Fig. 6: Semaphorin–Plexin–Mical repulsion combines with profilin to drive F-actin remodeling and guidance of growth cones.

a–c Neuronal overexpression of profilin (ELAV-GAL4/+, UAS:profilin (1M)/+ (n = 100 hemisegments in 10 animals)), but not profilin^Y6D (ELAV-GAL4/+, UAS:profilin^Y6D (5F)/+ (n = 120 hemisegments in 12 animals)), generates highly penetrant axon guidance defects (arrows), including that axons project away from the CNS (arrowheads). Neuronal driver only = ELAV-GAL4/+ (n = 100 hemisegments in 10 animals). ****p < 0.0001; Χ² test. d Neuronal overexpression of high levels (2 copies (2x)) of profilin^Y6D (ELAV-GAL4/ELAV-GAL4, UAS:profilin^Y6D (5F)/UAS:profilin^Y6D (5F) (n = 43 animals)) generates Mical^−/− and profilin^−/− double mutant-like CNS axon guidance defects. Scoring as in Fig. 5e. Neuronal driver only (2x) = ELAV-GAL4/ELAV-GAL4 (n = 12 animals). ****p < 0.0001; Χ² test. e Neuronal overexpression of profilin increases Mical-dependent axon guidance defects and SelR neuronal overexpression rescues these effects. Neuronal Mical⁺⁺⁺ (UAS:Mical/+, ELAV-GAL4/+ (n = 54 animals)): alters the guidance of axons in the 3rd (3) and 2nd (2) CNS longitudinal bundles, including that axons are abnormally separated from one another/present in thinner bundles and project away from the CNS (e.g., arrowheads). See also refs. ^9,56. These defects contrast to loss of Mical (Mical^−/−), or Mical and profilin double mutants (Mical^−/− & profilin^−/−), in which axons are more thickly bundled (e.g., Fig. 5e, Class III, arrowheads). Neuronal Mical⁺⁺⁺ & profilin⁺⁺⁺ (UAS:Mical/+, UAS:profilin (1M)/+, ELAV-GAL4/+ (n = 88 animals)): significantly increases the severity of guidance defects (****p < 0.0001; Χ² test)—now predominantly affecting all three (3, 2, 1) longitudinal bundles (image and graph, red) and generating axons that are even more separated from one another/present in thinner bundles. Neuronal Mical⁺⁺⁺ & profilin⁺⁺⁺ & SelR⁺⁺⁺ (UAS:Mical/+, UAS:profilin (1M)/+, ELAV-GAL4/+, UAS:SelR/+ (n = 57 animals)): SelR neuronal overexpression significantly suppresses the Neuronal Mical⁺⁺⁺ & profillin⁺⁺⁺ defects (****p < 0.0001; Χ² test), such that a more normal pattern of axons is observed in all three longitudinal bundles (3, 2, 1). Neuronal profilin⁺⁺⁺ = UAS:profilin (1M)/+, ELAV-GAL4/+ (n = 35 animals; see panel a and graph). f Mical (Neuronal Mical⁺⁺⁺=UAS:Mical^ΔPIR/+, RN2-GAL4/+, UAS:^GFPActin/+ (n = 27 growth cones in 5 animals)) significantly increases F-actin (red) remodeling in growth cones⁹—and reducing profilin (Neuronal Mical⁺⁺⁺ & profilin^+/− = UAS:Mical^ΔPIR/+, RN2-GAL4/+, UAS:^GFPActin/+, chic²²¹/+ (n = 65 growth cones in 12 animals)) decreases this remodeling. Means ± SEM. ****p < 0.0001; unpaired t-test (two-tailed). For a–f, ≥2 independent experiments gave similar results. Source data for Fig. 6 are provided as a Source Data file.