Electrophysiological TEVC analysis of Myo15 loss-of-function, PhTx treatment of Myo15−/−mutant, and sustained memory consolidation for Myo15 knock-down. (A–N) TEVC electrophysiological recordings of Myo15 RNAi–mediated knock-down, (A–G, M, and N) evoked, (H–L) spontaneous release. (A) eEPSC traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (B) eEPSC amplitude (control −55.43 ± 2.00 nA, n = 22; Myo15-RNAi −58.92 ± 4.71 nA, n = 24). (C) Rise time (control 1.27 ± 0.04 ms, n = 22; Myo15-RNAi 1.18 ± 0.04 ms, n = 24). (D) Decay time τ (control 5.2 ± 0.17 ms, n = 23; Myo15-RNAi 4.11 ± 0.24 ms, n = 24). (E) Charge (control −484.5 ± 23.03 pC, n = 22; Myo15-RNAi −476.4 ± 52.17 pC, n = 24). (F) 10-ms paired-pulse traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (G) Paired-pulse ratio 10 ms (control 0.92 ± 0.04 ms, n = 22; Myo15-RNAi 0.98 ± 0.06, n = 24). (H) mEPSC traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 2 nA, 200 ms. (I) mEPSC amplitude (control −0.70 ± 0.02 nA, n = 23; Myo15-RNAi −0.60 ± 0.02 nA, n = 24). (J) mEPSC rise time (control 2.13 ± 0.16 ms, n = 23; Myo15-RNAi 1.87 ± 0.16 ms, n = 24). (K) mEPSC decay time τ (control 5.95 ± 0.19 ms, n = 19; Myo15-RNAi 4.61 ± 0.21 ms, n = 23). (L) mEPSC frequency (control 0.97 ± 0.12 Hz, n = 23; Myo15-RNAi 1.08 ± 0.09 Hz, n = 24). (M) 30 ms paired-pulse traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (N) Paired-pulse ratio 30 ms (control 1.29 ± 0.03 ms, n = 22; Myo15-RNAi 1.26 ± 0.03, n = 24). (O–b) TEVC electrophysiological recordings of Myo15ΔMyTH4 (1+2) deletion mutant and wild-type NMJs, (O–U, a, and b) evoked, (V–Z) spontaneous release. (O) eEPSC traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (P) eEPSC amplitude (wild type −61.25 ± 3.48 nA, n = 16; Myo15ΔMyTH4 (1+2) −66.16 ± 6.46 nA, n = 16). (Q) Rise time (wild type 1.23 ± 0.05 ms, n = 16; Myo15ΔMyTH4 (1+2) 1.00 ± 0.03 ms, n = 16). (R) Decay time t (wild type 4.80 ± 0.21 ms, n = 16; Myo15ΔMyTH4 (1+2) 4.23 ± 0.10 ms, n = 16). (S) Charge (wild type −526.4 ± 39.29 pC, n = 16; Myo15ΔMyTH4 (1+2) −527.2 ± 67.47 pC, n = 16). (T) 10 ms paired-pulse traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (U) Paired-pulse ratio 10 ms (wild type 0.78 ± 0.04 ms, n = 16; Myo15ΔMyTH4 (1+2) 0.75 ± 0.03, n = 16). (V) mEPSC traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 1 nA, 200 ms. (W) mEPSC amplitude (wild type −0.65 ± 0.02 nA, n = 16; Myo15ΔMyTH4 (1+2) −0.61 ± 0.02 nA, n = 16). (X) mEPSC rise time (wild type 2.27 ± 0.13 ms, n = 16; Myo15ΔMyTH4 (1+2) 1.64 ± 0.16 ms, n = 16). (Y) mEPSC decay time t (wild type 5.53 ± 0.19 ms, n = 16; Myo15ΔMyTH4 (1+2) 4.96 ± 0.21 ms, n = 16). (Z) mEPSC frequency (wild type 1.96 ± 0.27 Hz, n = 16; Myo15ΔMyTH4 (1+2) 1.72 ± 0.16 Hz, n = 16). (a) 30-ms paired-pulse traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (b) Paired-pulse ratio 30 ms (wild type 1.04 ± 0.03 ms, n = 16; Myo15ΔMyTH4 (1+2) 0.97 ± 0.01, n = 15). (c) Confocal images of myo15−/− mutant and control NMJs treated with