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. 2021 Sep 20;10:e67261. doi: 10.7554/eLife.67261

Figure 6. SYT7 is mislocalized and destabilized when amino-terminal cleavage is blocked.

(a) Representative super-resolution maximum z-projections of rat hippocampal neurons transduced with LAMP1-msGFP (cyan), fixed for immunocytochemistry (ICC), and stained for synaptotagmin 1 (SYT1) (yellow) and the axon initial segment (AIS) (magenta). Four separate conditions were imaged: control neurons, neurons grown for 10–12 days in 0.5 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), neurons exposed to 2-bromopalmitate (2-BP) for 3 hr before imaging, and neurons exposed to a combination treatment of DAPT and 2-BP. (b) Same as in panel (a), but instead of anti-SYT1 staining, neurons were transduced with SYT7α-HaloTag and reacted with JF549 during overnight primary antibody incubation to monitor SYT7α localization. Scale bar = 10 μm. (c) Illustration of the model neuron and compartments assayed for SYT7α-HaloTag colocalization. (d) Bar graph showing changes in colocalization of SYT7α-HaloTag/JF549 and labeled organelles (M6PR and sortilin label post-Golgi vesicles). Quantified by taking the difference of the PCC between DAPT-treated and control neurons in each condition. Values are means +/- error propagated SEM from at least three separate experiments for each condition. (e) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and when grown in 0.5 μM DAPT. Cultures were labeled with JF635 at 13 DIV and then robustly washed with conditioned media. The disappearance of labeled SYT7α-HaloTag/JF635 from the gel can be used to calculate protein half-life. Control SYT7α-HaloTag/JF635 runs between 75 and 100 kDa, while DAPT-treated SYT7α-HaloTag/JF635 runs slightly higher because cleavage of the amino-terminus is blocked. Trichloroethanol (TCE) staining was used as a loading control. (f) Normalized intensity of SYT7α-HaloTag/JF635 plotted as the fraction of total control SYT7α-HaloTag/JF635 against days post-wash. Values are means +/- SEM from three independent experiments. Single exponential functions were fitted to control (black) and DAPT (red) conditions. The tau for control SYT7α-HaloTag/JF635 is 9.5 days, while the tau for DAPT-treated SYT7α-HaloTag/JF635 is 3.3 days.

Figure 6.

Figure 6—figure supplement 1. Treatment with DAPT does not recapitulate SYT7KO synaptic phenotypes in vitro.

Figure 6—figure supplement 1.

(a) Average iGluSnFR ΔF/F0 traces from high-frequency stimulation (HFS) of wild-type (WT) (black, n = 9) and WT + 0.5 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (gray, n = 11)-treated mouse hippocampal neurons, from two independent experiments. Samples were field stimulated at 20 Hz for 2.5 s (50 action potentials (APs)). (b) Fraction of active synapses (synapses releasing peak glutamate above baseline, >4 SD above noise) as a function of stimulation number during HFS. Values are means +/- SEM, p = ns by two-way analysis of variance (ANOVA) comparing genotypes. (c) Synchronous fraction of iGluSnFR ΔF/F0 peaks (synchronous peaks within 10 ms of stimulus) as a function of stimulation number during HFS. Values are means +/- SEM, ****p<0.0001 by two-way ANOVA comparing genotypes. (d) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and when grown in 0.5 μM DAPT. (e) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with palmitoylation site mutant (3x Cys -> 3x Ala) SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and then grown in 0.5 μM DAPT. Palmitoylation mutants are not stable in control conditions, presumably because they are cleaved by γ-secretase and degraded.