Organization of signaling complexes at excitatory synapses by membrane-associated guanylate kinase (MAGUK) proteins regulates synapse development, plasticity, senescence and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches and in vivo models that the trans-synaptic organizing protein ephrin-B3 controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a newly identified mitogen-associated protein kinase (MAPK)-dependent phosphorylation site on ephrin-B3, Ser332. Unphosphorylated ephrin-B3 was enriched at synapses, and interacted directly with and stabilized PSD-95 at synapses. Activity-induced phosphorylation of Ser332 dispersed ephrin-B3 from synapses, prevented the interaction with PSD-95 and enhanced the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity.
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We would like to thank W. Zhou for comments on FRAP data analysis and the students and faculty of the Neurobiology course (2009–2010) at the Marine Biological Laboratory for help with pilot experiments. This work was supported by grants from NIDA (DA022727) and NIMH (MH086425 and MH100093) to M.B.D.
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Representative time sequence of a FRAP experiment from a control neuron transfected with PSD-95-GFP at DIV0. Imaging was performed at DIV10 and images were acquired once every minute for 60 minutes. Bleached PSD-95-GFP puncta, shown in the white circle, exhibits poor recovery over the course of one hour, indicating low mobility of PSD-95-GFP in control neurons. For quantification see figure 5c. Scale bar: 3 μm.
Representative time sequences of FRAP experiments from neurons transfected with PSD-95-GFP and either control (pSuper) or ephrin-B3 shRNA constructs at DIV0. At DIV10 neurons were loaded with FM 4-64 dye to mark pre-synaptic release sites. PSD-95-GFP puncta that co-localized with FM 4-64 dye were designated as synaptic, while PSD-95-GFP puncta that did not co-localize with FM 4-64 were designated as non-synaptic. In both transfection conditions, synaptic and non-synaptic puncta were selected for photobleaching (shown in circles) and their recovery was measured for 20 minutes, acquiring images every 20 seconds. Knockdown of endogenous ephrin-B3 resulted in increased recovery of synaptic and non-synaptic puncta compared to the control condition, indicating higher mobile PSD-95-GFP fractions in ephrin-B3 shRNA transfected neurons. Results are quantified in figure 6e, f. Scale bar: 2 μm.
Representative time sequences of FRAP experiments from neurons co-transfected with PSD-95-GFP and either control (pSuper) or ephrin-B3 shRNA at DIV0. Expression of ephrin-B3 after shRNA knockdown was rescued with the indicated shRNA-resistant ephrin-B3 wild type and mutant constructs that regulate interaction with PSD-95. FRAP experiments were performed at DIV10 and the recovery of bleached PSD-95-GFP (shown in circles) was followed for 20 minutes, acquiring images every 20 seconds. Knock down of ephrin-B3 resulted in higher recovery of PSD-95-GFP compared to control. Wild type flag-ephrin-B3 and non-phosphorylable flag-ephrin-B3 S332A, which show normal interaction with PSD-95, rescued PSD-95-GFP mobility to control levels. In contrast, flag-ephrin-B3 L293A and phosphomimetic flag-ephrin-B3 S332D, which exhibit reduced interaction with PSD-95, did not rescue PSD-95-GFP mobility. Thus, domains in ephrin-B3 that regulate the interaction with PSD-95 are required for the control of PSD-95 mobility. Results are quantified in figure 7b-g. Scale bar: 3 μm.
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Cellular and Molecular Life Sciences (2019)