Fig. 3

Mutation of the phosphorylation site S499 of FMRP alters its association with TDP-43 and subsequent spine localization of the TDP-43 granules. A IP analysis using anti-GFP was carried out with HEK293T cell lysates co-expressing RFP-TDP-43 plus GFP–FMRP (WT), GFP–FMRP (S499D), or GFP–FMRP (S499A) followed by Western blotting using anti-RFP and anti-GFP. (a) Left, representative Western blotting patterns showing the differential association of TDP-43 with FMRP (WT), phosphomimetic FMRP (S499D), and dephosphomimetic FMRP (S499A), respectively. Input panels on the right show the expression levels of different exogenous proteins in the transfected HEK293T cells. (b) Bar diagram from three independent IP experiments using anti-GFP showing the average fold enrichment of RFP-TDP-43 protein relative to the IgG control after normalization with amount of GFP-FMRP. Error bars represent SEM. Student’s t-test was performed to compare the mean with the corresponding IgG control and indicated as ***p < 0.0001. One-way ANOVA was used to compare fold enrichment of RFP-TDP-TDP-43 in Wt and mutant GFP-FMRP-expressing cells, *q < 0.05 (not shown in the figure). B Co-localization of RFP-TDP-43 with GFP-tagged WT or mutant FMRP proteins in the dendrites and with endogenous myosin V protein in the spine, respectively, of DIV 14 primary hippocampal neurons. The co-IF analysis was performed using anti-RFP, anti-GFP, and anti-myosin V. (a) Representative confocal microscopy images showing the co-localizations of RFP-TDP-43 puncta with WT GFP-FMRP and mutant GFP-FMRP in the dendrites or with myosin V in the spine, as indicated by the arrows. Magnified pictures of specific spine regions are shown in the lower panels. Scale bars, 2 µm. White dotted lines represent the boundaries of the dendrites and spine regions as determined from the corresponding DIC images shown in Supplementary Fig. S9A. Note that, in primary neurons co-expressing GFP-FMRP(S499A) and RFP-TDP-43, dendritic transport of TDP-43 granules in the anterograde direction was severely restricted presumably due to loss of the association between TDP-43 and FMRP [43]. For unbiased comparison, the dendritic spine regions near soma were selected for analysis under all three experimental conditions. (b, c) Statistical analysis of the co-localization (%) represented by the bar diagrams. The data are derived from three sets of independent experiments (N = 3). Technical repeats, n = 26–30 dendritic and spine regions. Error bars are SEM. Student’s t-test was carried out to compare the means and indicated as **p < 0.001, ***p < 0.0001. One-way ANOVA was used to compare Wt and different mutants, *q < 0.05 (a), ***q < 0.0001 (b), (not shown in the figure)