Figure 4. Tomosyn inhibits release independent of Synaptotagmin 1 and 7.

(A) Average evoked response in 2.0 mM Ca²⁺ for control and tomosyn^NA1 (left) compared to representative traces of syt1 nulls (Syt1^N13/AD4) and tomosyn/syt1 double mutants (Tom^NA1;Syt1^N13/AD4, right). (B) Quantification of average evoked response area (nA*msec) per NMJ in 2.0 mM Ca²⁺ (syt1^N13/AD4: 37.22, 37.13 ± 5.139, n = 12; tomosyn^NA1/syt1^N13/AD4: 66.59, 69.05 ± 4.471, n = 10; p = 0.0002; ≥ 5 larvae per group). (C) Average EJC response in 2.0 mM Ca²⁺. (D) Log normalized average evoked response in 2.0 mM Ca²⁺. (E) Normalized cumulative charge transfer for the average evoked response in 2.0 mM Ca²⁺. (F) The slow component of evoked release was identified by fitting a double logarithmic function to the average normalized cumulative charge transfer per NMJ in 2.0 mM Ca²⁺ and plotted as a percent of total charge transfer (syt1^N13/AD4: 43.69, 44.55 ± 3.717, n = 11; tomosyn^NA1/syt1^N13/AD4: 75.38, 76.72 ± 2.295, n = 10; p < 0.0001; ≥ 5 larvae per group). (G) Representative mEJC traces. (H) Quantification of mEJC rate (Hz) per NMJ (syt1^N13/AD4: 5.567, 6.192 ± 0.7904, n = 12; tomosyn^NA1, syt1^N13/AD4: 17.17, 17.17 ± 1.034, n = 10; p < 0.0001; ≥ 5 larvae per group). (I) Average evoked response in 0.3 mM Ca²⁺ of control (Syt7^M1 control), syt7 null (Syt7^M1), tomosyn null (tomosyn^NA1), and tomosyn/syt7 double null (tomosyn^NA1;;;Syt7^M1). (J) Quantification of average evoked response area (nA*msec) per NMJ in 0.3 mM Ca²⁺ (control: 437.4, 437.4 ± 36.11, n = 8; syt7^M1: 844.6, 840.4 ± 33.77, n = 9; tomosyn^NA1: 1602, 1627 ± 94.19, n = 8; tomosyn^NA1, syt7^M1: 1920, 1923 ± 108.4, n = 8; p < 0.0001; ≥ 5 larvae per group). (K) Representative mEJC traces. (L) Quantification of mEJC rate per NMJ (Hz) (control: 1.056, 1.070 ± 0.1290, n = 8; syt7^M1: 1.617, 1.569 ± 0.161, n = 8; tomosyn^NA1: 6.256, 6.404 ± 0.7475, n = 8; tomosyn^NA1, syt7^M1: 5.092, 5.304 ± 0.3292, n = 8; p < 0.0001; ≥ 5 larvae per group). Complete data for this figure provided in Figure 4—source data 1.

Figure 4—figure supplement 1. Tomosyn inhibits release in a Ca²⁺-independent mechanism.

(A) Quantification of average EJC peak amplitude (nA) per NMJ across a range of Ca²⁺ concentrations, including 0.1 mM (control: 0.7351, 0.8639 ± 0.1197, n = 12; tomosyn^NA1: 2.846, 3.779 ± 0.7682, n = 12; p = 0.0011; ≥ 7 larvae per group), 0.2 mM (control: 13.29, 14.14 ± 1.591, n = 12; tomosyn^NA1: 35.03, 33.49 ± 2.207, n = 13; p < 0.0001; ≥ 8 larvae per group), 0.25 mM (control: 22.87, 24.31 ± 1.876, n = 12; tomosyn^NA1: 54.26, 51.66 ± 2.273, n = 12; p < 0.0001; ≥ 7 larvae per group), 0.3 mM (control: 55.30, 51.78 ± 3.522, n = 17; tomosyn^NA1: 83.74, 83.74 ± 3.378, n = 128 p < 0.0001; ≥ 10 larvae per group), 0.4 mM (control: 73.90, 78.32 ± 5.599, n = 14; tomosyn^NA1: 114.7, 115.5 ± 3.090, n = 12; p < 0.0001; ≥ 10 larvae per group), 0.5 mM (control: 97.09, 92.87 ± 6.413, n = 12; tomosyn^NA1: 128.6, 130.7 ± 4.415, n = 12; p < 0.0001; ≥ 10 larvae per group), 0.75 mM (control: 139.3, 130.2 ± 11.49, n = 10; tomosyn^NA1: 159.8, 164.7 ± 7.758, n = 10; p = 0.0229; ≥ 7 larvae per group), and 1.5 mM (control: 150.5, 159.8 ± 8.010, n = 12; tomosyn^NA1: 191.2, 198.1 ± 8.781, n = 12; p = 0.0039; ≥ 7 larvae per group). (B) Log-log plot of evoked response peak amplitude across the Ca²⁺ range, fit with a four-parameter logistic regression. The Hill slope is not significantly different between groups (control: 3.08 ± 0.48; tomosyn^NA1: 2.97 ± 0.28; extra sum-of-squares F test, p = 0.8272). (C) Average evoked response in Ca²⁺ concentrations where first evoked response amplitude is similar between control and tomosyn^NA1 (0.3 mM for control, 0.25 mM for tomosyn^NA1). (D) Quantification of average EJC amplitude (nA) per NMJ (control, 0.3 mM Ca²⁺: 65.81, 64.44 ± 3.566, n = 8; tomosyn^NA1, 0.25 mM Ca²⁺: 74.02, 72.58 ± 2.346, n = 8; p = 0.0773; ≥ 7 larvae per group). (E) Representative paired-pulse recordings with interstimulus intervals (ISI) of 25 msec, 50 msec, and 75 ms. (F) Quantification of average facilitation index per NMJ, calculated as the fold change in evoked peak current amplitude between pulse 1 and pulse 2. Three different ISIs were tested, including 25 ms (control, 0.3 mM Ca²⁺: 1.214, 1.216 ± 0.03225, n = 8; tomosyn^NA1, 0.25 mM Ca²⁺: 1.463, 1.455 ± 0.02237, n = 8; p < 0.0001; ≥ 7 larvae per group), 50 ms (control, 0.3 mM Ca²⁺: 1.256, 1.244 ± 0.02428, n = 8; tomosyn^NA1, 0.25 mM Ca²⁺: 1.349, 1.386 ± 0.03265, n = 8; p = 0.0035; ≥ 7 larvae per group) and 75 msec (control, 0.3 mM Ca²⁺: 1.197, 1.189 ± 0.02715, n = 8; tomosyn^NA1, 0.25 mM Ca²⁺: 1.239, 1.240 ± 0.01851, n = 8; p = 0.1396; ≥ 7 larvae per group). Complete data provided in Figure 4—figure supplement 1—source data 1.