Article Figures & Data
Figures
- Figure 1.
Monitoring activity-dependent changes in [ATP]syn using a synaptic FRET-based ATP sensor. A, Schematic structure of the synapticly targeted FRET-based ATP sensor SypI-ATeam1.03. ATeam1.03 was fused to the C-terminal of synaptophysin I, thus targeting it to synaptic vesicles. B, SypI-ATeam1.03 expressed in WT hippocampal neurons. Notice its synaptic localization. C, SypI-ATeam1.03 expressing neurons were immunostained for synaptobrevin 2 as a marker of synaptic vesicles. Both the mseCFP (blue) and cpmVenus (green) components of SypI-ATeam1.03 colocalize with endogenous synaptobrevin 2 (red and see merged image at bottom), illustrating that SypI-ATeam1.03 is targeted to presynaptic terminals. D, The SypI-ATeam1.03 FRET ratio (FR, FcpmVen/FmseCFP) was calculated in neurons at rest, after 90 s stimulation at 10 Hz, and finally 3 and 10 min after cessation of stimulation (black bars). FR was reduced to 82.7 ± 2% by stimulation and gradually recovered back to 100 ± 2% 10 min after cessation of stimulation. Shown are mean ± SEM values normalized by the initial FR. n = 5/84 coverslips/boutons. Comparisons with prestimulation values were performed by nonparametric Kruskal–Wallis ANOVA, followed by Dunn's post hoc analysis. ***p < 0.001, ns: p > 0.05. SypI-ATeam1.03R122K/R126K, which is insensitive to ATP, did not show a significant change in FR after stimulation or during the recovery period (gray bars). n = 4/96. ns: p > 0.05. E, Fluorescence of cpmVen within SypI-ATeam1.03, elicited directly with the 514 nm laser line, was not affected by stimulation. n = 1/30. ns: p > 0.05. F, Representative confocal images of boutons of a neuron infected with SypI-ATeam1.03. G, Pseudocolor-coded fluorescent images of cpmVen (left) and mseCFP (right) in a resting bouton before (top) and after (bottom) cpmVen was bleached. Note the increase in mseCFP fluorescence after cpmVen photobleaching. H, FRET efficiency (Em) of SypI-ATeam1.03 in boutons decreased from 0.23 ± 0.02 to 0.15 ± 0.02 by stimulation. n = 1/41 and 1/35, respectively. Two-tailed unpaired Student's t test. **p = 0.007.
- Figure 2.
Mutating the ATP-binding site in synapsin IIa does not overtly alter its structure. A, Domain structure of synapsins IIa and Ia. K marks the location within the C domain of K270/K269 in synapsins IIa/Ia, respectively, both of which participate in the coordination of ATP binding. S marks the location of S10/S9 in synapsins IIa/Ia, respectively, which is the consensus PKA/CaMKI phosphorylation site found within domain A. B, Alignment of a 31 aa segment of the well conserved C-domain of synapsin II and synapsin I illustrates the preservation of Lys270/Lys269 (SynII/SynI; underlined and highlighted in yellow) and of its neighboring residues: red, identical amino acid; green, similar polarity. C, Superposition of the crystal structure (PDB-ID: 1I7N) of the C domain of synapsin II (white ribbon, residues 113–420) and the predicted effect of the K270Q mutation (green ribbon, modeled using SWISS-MODEL). For clarity, only the lysine (K, cyan) and glutamine (Q, red) side chains are shown. Left, Whole C domain; right, zoom-in on mutation site. The original and modeled structures overlap. D, Western blot (anti synapsin II) of untransfected HEK cells or HEK cells expressing EGFP-SynIIa or EGFP-SynIIa-K270Q. Both constructs produce a fusion protein of the expected combined molecular weight of synapsin IIa and EGFP (75 + 28 KDa, respectively). A weak lighter band, presumably a degradation product, was present to a similar extent for both constructs.
- Figure 3.
