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. 2017 Dec 6;208(2):705–716. doi: 10.1534/genetics.117.300538

Figure 3.

Figure 3

Wild-type and R979A mutant Syd-1 colocalize with Brp and cause similar increases in NMJ bouton number and Nrx-1 levels when expressed in wild-type motorneurons. (A–B’) Third-instar larval NMJ boutons on muscle 4 stained with anti-HRP (blue), anti-Brp (magenta), and anti-FLAG (green). Bar, 5 µm. (A and A’) A wild-type animal in which motorneurons express wild-type full-length FLAG-tagged Syd-1 driven by BG380-Gal4. Like endogenous Syd-1, tagged wild-type Syd-1 forms puncta that are adjacent to, and partly overlapping with, the AZ protein Brp. (B and B’) A wild-type animal in which motorneurons express R979A mutant full-length FLAG-tagged Syd-1 (Syd-1RA) driven by BG380-Gal4. The punctate pattern and levels of Syd-1RA resemble those of Syd-1wt. (C–E) Third-instar larval NMJs at muscles 6/7 in abdominal segment 3 stained with anti-HRP (green). Bar, 25 µm. All genotypes also contain the motorneuron driver BG380-Gal4, and were costained with anti-Dlg. (C) Wild type. (D) Wild type in which motorneurons express wild-type full-length Syd-1. (E) Wild type in which motorneurons express R979A mutant full-length Syd-1. (F) Quantification of the number of boutons on muscles 6/7 in abdominal segment 3 per muscle area. Overexpressing wild-type (114.9 ± 6.4, n = 10) or R979A mutant Syd-1 (117.8 ± 5.3, n = 7) causes similar, significant increases in bouton number (P = 0.02 and 5.9 × 10−4, respectively) compared to the wild-type control (BG380-Gal4 alone: 96.9 ± 1.8, n = 9). Error bars represent SEM. n = animals. * P < 0.05, *** P < 0.001, based on two-tailed t-tests. (G–I”) Third-instar larval NMJ boutons on muscle 4 stained with anti-HRP (blue) and anti-Nrx-1 (green). Bar, 5 µm. All genotypes also contain the motorneuron driver BG380-Gal4. (G–G”) Wild type. (H–H”) Wild type in which motorneurons express wild-type Syd-1. (I–I”) Wild type in which motorneurons express R979A mutant Syd-1. (J) Quantification of anti-Nrx-1 staining. Overexpressing wild-type (155 ± 12.4%, n = 14) or R979A mutant Syd-1 (156 ± 9.4%, n = 13) significantly increases Nrx-1 levels in NMJ boutons (P = p = 9.2 × 10−4 and 3.1 × 10−4, respectively) compared to the wild-type control (BG380-Gal4 alone: 100 ± 8.4% n = 15). Error bars represent SEM. n = animals. ns, not significant; * P < 0.05, ** P < 0.01, based on one-tailed t-tests.