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. 2013 Mar 13;33(11):4726–4740. doi: 10.1523/JNEUROSCI.4191-12.2013

Figure 3.

Figure 3.

Sumoylation of MEF2A drives the suppression of orphan presynaptic sites. A, Lysates of cerebella from rat pups at postnatal days 2, 6, 10, 14, 18, and 22 were immunoblotted with the MEF2A or ERK1/2 antibody. MEF2A runs as a doublet at 55 kDa, and the sumoylated form of MEF2A runs as an ∼100 kDa band (Shalizi et al., 2006). The levels of the sumoylated MEF2A increased during the second and third postnatal weeks in the rat cerebellum. B, Granule neurons were transfected with the GFP-synapsin expression plasmid together with the U6/mef2a or control U6 RNAi plasmid and an expression plasmid encoding MEF2A-WT, MEF2A-RES, MEF2A-RES K403R, or a control vector and analyzed as in Figure 2B. MEF2A knockdown increased the density of orphan PSD95-unapposed synapsin clusters (p < 0.01, ANOVA followed by Fisher's PLSD post hoc test, n = 3). Expression of MEF2A-RES, but neither MEF2A-WT nor MEF2A-RES K403R, decreased orphan synapsin cluster density in the background of MEF2A RNAi (p < 0.01, ANOVA followed by Fisher's PLSD post hoc test, n = 3). C, Granule neurons were transfected with the GFP-Munc13 expression plasmid together with the U6/mef2a or control U6 RNAi plasmid and an expression plasmid encoding MEF2A-WT, MEF2A-RES, MEF2A-RES K403R, or a control vector and analyzed as in Figure 2B. MEF2A knockdown increased the density of orphan Munc13 clusters (p < 0.001, ANOVA followed by Fisher's PLSD post hoc test, n = 3). Expression of MEF2A-RES, but neither MEF2A-WT nor MEF2A-RES K403R, reduced orphan Munc13 cluster density in the background of MEF2A RNAi (p < 0.001, ANOVA followed by Fisher's PLSD post hoc test, n = 3). D, Granule neurons were transfected with the GFP-synapsin expression plasmid together with an expression plasmid encoding MEF2A-SUMO or the control vector and analyzed as in Figure 2B. Arrowheads denote orphan PSD95-unapposed synapsin clusters. Double arrowheads indicate synapsin/PSD95 coclusters. Expression of MEF2A-SUMO in neurons decreased the density of orphan PSD95-unapposed synapsin clusters (p < 0.05, t test, n = 3), but had little or no effect on the density of synapsin/PSD95 coclusters. Scale bar, 10 μm. E, Granule neurons were transfected with the GFP-Munc13 expression plasmid together with an expression plasmid encoding MEF2A-SUMO or a control vector and analyzed as in Figure 2B. Expression of MEF2A-SUMO decreased the density of orphan PSD95-unapposed Munc13 clusters (p < 0.05, t test, n = 3), but failed to significantly reduce the density of Munc13/PSD95 coclusters. F, Granule neurons transfected with the GFP-synapsin expression plasmid together with the MEF2A-SUMO expression plasmid or the control vector were analyzed as in Figure 2D. Expression of MEF2A-SUMO decreased the density of orphan PSD95-unapposed synapsin/Munc13 coclusters in granule neurons (p < 0.05, t test, n = 3), but had little or no effect on the density of synapsin/Munc13/PSD95 coclusters. G, Granule neurons transfected with the GFP-synapsin expression plasmid together with an expression plasmid encoding MEF2A-SUMO or the control vector were analyzed for synaptic vesicle recycling as in Figure 2E. Expression of MEF2A-SUMO decreased the density of orphan NR1-unapposed synapsin/FM4-64 coclusters (p < 0.05, t test, n = 3), but failed to significantly reduce the density of synapsin/FM4-64/NR1 coclusters. H, Granule neurons were transfected with the GFP-synapsin expression plasmid together with an expression plasmid encoding MEF2A-WT, MEF2A-K403R, or the control vector and analyzed as in Figure 2B. Expression of MEF2A-WT or MEF2A-K403R in neurons had little or no effect on the density of orphan PSD95-unapposed and PSD95-apposed synapsin clusters.