Mutating the ATP-binding site affects the association of synapsin IIa with the vesicle cluster. A, EGFP-SynIIa (left, green) or EGFP-SynIIa-K270Q (right) were coexpressed with TagBFP (blue) to assess their synaptic targeting capability. Merged images (bottom) illustrate that both proteins form a punctate pattern along the axons, consistent with synaptic localization. B, Synaptic targeting of both proteins was quantified by calculating their “targeting factor” (see Materials and Methods section). Both proteins are enriched in synaptic puncta (i.e., their targeting factor is larger than 0, one-sample Wilcoxon signed rank test, p < 0.0001), but targeting of the K270Q mutant is weaker (Mann–Whitney nonparametric test, ***p < 0.0001). Shown are mean ± SEM values, n = 19 and 20 images for EGFP-SynIIa and EGFP-SynIIa-K270Q, respectively, ∼20 synapses analyzed in each. C, EGFP-SynIIa and EGFP-SynIIa-K270Q both form synaptic puncta at rest (Ctl, left), which disperse into the adjacent axonal segments following 1 min of depolarization using hyperkalemic saline (middle). Recovery to the original distribution is achieved after 10 min in normal saline (right). D, Quantification of C. The initial width of EGFP-SynIIa-K270Q is larger, but during stimulation, the final width equalizes to that of EGFP-SynIIa. The initial width fully recovers after repolarization. n = 24 and 20 image sets (control, stimulated, recovered) for EGFP-SynIIa and EGFP-SynIIa-K270Q, respectively, ∼20 matching synapses analyzed per image. Two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. Comparisons within EGFP-SynIIa: Ctl versus Stim, ***p ≪ 0.001; Stim versus Rec, ***p ≪ 0.001; Ctl versus Rec, p = 0.98. Comparisons within EGFP-SynIIa-K270Q: Ctl versus Stim, **p = 0.006; Stim versus Rec, ***p = 0.0005; Ctl versus Rec, p = 0.61. Comparison within Ctl: EGFP-SynIIa versus EGFP-SynIIa-K270Q, ***p < 0.001; comparison within Stim: EGFP-SynIIa versus EGFP-SynIIa-K270Q, p = 0.69.
- Figure 4.
The K270Q mutation increases the capability of synapsin IIa to cluster vesicles at the presynaptic terminal. A, TKO neurons were induced to express EGFP-SynIIa or EGFP-SynIIa-K270Q (green) and were subsequently processed for synaptobrevin 2 immunofluorescence (red). B, Density of synaptic vesicles within presynaptic terminals was assessed by measuring the synaptobrevin 2 immunofluorescence intensity at the center of mass of the synaptic puncta. The values measured in each imaging session were normalized by the mean values recorded in TKO neurons. Only synapses negative for GAD65 were included in the analysis. Venus, as a control, had no effect on the vesicle density, SynIIa increased it, and the K270Q mutant had a significantly larger effect. n = 39, 10, 30, and 32 independent images (respectively), each including >30 synapses. Values were compared by one-way ANOVA followed by Tukey's post hoc analysis. TKO versus Venus, p = 0.99; Venus versus SynIIa, *p = 0.05; Venus versus SynIIa-K270Q, ***p < 0.001; SynIIa versus SynIIa-K270Q, ***p = 0.0004. C, Relationship between the synapsin IIa content in each synapse and the density of vesicles therein was termed the clustering index (CI). This value was calculated per each image by plotting the syb2 versus EGFP fluorescence intensities and extracting the slope. Shown are representative analyses of single images depicting neurons expressing EGFP-SynIIa (red) or EGFP-SynIIa-K270Q (blue). D, Left, Distribution of clustering index values calculated for endogenous syb2 in neurons expressing EGFP-SynIIa and EGFP-SynIIa-K270Q (n = 25, 30 independent images, respectively). Symbols, Single-image clustering indice; bars, 10–90% percentiles; box, 25–75% percentiles; middle bar, median. The clustering index of the K270Q mutant is significantly larger than that of EGFP-synapsin IIa; Mann–Whitney nonparametric test, ***p ≪ 0.001. Normality of the distribution was tested using the Shapiro–Wilk test, p < 0.01. Right, The clustering index values were also calculated for endogenous synaptophysin I. n = 11 and 16 images, *p = 0.025. Similarly, the clustering index for the K270Q mutant was larger.
- Figure 5.