PhTx (fire LUT), scale bar 2 µm. (d) Quantification of c. BRP mean pixel intensity (wild type - PhTx 100.0 ± 7.59%, n = 11; + PhTx 203.2 ± 17.60%, n = 12; Myo15−/− mutant - PhTx 100.0 ± 10.35%, n = 11; + PhTx 111.8 ± 17.76%, n = 8. (e) mEPSP traces of Myo15 knock-down and control NMJs with (red) and without (black) PhTx treatment for 10 min, scale bar 5 mV, 500 ms. (f) mEPSP traces of Myo15 knock-down and control NMJs with (red) and without (black) PhTx treatment for 30 min, scale bar 2 mV, 500 ms. (g) Quantification of MTM 1 h of Gal80ts;ok107-Gal4>Myo15-RNAi101729 without induction (restrictive 18°C temperature, control driver line PI 0.39 ± 0.061, Myo15 knock-down (RNAi101729) PI 0.50 ± 0.054, control RNAi PI 0.43 ± 0.052, F(2,23) = 0.9187, P = 0.4145, n = 8). (h) Flies expressing RNAi-Myo15101729 in the adult MB lobes present normal olfactory acuity to octanol (control driver line OA 0.52 ± 0.060, Myo15 knock-down (RNAi101729) OA 0.383 ± 0.0598, control RNAi OA 0.542 ± 0.085; F(2,17) = 1.530, P = 0.2486, n = 6) and methylcyclohexanol (control driver line OA 0.55 ± 0.0629, Myo15 knock-down (RNAi101729) OA 0.728 ± 0.0682, control RNAi OA 0.740 ± 0.0879; F(2,17) = 2.046, P = 0.1639, n = 6). (i) Those flies present normal electrical shock reactivity (control driver line reactivity 0.79 ± 0.045, Myo15 knock-down (RNAi101729) reactivity 0.855 ± 0.039, control RNAi reactivity 0.868 ± 0.0566; F(2,17) = 0.7134, P = 0.5059, n = 6). (j) Quantification of MTM 1 h for flies expressing RNAi-Myo15104606 in the adult MB lobes (control driver line PI 0.21 ± 0.016, Myo15 knock-down (RNAi104606) PI 0.05 ± 0.054, control RNAi PI 0.29 ± 0.046; F(2,23) = 8.370, P = 0.0021, n = 8, post hoc Tukey’s multiple comparisons test, Gal80ts;ok107-Gal4/+ versus Gal80ts;ok107-Gal4/RNAi104604, *P < 0.05, +/RNAi104604 versus Gal80ts;ok107-Gal4/RNAi104604, **P < 0.01). (k) Quantification of STM for those flies (control driver line PI 0.18 ± 0.008, Myo15 knock-down (RNAi104606) PI 0.18 ± 0.047, control RNAi PI 0.25 ± 0.028; F(2,17) = 1.414, P = 0.2738, n = 6). (l) Flies expressing RNAi-Myo15104606 in the adult MB lobes present normal olfactory acuity (OA) to octanol (control driver line OA 0.56 ± 0.083, Myo15 knock-down (RNAi104606) OA 0.403 ± 0.0577, control RNAi OA 0.477 ± 0.065; F(2,17) = 1.333, P = 0.2933, n = 6) and methylcyclohexanol (control driver line OA 0.60 ± 0.0733, Myo15 knock-down (RNAi104606) OA 0.713 ± 0.107, control RNAi OA 0.642 ± 0.093; F(2,17) = 0.3771, P = 0.6922, n = 6). (m) Those flies present normal electrical shock reactivity (control driver line reactivity 0.87 ± 0.029, Myo15 knock-down (RNAi104606) reactivity 0.865 ± 0.0425, control RNAi reactivity 0.843 ± 0.0189; F(2,17) = 0.2614, P = 0.7734, n = 6). All data are provided as mean ± SEM. (A–b and d–f)n = NMJs with 1–2 NMJs/animal, four to six animals, g–m: n = cohort of ∼50 adult flies, 5–8 cohorts measured. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant. PI, performance index.
Figure S5.

