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

Figure 2.

Figure 2

Wild-type Rac1, unlike Rac1V12, requires wild-type Syd-1 levels in order to promote NMJ growth. (A–F) Third-instar larval NMJs at muscle 4 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. (A) Wild type. (B) syd-1CD/+ heterozygote. (C) syd-1CD/syd-1w46 mutant. (D) Wild-type animal in which motorneurons express wild-type Rac1 (Rac1wt). (E) syd-1CD/+ heterozygote in which motorneurons express Rac1wt. (F) syd-1CD/syd-1w46 mutant in which motorneurons express Rac1wt. (G) Quantification of the number of boutons on muscle 4 in abdominal segment 3 per muscle area. Loss of one copy of syd-1 does not cause a decrease in bouton number (36.3 ± 1.3, n = 14) compared to wild type (BG380-Gal4 alone: 35.7 ± 1.9, n = 12). By contrast, while overexpressing wild-type Rac1 (49.5 ± 3.4, n = 12) significantly increases bouton number compared to the wild-type control (P = 0.0011), loss of one copy of syd-1 prevents this increase (36.8 ± 1.0, n = 17, P = 1.9 × 10−8 compared to wild-type Rac1 in wild type). By contrast, loss of one copy of syd-1 has no effect on the increase in bouton number caused by overexpression of a constitutively-active form of Rac1, Rac1V12 (Rac1V12 alone: 58.5 ± 3.5, n = 8 (P = 4.8 × 10−5 compared to wild type); Rac1V12 in syd-1/+: 56.0 ± 2.5, n = 12). Complete loss of syd-1 impairs the ability of wild-type Rac1 (21.4 ± 1.6, n = 14, P = 1.9 × 10−8) or Rac1V12 (36.6 ± 4.2, n = 11, P = 1.4 × 10−4) to increase bouton number. Error bars represent SEM. n = animals. ns, not significant; *** P < 0.001, based on two-tailed t-tests. (H) Quantification of the number of boutons on muscles 6/7 in abdominal segment 3 per muscle area. We observed the same effects on bouton number as at both muscle 4. Wild type (105.4 ± 3.1, n = 14) and syd-1/+ heterozygotes (108.0 ± 5.7, n = 14) have similar numbers of boutons. Overexpressing either wild-type Rac1 (133.0 ± 5.7, n = 14, P = 1.3 × 10−4) or Rac1V12 (141.0 ± 6.5, n = 8, P = 2.6 × 10−5) causes an increase in bouton number. Removing one copy of syd-1 prevents wild-type Rac1 from increasing bouton number (107.4 ± 8.1, n = 14, P = 0.0015 compared to wild-type Rac1 in wild type) but has no effect on Rac1V12 (146.0 ± 5.2, n = 17). Complete loss of syd-1 impairs the ability of wild-type Rac1 (86.8 ± 5.0, n = 15, P = 1.5 × 10−6) or Rac1V12 (93.5 ± 7.8, n = 14, P = 5.3 × 10−4) to increase bouton number. Error bars represent SEM. n = animals. ns, not significant; * P < 0.05, * P < 0.01, *** P < 0.001, based on two-tailed t-tests.