Figure 3.
Figure 3. Dynamin FlAsH-FALI is specific. (A–C) Labeling of controls (dicer-2/+;; nSybGal4/+; A) and larvae expressing shi RNAi (dicer-2/+; shi RNAi/+; nSybGal4/+; B) third instar larval boutons with anti-Dynamin (Dyn) and anti-HRP and quantification of boutonic anti-Dynamin labeling intensity. Error bars show SEMs; t test: **, P < 0.001 (n = 10 NMJs from five larvae per genotype). (D–F) FM 1-43 dye uptake measured after 5 min of stimulation with KCl in controls (dicer-2/+;; nSybGal4/+; D), larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+; E) and in heterozygous mutant shi larvae that express RNAi to shi (shi12-12B/dicer-2; shi RNAi/+; nsybGAL4/+; F). (G and H) Quantification of the number of FM 1-43–labeled accumulations (Accum.) per boutonic area (G) and relative FM 1-43 labeling intensity (int.; H) in controls (dicer-2/+;; nSybGal4/+), in larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+), and in heterozygous mutant shi larvae that express RNAi to shi (shi12-12B/dicer-2; shi RNAi/+; nsybGAL4/+). Error bars show SEMs; ANOVA (post hoc Tukey’s test): **, P < 0.001. In G, n = 24, 36, and 60 boutons from three, seven, and five animals. In H, n = 24, 36, and 20 boutons from three, five, and four animals. (I) Strategy used to generate a genomic Endo-4C construct. The 4C is inserted between the BAR and SH3 domain. ATG is the start codon. (J–L) FM 1-43 labeling in w and w; endo-4C; endo1 animals treated (+) or not treated (−) with FlAsH for 10 min and/or illumination for 5 min (±). All preparations were stimulated with KCl in the presence of FM 1-43 for 1 min, washed, and imaged. (M) Quantification of FM 1-43 labeling intensity after loading of w and w; endo-4C; endo1 before and after FALI normalized to the w control. Error bars show SEMs; ANOVA (post hoc Tukey’s test): ***, P < 0.0001. n = 24, 60, and 64 boutons from six, four, and seven animals. Bars, 5 µm.

Dynamin FlAsH-FALI is specific. (A–C) Labeling of controls (dicer-2/+;; nSybGal4/+; A) and larvae expressing shi RNAi (dicer-2/+; shi RNAi/+; nSybGal4/+; B) third instar larval boutons with anti-Dynamin (Dyn) and anti-HRP and quantification of boutonic anti-Dynamin labeling intensity. Error bars show SEMs; t test: **, P < 0.001 (n = 10 NMJs from five larvae per genotype). (D–F) FM 1-43 dye uptake measured after 5 min of stimulation with KCl in controls (dicer-2/+;; nSybGal4/+; D), larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+; E) and in heterozygous mutant shi larvae that express RNAi to shi (shi12-12B/dicer-2; shi RNAi/+; nsybGAL4/+; F). (G and H) Quantification of the number of FM 1-43–labeled accumulations (Accum.) per boutonic area (G) and relative FM 1-43 labeling intensity (int.; H) in controls (dicer-2/+;; nSybGal4/+), in larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+), and in heterozygous mutant shi larvae that express RNAi to shi (shi12-12B/dicer-2; shi RNAi/+; nsybGAL4/+). Error bars show SEMs; ANOVA (post hoc Tukey’s test): **, P < 0.001. In G, n = 24, 36, and 60 boutons from three, seven, and five animals. In H, n = 24, 36, and 20 boutons from three, five, and four animals. (I) Strategy used to generate a genomic Endo-4C construct. The 4C is inserted between the BAR and SH3 domain. ATG is the start codon. (J–L) FM 1-43 labeling in w and w; endo-4C; endo1 animals treated (+) or not treated (−) with FlAsH for 10 min and/or illumination for 5 min (±). All preparations were stimulated with KCl in the presence of FM 1-43 for 1 min, washed, and imaged. (M) Quantification of FM 1-43 labeling intensity after loading of w and w; endo-4C; endo1 before and after FALI normalized to the w control. Error bars show SEMs; ANOVA (post hoc Tukey’s test): ***, P < 0.0001. n = 24, 60, and 64 boutons from six, four, and seven animals. Bars, 5 µm.

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