Mutating the ATP-binding site does not affect vesicle redistribution at rest, during synaptic activity, or during depletion of cellular ATP. A, Hyperkalemic depolarization induced widening of SypI-EGFP puncta in neurons also coexpressing either TagBFP-SynIIa or TagBFP-SynIIa-K270Q. The extent of widening and its recovery were similar in both cases. n = 19 and 20 image sets (control, stimulated, recovered), respectively, ∼20 matching synapses analyzed per image. Two-way repeated-measures ANOVA, p = 0.76. B, Depletion of ATP does not induce acute redistribution of vesicles. Top, [ATP]syn was assessed by measuring the acceptor/donor FR of SypI-ATeam1.03 (black symbols). n = 8 experiments, >20 synapses each. Incubation in ATP-depleting saline induced a progressive decrease in FR down to the level measured using the ATP-insensitive construct SypI-ATeam1.03R122K/R126K (gray symbols, n = 3 experiments, >20 synapses each). Bottom, The width of SypI-TagBFP puncta is unchanged after 30 min of incubation in ATP-depleting saline. n = 15, 16, and 17 image pairs for neurons coexpressing Venus, EGFP-SynIIa, or EGFP-SynIIa-K270Q, respectively, ∼20 synapses analyzed per pair. Two-way repeated-measures ANOVA followed by Tukey's post hoc analysis, p = 0.12, 0.62, and 0.17, respectively. C, Vesicle sharing in TKO neurons expressing SypI-EGFP or those coexpressing SypI-EGFP and either TagBFP-SynIIa or TagBFP-SynIIa-K270Q, was assessed by measuring the recovery of SypI-EGFP after photobleaching single synapses. Shown is a representative experiment in a neuron expressing the TagBFP-SynIIa-K270Q. Arrowhead, Bleached synapse. Elapsed time is indicated in minutes, starting with bleaching (t = 0′). SypI-EGFP fluorescence recovered gradually and partially within 35 min. D, Quantification of FRAP experiments illustrating that both TagBFP-SynIIa and TagBFP-SynIIa-K270Q restricted vesicle sharing in TKO neurons to a similar extent. Recovery was compared by repeated-measures ANOVA, n = 18, 33, and 24 experiments, respectively. TKO versus SynIIa, ***p = 0.001; TKO versus SynIIa-K270Q, *p = 0.048; SynIIa versus SynIIa-K270Q, p = 0.37.
- Figure 6.
Effect of synapsin IIa and its mutant on basal synaptic properties. A, Averaged EPSCs elicited by brief depolarization commands in autaptic synapsin TKO neurons. Shown are control TKO neurons expressing Venus (black, n = 14), EGFP-SynIIa (red, n = 15), and EGFP-SynIIa-K270Q (blue, n = 22). The action potential current preceding the EPSC was blanked for the purpose of clarity. Superposition of averaged normalized EPSCs (right traces) illustrates that they fully overlap. B, Quantification of mean EPSC amplitudes. Mean ± SEM, 848 ± 7.6 pA, 743 ± 7.1 pA, and 1092 ± 12.3 pA, n = 14, 18, and 22, respectively. Notice that both SynIIa and its K270Q mutant do not differ from control, although they do differ from each other. One-way ANOVA followed by Tukey's post hoc analysis: control versus EGFP-SynIIa, p = 0.78; control versus EGFP-SynIIa-K270Q, p = 0. 24; EGFP-SynIIa versus EGFP-SynIIa-K270Q, *p = 0.038. C, The K270Q mutation reduces the effect of synapsin IIa on frequency modulation of release. Neurons were stimulated at 10 Hz and the responses to five subsequent stimuli were monitored. Shown are representative recordings, as indicated. Control neurons (Venus) and neurons expressing EGFP-SynIIa-K270Q exhibited depression, whereas neurons expressing EGFP-SynIIa exhibited facilitation. D, Quantification of frequency-modulation. n = 11, 8, and 11, respectively. Repeated-measures ANOVA followed by Tukey's post hoc analysis. Control versus EGFP-SynIIa, **p = 0.006; control versus EGFP-SynIIa-K270Q, p = 0.99; EGFP-SynIIa versus EGFP-SynIIa-K270Q, **p = 0.008.
- Figure 7.
