Figure 6.
Role of KIF13A in LTP-induced endosomal redistribution. (A) Representative images of KIF13A-YFP (magenta) and mCherry-FIP2 (green) fluorescence in transfected HEK-293T cells 24 h after transfection. Scale bars, 10 µm. (B) Quantification of colocalization between KIF13A-YFP and mCherry-FIP2 in clustered areas compared with the expected colocalization for a random distribution of the proteins (percentage of mCherry-FIP2 and KIF13A-YFP clustered area over whole-cell area, respectively). Plots represent percentage of the area for each individual cell and show mean ± SEM for each analysis. n = 16 cells from two independent experiments; P < 0.0001, Wilcoxon test. (C) Quantification of colocalization between KIF13A-YFP and mCherry-FIP2 clusters from the experiments described in A using PCC an MOC. Individual Manders coefficients (M1 and M2, KIF13A and FIP2, respectively) are also shown. Plots represent correlation coefficient for each individual cell and show mean ± SEM for each analysis. n = 16 cells from two independent experiments. (D) Representative images of a cLTP experiment showing dendritic branches from an organotypic hippocampal slice infected with GFP-FIP2 (green) together with a control lentivirus (control; left panels) or a lentivirus expressing shKIF13A (right panels), both expressing mCherry (red). Arrows indicate coinfected dendrites. Scale bars, 10 µm. (E) Quantifications of cluster density per dendrite for each condition before and after cLTP induction. Analysis of cluster density was performed blind with respect to the lentivirus identity. Gray lines represent each analyzed dendrite, and black and red symbols show the mean ± SEM for each condition. n = 24 (control) and n = 13 (shKIF13A) dendrites from 10 and 6 independent experiments, respectively; P = 0.005, Wilcoxon test. (F) Cumulative probability distribution of cluster area of GFP-FIP2 for each condition described in D and E. Lighter lines represent baseline conditions. Darker lines represent conditions after cLTP induction. Gray lines represent data from slices infected with the control lentivirus (lacking shRNA sequence). Red lines represent data from slices expressing shKIF13A. P value < 0.0001 (Kolmogorov–Smirnov test) compares baseline and 15-min LTP in control conditions. Cluster size did not change in dendrites lacking KIF13A (shKIF13A; P = 0.92, Kolmogorov–Smirnov test).

Role of KIF13A in LTP-induced endosomal redistribution. (A) Representative images of KIF13A-YFP (magenta) and mCherry-FIP2 (green) fluorescence in transfected HEK-293T cells 24 h after transfection. Scale bars, 10 µm. (B) Quantification of colocalization between KIF13A-YFP and mCherry-FIP2 in clustered areas compared with the expected colocalization for a random distribution of the proteins (percentage of mCherry-FIP2 and KIF13A-YFP clustered area over whole-cell area, respectively). Plots represent percentage of the area for each individual cell and show mean ± SEM for each analysis. n = 16 cells from two independent experiments; P < 0.0001, Wilcoxon test. (C) Quantification of colocalization between KIF13A-YFP and mCherry-FIP2 clusters from the experiments described in A using PCC an MOC. Individual Manders coefficients (M1 and M2, KIF13A and FIP2, respectively) are also shown. Plots represent correlation coefficient for each individual cell and show mean ± SEM for each analysis. n = 16 cells from two independent experiments. (D) Representative images of a cLTP experiment showing dendritic branches from an organotypic hippocampal slice infected with GFP-FIP2 (green) together with a control lentivirus (control; left panels) or a lentivirus expressing shKIF13A (right panels), both expressing mCherry (red). Arrows indicate coinfected dendrites. Scale bars, 10 µm. (E) Quantifications of cluster density per dendrite for each condition before and after cLTP induction. Analysis of cluster density was performed blind with respect to the lentivirus identity. Gray lines represent each analyzed dendrite, and black and red symbols show the mean ± SEM for each condition. n = 24 (control) and n = 13 (shKIF13A) dendrites from 10 and 6 independent experiments, respectively; P = 0.005, Wilcoxon test. (F) Cumulative probability distribution of cluster area of GFP-FIP2 for each condition described in D and E. Lighter lines represent baseline conditions. Darker lines represent conditions after cLTP induction. Gray lines represent data from slices infected with the control lentivirus (lacking shRNA sequence). Red lines represent data from slices expressing shKIF13A. P value < 0.0001 (Kolmogorov–Smirnov test) compares baseline and 15-min LTP in control conditions. Cluster size did not change in dendrites lacking KIF13A (shKIF13A; P = 0.92, Kolmogorov–Smirnov test).

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