Electrophysiological TEVC analysis of Myo15 loss-of-function, PhTx treatment of Myo15 −/− mutant, and sustained memory consolidation for Myo15 knock-down. (A–N) TEVC electrophysiological recordings of Myo15 RNAi–mediated knock-down, (A–G, M, and N) evoked, (H–L) spontaneous release. (A) eEPSC traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (B) eEPSC amplitude (control −55.43 ± 2.00 nA, n = 22; Myo15-RNAi −58.92 ± 4.71 nA, n = 24). (C) Rise time (control 1.27 ± 0.04 ms, n = 22; Myo15-RNAi 1.18 ± 0.04 ms, n = 24). (D) Decay time τ (control 5.2 ± 0.17 ms, n = 23; Myo15-RNAi 4.11 ± 0.24 ms, n = 24). (E) Charge (control −484.5 ± 23.03 pC, n = 22; Myo15-RNAi −476.4 ± 52.17 pC, n = 24). (F) 10-ms paired-pulse traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (G) Paired-pulse ratio 10 ms (control 0.92 ± 0.04 ms, n = 22; Myo15-RNAi 0.98 ± 0.06, n = 24). (H) mEPSC traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 2 nA, 200 ms. (I) mEPSC amplitude (control −0.70 ± 0.02 nA, n = 23; Myo15-RNAi −0.60 ± 0.02 nA, n = 24). (J) mEPSC rise time (control 2.13 ± 0.16 ms, n = 23; Myo15-RNAi 1.87 ± 0.16 ms, n = 24). (K) mEPSC decay time τ (control 5.95 ± 0.19 ms, n = 19; Myo15-RNAi 4.61 ± 0.21 ms, n = 23). (L) mEPSC frequency (control 0.97 ± 0.12 Hz, n = 23; Myo15-RNAi 1.08 ± 0.09 Hz, n = 24). (M) 30 ms paired-pulse traces of Myo15 knock-down (red) and control (black) NMJs, scale bar 10 nA, 10 ms. (N) Paired-pulse ratio 30 ms (control 1.29 ± 0.03 ms, n = 22; Myo15-RNAi 1.26 ± 0.03, n = 24). (O–b) TEVC electrophysiological recordings of Myo15ΔMyTH4 (1+2) deletion mutant and wild-type NMJs, (O–U, a, and b) evoked, (V–Z) spontaneous release. (O) eEPSC traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (P) eEPSC amplitude (wild type −61.25 ± 3.48 nA, n = 16; Myo15ΔMyTH4 (1+2) −66.16 ± 6.46 nA, n = 16). (Q) Rise time (wild type 1.23 ± 0.05 ms, n = 16; Myo15ΔMyTH4 (1+2) 1.00 ± 0.03 ms, n = 16). (R) Decay time t (wild type 4.80 ± 0.21 ms, n = 16; Myo15ΔMyTH4 (1+2) 4.23 ± 0.10 ms, n = 16). (S) Charge (wild type −526.4 ± 39.29 pC, n = 16; Myo15ΔMyTH4 (1+2) −527.2 ± 67.47 pC, n = 16). (T) 10 ms paired-pulse traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (U) Paired-pulse ratio 10 ms (wild type 0.78 ± 0.04 ms, n = 16; Myo15ΔMyTH4 (1+2) 0.75 ± 0.03, n = 16). (V) mEPSC traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 1 nA, 200 ms. (W) mEPSC amplitude (wild type −0.65 ± 0.02 nA, n = 16; Myo15ΔMyTH4 (1+2) −0.61 ± 0.02 nA, n = 16). (X) mEPSC rise time (wild type 2.27 ± 0.13 ms, n = 16; Myo15ΔMyTH4 (1+2) 1.64 ± 0.16 ms, n = 16). (Y) mEPSC decay time t (wild type 5.53 ± 0.19 ms, n = 16; Myo15ΔMyTH4 (1+2) 4.96 ± 0.21 ms, n = 16). (Z) mEPSC frequency (wild type 1.96 ± 0.27 Hz, n = 16; Myo15ΔMyTH4 (1+2) 1.72 ± 0.16 Hz, n = 16). (a) 30-ms paired-pulse traces of Myo15ΔMyTH4 (1+2) deletion mutant (red) and wild-type (black) NMJs (black), scale bar 10 nA, 10 ms. (b) Paired-pulse ratio 30 ms (wild type 1.04 ± 0.03 ms, n = 16; Myo15ΔMyTH4 (1+2) 0.97 ± 0.01, n = 15). (c) Confocal images of myo15−/− mutant and