Mutating the ATP-binding site annuls the effect of synapsin IIa on synaptic depression. A, Neurons were stimulated 900 times at 10 Hz and their synaptic responses were recorded. Shown are representative responses for control neurons expressing Venus (control, black) and for TKO neurons expressing EGFP-SynIIa (red) or its K270Q mutant (blue). Every 15th response in the stimulation train is shown. B, Normalized end-point subtracted semilog plots of mean ± SEM EPSC responses to stimulation trains as in A (n = 11, 7, and 11, respectively). Depression is deferred by the expression of EGFP-SynIIa, but not of EGFP-SynIIa-K270Q, compared with the Venus control. Furthermore, the rate of depression, once initiated, is slower for EGFP-SynIIa than for the K270Q mutant. For clarity, symbols and error bars are shown for every 10th stimulus, and down to ∼5% of the initial amplitude. Solid lines denote single-exponent fits applied starting 5, 10, and 9 s after the first EPSC, respectively. C, Average time constant of synaptic depression, calculated by fitting the EPSC amplitudes with a single-exponent decay function as shown in B. One-way ANOVA followed by Tukey's post hoc analysis. Control versus EGFP-SynIIa, ***p = 0.0008; control versus EGFP-SynIIa-K270Q, p = 0.39; EGFP-SynIIa versus EGFP-SynIIa-K270Q, *p = 0.014. D, Because synaptic depression follows complex kinetics, the t1/2 of depression was calculated. EGFP-SynIIa delays depression, whereas the K270Q mutant does not. One-way ANOVA followed by Tukey's post hoc analysis. Control versus EGFP-SynIIa, ***p = 0.0005; control versus EGFP-SynIIa-K270Q, p = 0.32; EGFP-SynIIa versus EGFP-SynIIa-K270Q, *p = 0.013. E, Cumulative charge transfer during EPSC trains. Although EGFP-SynIIa significantly increased the charge transfer throughout the train, the K270Q mutant was ineffective in this respect. n = 6, 6, and 8, respectively. Repeated-measures ANOVA using every fifth EPSC value within the 200th-500th stimuli range, followed by Tukey's post hoc analysis. Control versus EGFP-SynIIa, *p < 0.049; control versus EGFP-SynIIa-K270Q, p = 0.99; EGFP-SynIIa versus EGFP-SynIIa-K270Q, *p = 0.033. F, Synapsin IIa progressively increases the ratio between the charge and amplitude of each EPSC during stimulation trains, consistent with a gradual desynchronization of synaptic release. The K270Q mutation annuls this effect. Repeated-measures ANOVA, followed by Tukey's post hoc analysis. Control versus EGFP-SynIIa, **p = 0.002; control versus EGFP-SynIIa-K270Q, p = 0.92; EGFP-SynIIa versus EGFP-SynIIa-K270Q, **p = 0.006.
- Figure 8.
Mutating the ATP-binding site in the C domain of synapsin IIa attenuates phosphorylation in site 1 within the A domain. A, Sequence homology of the A domains of synapsin II and synapsin I. Red, Identities; yellow background, phosphorylation site 1. B, An antibody for phosphorylation site 1 (S9) in synapsin I interacts specifically with the homologous site in synapsin IIa (S10). Neurons from synapsin TKO mice, which lack all synapsins, were induced to express TagBFP-SynIIa (left column) and were immunostained for phospho-S10 (IF P-S10, right column). Neurons treated with H89 (top row) exhibited background immunofluorescence, whereas depolarized neurons (bottom row) exhibited robust P-S10 immunofluorescence. C, Comparison of the extent of phosphorylation of TagBFP-SynIIa and its K270Q mutant. Although the phosphorylation ratio in H89-treated samples was similar, it was significantly lower for the K270Q mutant both in resting and in stimulated neurons. n = 21, 28, 26, 29, 30, and 32 independent images per condition, respectively, per column from left to right, one-way ANOVA followed by Tukey's post hoc analysis comparing TagBFP-SynIIa versus TagBFP-SynIIa-K270Q in H89, p = 0.11; the same in resting neurons, ***p≪0.001; and the same in stimulated neurons, **p = 0.004. D, Clustering index measured using immunolabeling of endogenous syb2 is significantly lower for EGFP-SynIIa-S10A than it is for EGFP-SynIIa-K270Q. n = 29 and 23 images for K270Q and S10A, respectively, p = 0.0001, Mann–Whitney nonparametric test. E, Semilogarithmic end-point subtracted plots of the mean normalized EPSC amplitudes elicited by 10 Hz stimulation in glutamatergic autaptic neurons expressing EGFP-SynIIa-K270Q (n = 11) or EGFP-SynIIa-S10A (n = 5). For clarity, symbols and error bars are shown for every 10th stimulus and down to ∼5% of the initial amplitude. Solid lines denote single-exponent fits applied in the indicated range. Synaptic responses depressed in a similar manner in both groups. F, Average time constant of synaptic depression for data shown in E. p = 0.44, Student's t test.
