Figure 6. PtdIns(3,4,5)P₃ interaction alters Sx1A mobility.

(a) Scheme illustrating the Sx1A^KARRAA mutation that is known to exhibit reduced affinity to PtdIns(3,4,5)P₃. The Sx1A^KARRAA mutation was transgenically expressed in Drosophila and sptPALM was carried out on live motor nerve terminals. (b,c) Comparison of MSDs and diffusion coefficient distributions of Sx1A-mEos2 and Sx1A^KARRAA-mEos2 (n=17 NMJ chains for Sx1A-mEos2 and 19 NMJ chains for Sx1A^KARRAA-mEos2; average of ∼2,600 trajectories analysed per NMJ chain). (d) The mobile to immobile ratio was 3.6±0.2 for Sx1A-mEos2 and 5.0±0.2 for Sx1A^KARRAA-mEos2. (e,f) Sx1A^KARRAA-mEos2 or Sx1A-mEos2 larvae with concomitant dTRPA1 expression were heated from 25 to 30 °C to stimulate neurotransmitter release. Note that the MSD of Sx1A^KARRAA-mEos2 was also not affected by stimulation. (g) The mobile to immobile ratio was not significantly altered: 6.7±0.5 at 25 °C to 6.7±0.3 at 30 °C (n=13 NMJ chains at 25 °C and 14 NMJ chains at 30 °C; average of ∼1,700 trajectories per NMJ chain). (h) Fixed larvae were analysed for SML. The average cluster radius of Sx1A^KARRAA-mEos2 (55.8±2.7 nm) compared with Sx1A-mEos2 (83.5±4.2 nm) was significantly different. Expressing dTRPA1 (25 °C, 61.5±3.3 nm) or elevating the temperature did not significantly alter the cluster size (30 °C, 63.7±2.9 nm). (i) The Sx1A^KARRAA-mEos2 average density was significantly lower (36.1±4.3 μm⁻²) than that of Sx1A-mEos2 (57.2±7.9 μm⁻²); this was unchanged by dTRPA1 expression (25 °C, 37.3±3.6/μm²) and stimulation (30 °C, 36.1±4.8 μm⁻²). Statistical tests were performed using Student’s t-test (two-tailed distribution, unpaired). *P<0.05, ***P<0.001. Mean±s.e.m. are plotted.