control NMJs treated with PhTx (fire LUT), scale bar 2 µm. (d) Quantification of c. BRP mean pixel intensity (wild type - PhTx 100.0 ± 7.59%, n = 11; + PhTx 203.2 ± 17.60%, n = 12; Myo15−/− mutant - PhTx 100.0 ± 10.35%, n = 11; + PhTx 111.8 ± 17.76%, n = 8. (e) mEPSP traces of Myo15 knock-down and control NMJs with (red) and without (black) PhTx treatment for 10 min, scale bar 5 mV, 500 ms. (f) mEPSP traces of Myo15 knock-down and control NMJs with (red) and without (black) PhTx treatment for 30 min, scale bar 2 mV, 500 ms. (g) Quantification of MTM 1 h of Gal80ts;ok107-Gal4>Myo15-RNAi101729 without induction (restrictive 18°C temperature, control driver line PI 0.39 ± 0.061, Myo15 knock-down (RNAi101729) PI 0.50 ± 0.054, control RNAi PI 0.43 ± 0.052, F(2,23) = 0.9187, P = 0.4145, n = 8). (h) Flies expressing RNAi-Myo15101729 in the adult MB lobes present normal olfactory acuity to octanol (control driver line OA 0.52 ± 0.060, Myo15 knock-down (RNAi101729) OA 0.383 ± 0.0598, control RNAi OA 0.542 ± 0.085; F(2,17) = 1.530, P = 0.2486, n = 6) and methylcyclohexanol (control driver line OA 0.55 ± 0.0629, Myo15 knock-down (RNAi101729) OA 0.728 ± 0.0682, control RNAi OA 0.740 ± 0.0879; F(2,17) = 2.046, P = 0.1639, n = 6). (i) Those flies present normal electrical shock reactivity (control driver line reactivity 0.79 ± 0.045, Myo15 knock-down (RNAi101729) reactivity 0.855 ± 0.039, control RNAi reactivity 0.868 ± 0.0566; F(2,17) = 0.7134, P = 0.5059, n = 6). (j) Quantification of MTM 1 h for flies expressing RNAi-Myo15104606 in the adult MB lobes (control driver line PI 0.21 ± 0.016, Myo15 knock-down (RNAi104606) PI 0.05 ± 0.054, control RNAi PI 0.29 ± 0.046; F(2,23) = 8.370, P = 0.0021, n = 8, post hoc Tukey’s multiple comparisons test, Gal80ts;ok107-Gal4/+ versus Gal80ts;ok107-Gal4/RNAi104604, *P < 0.05, +/RNAi104604 versus Gal80ts;ok107-Gal4/RNAi104604, **P < 0.01). (k) Quantification of STM for those flies (control driver line PI 0.18 ± 0.008, Myo15 knock-down (RNAi104606) PI 0.18 ± 0.047, control RNAi PI 0.25 ± 0.028; F(2,17) = 1.414, P = 0.2738, n = 6). (l) Flies expressing RNAi-Myo15104606 in the adult MB lobes present normal olfactory acuity (OA) to octanol (control driver line OA 0.56 ± 0.083, Myo15 knock-down (RNAi104606) OA 0.403 ± 0.0577, control RNAi OA 0.477 ± 0.065; F(2,17) = 1.333, P = 0.2933, n = 6) and methylcyclohexanol (control driver line OA 0.60 ± 0.0733, Myo15 knock-down (RNAi104606) OA 0.713 ± 0.107, control RNAi OA 0.642 ± 0.093; F(2,17) = 0.3771, P = 0.6922, n = 6). (m) Those flies present normal electrical shock reactivity (control driver line reactivity 0.87 ± 0.029, Myo15 knock-down (RNAi104606) reactivity 0.865 ± 0.0425, control RNAi reactivity 0.843 ± 0.0189; F(2,17) = 0.2614, P = 0.7734, n = 6). All data are provided as mean ± SEM. (A–b and d–f)n = NMJs with 1–2 NMJs/animal, four to six animals, g–m: n = cohort of ∼50 adult flies, 5–8 cohorts measured. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant. PI, performance index.

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