Figure 4: SUMOylation of SynIa modulates its presynaptic targeting and reclustering at the presynapse following stimulation. | Nature Communications

Figure 4: SUMOylation of SynIa modulates its presynaptic targeting and reclustering at the presynapse following stimulation.

From: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation

Figure 4

(a) SUMOylation is required for presynaptic targeting. WT (n=12) or C603S (dead mutant, n=12) SENP1-mCherry was overexpressed in DIV 9–10 hippocampal neurons. Cells were fixed 7 days later and immunostained with anti-SynI and anti-bassoon (presynaptic marker) antibodies. Scale bar, 5 μm. (b) Quantification of a. Co-localizing SynIa and bassoon-positive puncta were counted and divided by the total number of puncta. Data presented as mean±s.e.m. **P<0.01 against C603S mutant, unpaired Student’s t-test. (c) SUMOylation of Syn1a is required for correct presynaptic targeting. Molecular replacement of endogenous SynIa in hippocampal neurons with GFP-SynIa WT (n=13) or GFP-SynIa K687R (n=13). Neurons were transfected at DIV 9–10 and fixed and imaged 7 days later using anti-bassoon as an active zone marker, and anti-VAMP2 as SV marker. Scale bar, 5 μm. (d) Quantification of c. Synaptic co-localization of SynIa with bassoon or VAMP2 positive puncta were counted and divided by the total number of puncta. Data presented as mean±s.e.m. ***P<0.001 against wild type with respect to the same marker, unpaired Student’s t-test. (e) DIV 9–10 hippocampal neurons transfected with wild type (n=9) or K687R (n=12) GFP-tagged SynIa and imaged 7 days later. Electric field stimulation (900 AP at 10 Hz) was applied after 60 s of baseline recording and the recovery followed for 300 s afterwards. Representative images of clusters of GFP-SynIa WT and GFP-SynIa K687R before, during and after stimulation. Colour scale in arbitrary fluorescent units. Scale bar, 5 μm. (f) Dispersion and reclustering profiles GFP-SynIa WT and GFP-SynIa K6887R fluorescence. The data are presented as mean ± s.e.m. of the changes in punctate fluorescence (ΔF/F0). n numbers as in e. (g) Histogram showing the mean ± s.e.m. of maximum dispersion at t=180 s. n numbers as in e. (h,i) The dispersion (t=60–150 s) and reclustering (t=150–420 s) profiles were fitted with exponential curves and time constants presented as mean±s.e.m. ***P<0.001 against wild type, Student’s t-test. n numbers as in e.

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