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. 2016 Dec 16;7:13660. doi: 10.1038/ncomms13660

Figure 7. Sx1A-mEos2 mobility is increased by co-expression of TeTx/LC in motor nerve terminals.

Figure 7

TeTx/LC was transiently expressed in Sx1A-mEos2-expressing larvae. Single-molecule tracking was then carried out on the motor nerve terminals. (a) Representative low-resolution image, sptPALM average intensity, diffusion coefficient and trajectory map of Sx1A-mEos2 in larvae with motor neuron expression of TeTx/LC (1,836 trajectories analysed). Scale bar, 3 μm. (b) Low-resolution image, sptPALM average intensity, diffusion coefficient and trajectory map of Sx1A-mEos2 in PC12 cells. (c–e) Blocking SNARE complex formation via TeTx/LC expression increased the MSD, the diffusion coefficient distribution and the ratio of the mobile to immobile fractions of Sx1A-mEos2 in live motor nerve terminals (Sx1A-mEos2 at the NMJ, 3.5±0.1; Sx1A-mEos2 with TeTx/LC expression at the NMJ, 8.6±0.8; and Sx1A-mEos2 in PC12 cells, 6.5±0.4). This increase in Sx1A-mEos2 mobility is comparable to that observed in PC12 cells (n=19 NMJ chains for Sx1A-mEos2-expressing larvae, n=14 NMJ chains for larvae also expressing TeTx/LC and n=23 cells for PC12; average of ∼3,000 trajectories in NMJ chains and ∼11,500 trajectories in PC12 cells). Comparisons were performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. *P<0.05, ***P<0.001. (f,g) TeTx/LC expression significantly decreased the Sx1A-mEos2 cluster size from 83.5±4.2 to 58.4±4.0 nm. The average Sx1A-mEos2 density also decreased significantly from 57.2±7.9 to 21.2±2.9 μm−2. Statistical tests were performed using the Mann–Whitney U-test ***P<0.001. (h) Western blot showed that VAMP2 was cleaved by TeTx/LC expression at 32 °C; at 25 °C, there was no TeTx/LC expression and VAMP2 remained intact. (i–k) Excitatory junction potentials in wild-type Drosophila larvae, Sx1A-mEos2 transgenic larvae with tubulin Gal80 suppression of TeTx/LC (25 °C) and Sx1A-mEos2 larvae with TeTx/LC expression (32 °C); the amplitude of evoked release decreased significantly on TeTx/LC expression (wild type, 35.3±0.2 mV; TeTx/LC suppression at 25 °C, 35.2±0.1 mV; and TeTx/LC expression at 32 °C, 2.6±0.2 mV). The quantal content decreased significantly with the expression of TeTx/LC (wild type, 58.1±1.0; TeTx/LC suppression at 25 °C, 46.8±3.2; and TeTx/LC expression at 32 °C, 3.1±0.3). Comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test *P<0.05, ***P<0.001. Mean±s.e.m. are plotted. WT, wild type.