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. 2023 Feb 2;12:e82568. doi: 10.7554/eLife.82568

Figure 3. The direct and selective transport of synaptic vesicle (SV) proteins is obscured by overexpression.

(A) A super-resolution MAX-projection image of 15 days in vitro (DIV) rat neurons expressing SYT1-HaloTag at high levels, as evidenced by the localization of SYT1-HaloTag in both axons and dendrites, along with the formation of filopodia-like structures in the somatodendritic compartment. The construct is visualized with JF549 HaloTag ligand. The boxed regions were expanded to show that the overexpressed protein accumulates on both the dendritic (i) and axonal (ii) plasma membrane (PM). Scale bar is 5 µm. (B) Graphs comparing the fluorescence intensity of the α-SYT1 antibody at synapses with or without tagged SYT1 reporter at low, intermediate, and high expression levels. We note that here, “x” represents the titer of virus (see Figure 3—figure supplement 1 for comparison of wild type (WT) vs. transduced protein) where 1x is slightly less than endogenous levels; average relative expression levels are shown in panels (B) and (C). Data were plotted as median with 95% confidence intervals and Mann-Whitney tests were run comparing native protein to native and tagged protein for each virus dose (p-value0.1x = 0.36, p-value1x = 0.20, p-value10x = 0.0004). Average relative expression levels are included on the graph. (C) The same as panel (B) but comparing the fluorescence intensity of SYB2 with and without expression of the SYB2 reporter. Data were analyzed as in panel (B) (p-value0.1x = 0.79, p-value1x = 0.17, p-value10x = <0.0001). Data from panels (B) and (C) represent 40 synapses per condition, collected from four total fields of view from two different litters. Mean values and descriptive statistics are found in Figure 3—source data 1. (D) MAX-projection images of 14–16 DIV rat neurons expressing the SYT1 reporter showing the transduction coverage achieved with each viral dose. Scale bar represents 150 µm. Images were adjusted individually, with linear brightness and contrast, to the brightest area of the image to aid in visualization. (E) The same as panel (D), but for the SYB2 reporter. Super-resolution optical sections of the SYT1 (F) and SYB2 (G) reporters at low, intermediate, and high expression levels, in axons and dendrites, demonstrate that as expression levels increase, SV proteins spillover into dendrites. Scale bar represents 2.5 µm. Corresponding axon and dendrite images, at each expression level, were adjusted with the same linear brightness and contrast settings.

Figure 3—source data 1. Descriptive statistics corresponding to Figure 3.
(A) Corresponds to Figure 3B. (B) Corresponds to Figure 3C.

Figure 3.

Figure 3—figure supplement 1. Expression levels of the SYT1 and SYB2 reporters as compared to native protein.

Figure 3—figure supplement 1.

(A) An immunoblot of 15 days in vitro (DIV) rat neurons expressing the SYT1 reporter at low, intermediate, and high levels using virus. Probing with an α-SYT1 antibody reveals that at the 10x viral dose, the exogenously expressed SYT1 reporter is present at much higher levels than the endogenous protein, confirming high expression. (B) Likewise, an immunoblot of rat neurons expressing the SYB2 reporter at various levels was probed with an α-SYB2 antibody to reveal the degree of overexpression compared to the native protein. Lower expression levels of SYT1 and SYB2 escaped detection due to the sparse transduction.