representative images of baseline, t = 10, and t = 30 are shown. Intensity presented in the fire LUT color scheme. Scale bar, 2.5 µm. (B) The endpoint of the experiment (30 min) was used to measure the cLTP-dependent increase in dendritic spine width. To calculate the increase in spine size, the final spine width was normalized to the baseline width for each spine. Mock: 1.024 ± 0.025, N = 98 spines across 15 cells; cLTP: 1.498 ± 0.056, N = 108 spines across 15 cells. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) The mean intensity of mScarlet-SNX27 was measured in the spines that remained in the same plane at each time point following cLTP (or mock) and normalized to the baseline for each individual spine. Mock: N = 98 spines across 15 cells (baseline: 1,000, cLTP: 0.989 ± 0.023, 0: 1.037 ± 0.023, 5: 1.037 ± 0.025, 10: 0.991 ± 0.028, 15: 1.032 ± 0.027, 20: 1.022 ± 0.031, 25: 1.015 ± 0.029, 30: 1.017 ± 0.028); cLTP: N = 108 spines across 15 cells (baseline: 1,000, cLTP: 1.117 ± 0.034, 0: 1.252 ± 0.057, 5: 1.347 ± 0.070, 10: 1.506 ± 0.073, 15: 1.523 ± 0.065, 20: 1.566 ± 0.066, 25: 1.651 ± 0.073, 30: 1.673 ± 0.064). Two-way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance, **P < 0.01, ****P < 0.001. Error bars are SEM. (D) The number of puncta containing mScarlet-SNX27 in 30 µm of dendritic spines at the indicated time points was quantified for the cLTP and mock conditions and normalized to the baseline for each dendrite. Mock: N = 15 dendrites (baseline: 1,000, 10: 1.020 ± 0.025, 30: 1.079 ± 0.028); cLTP: N = 15 dendrites (baseline: 1,000, 10: 1.312 ± 0.049, 30: 1.544 ± 0.074). Three independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance, ****P <0.001. Error bars are SEM. (E) DIV16 hippocampal neurons were co-transfected with mScarlet-SNX27 and either an empty pRK5 vector or pRK5-HA-CaMKII-T286D. 24 h later, neurons were fixed, permeabilized, and incubated with an anti-HA antibody. 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. Ctrl: 0.775 ± 0.035, N = 30 neurons; CaMKII-T286D: 0.909 ± 0.041, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, *P < 0.05. Error bars are SEM. (F) Same as E, but the number of puncta containing mScarlet-SNX27 in 30 µm of dendritic spines was quantified. Ctrl: 0.257 ± 0.016, N = 30 neurons; CaMKII-T286D: 0.365 ± 0.018, N = 30 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. DIV, days in vitro.

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