Volume 86, Issue 3, Pages 711-725 (May 2015) Molecular Remodeling of the Presynaptic Active Zone of Drosophila Photoreceptors via Activity-Dependent Feedback  Atsushi Sugie, Satoko Hakeda-Suzuki, Emiko Suzuki, Marion Silies, Mai Shimozono, Christoph Möhl, Takashi Suzuki, Gaia Tavosanis  Neuron  Volume 86, Issue 3, Pages 711-725 (May 2015) DOI: 10.1016/j.neuron.2015.03.046 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Redistribution of the AZ Structural Component Brp in R8 Photoreceptors after Extended Exposure to Light (A) Scheme of the Drosophila visual system. The outer photoreceptors R1–R6 (blue) terminate in the lamina. The inner photoreceptors R7 (red) and R8 (green) project axons into the medulla reaching to layers M6 and M3, respectively. The dotted box indicates the area shown in the scheme and in (B). (B) Projection of a confocal stack through the medulla layers M1–M6, showing in R8s Brp-GFP BAC puncta (green) generated with STaR (Chen et al., 2014) using senseless-FLP recombinase. Anti-CadN antibody (blue) highlights the medulla layers, and mAb24B10 (red) highlights the R7 and R8 photoreceptor axons in this and following panels. Scale bar, 10 μm. (C) Light exposure protocols after eclosion. DD, continuous darkness; LD, 12-hr light/12-hr dark; LL, constant exposure to light. Fly brains were dissected at the times indicated by the brackets. (D–F) Projections of confocal stacks through the M1–M3 medulla layers showing Brp-GFP BAC puncta (green) in a single R8 axon of flies exposed to DD (D), LD (E), or LL (F). Scale bar, 5 μm. (G) Boxplot comparing the total number of Brp-GFP BAC puncta per axon in DD, LD, and LL. In this and all following boxplots, the boxes show median and interquartile range; the whiskers show the minima/maxima. The number of Brp-GFP BAC puncta in LL (227 axons/11 brains) is reduced compared to LD (107/5) or DD (83/7). In this and all following boxplots ∗∗∗p < 0.0001, n.s. p > 0.05; unpaired t test with Welch’s correction two-tailed. See also Figures S1 and S2. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 The Number of T-Bars Is Reduced in LL, but Their Size Is Not Modified (A–D) Electron micrographs of cross-sections of R7/8 photoreceptor axons in the medulla of flies exposed to DD (A and C) or LL (B and D). The boxed region in (A) and (B) contains a T-bar and is magnified in (C) and (D), respectively. (E–H) Electron micrographs of randomly selected cross-sections of a lamina cartridge of flies exposed to LD (E and G) or LL (F and H). Arrows in (E) and (F) indicate the T-bars in the synaptic terminals of the six photoreceptor cells (shaded in blue). (G) and (H) show T-bars in R1–R6 in lamina of flies exposed to LD (G) or LL (H). Arrowheads indicate the T-bar platform ends (C, D, G, and H). The dotted lines indicate a radius of 200 nm and 400 nm from the T-bar center, respectively (G and H). Scale bars in (A), (B), (E), and (F), 1 μm; (C), (D), (G), and (H), 200 nm. (I–L) Bar graphs comparing the length of T-bar platforms (DD, n = 73 T-bars/3 brains; LL, n = 72/3) in the medulla (I), the number of T-bars (LD, n = 54 cartridges/3 brains; LL, n = 59/2) (J), the length of T-bar platforms (LD, n = 89 T-bars/3 brains; LL, n = 94/2) (K), and the number of SVs in 0–200-nm or 200–400-nm distance from the T-bar center (LD, n = 89/3; LL, n = 94/2) (L) in the lamina. The number of T-bars in the lamina was reduced in LL. Mean values are shown, and error bars represent SD. n.s. p > 0.05; ∗∗p < 0.005; unpaired t test with Welch’s correction two-tailed. See also Figure S3. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 GFP-DLiprin-α and GFP-DRBP, but Not GFP-DSyd-1 or Cac-GFP, Are Delocalized in LL (A, C, E, and G) Brp-short-cherry puncta in a single R8 (green in central and left panels) and GFP-DLiprin-α (A), GFP-DRBP (C), GFP-DSyd-1 (E), or Cac-GFP (G) (magenta in left and right panels), respectively. White arrows in (A) indicate puncta with Brp-short-cherry, but no detectable GFP-DLiprin-α; yellow arrows represent GFP-DLiprin-α puncta with no detectable Brp-short-cherry. (B, D, F, and H) GFP-DLiprin-α (B), GFP-DRBP (D), GFP-DSyd-1 (F), or Cac-GFP (H) (magenta) in a single R8 axon in LD or LL. GFP-DLiprin-α puncta are largely lost in LL yielding a diffuse signal (B). GFP-DRBP puncta are reduced in number, and a diffuse signal becomes visible (D). GFP-DSyd-1 (F) or Cac-GFP (H) puncta are clearly identified also in LL. Scale bar, 5 μm. (I) Scheme of analyzed AZ components. (J) Boxplot comparing the number of GFP-DLiprin-α, GFP-DRBP, GFP-DSyd-1, or Cac-GFP puncta per axon in LD and LL. The number of GFP-DLiprin-α puncta (LD, n = 67 axons/7 brains; LL, n = 52/6) and the number of GFP-DRBP puncta (LD, n = 70/7; LL, n = 83/6) are reduced in LL, but GFP-DSyd-1 (LD, n = 48/5; LL, n = 69/7) or Cac-GFP (LD, n = 31/5; LL, n = 27/5) puncta number was not modified. (K) The level of diffuse signal of GFP-DLiprin-α and DRBP-GFP detected in R8 axons (see Figure S1J) is increased in LL, while no changes are observed for GFP-DSyd-1 or Cac-GFP. Mean values are shown, and error bars represent SD. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Neural Activity in Histamine Chloride Channel-Positive Cells Controls the Delocalization of Brp in Photoreceptors (A–H) Brp-GFP BAC puncta (green in left panels, gray in right panels) in a single R8 of norpA7 mutant flies maintained in LD (A) or LL (B), upon expression of UAS-Ork1Δ-C with Rh6-Gal4 in LD (C) or LL (D), upon expression of UAS-TrpA1 with Rh6-Gal4 in DD at 25°C (E) or 29°C (F), or upon expression of UAS-shits1 with Rh6-Gal4 in LL at 25°C (G) or 29°C (H). (I) Boxplot comparing the total number of Brp-GFP BAC puncta: norpA7 mutant in LD (n = 71 axons/7 brains) and LL (n = 97/10); UAS-Ork1Δ-C in LD (n = 51/7) and LL (n = 49/9); UAS-TrpA1 in DD at 25°C (n = 85/8), 29°C (n = 78/8), and in control without UAS-TrpA1 in DD at 29°C (n = 48/5); UAS-shits1 in LL at 25°C (n = 71/7), 29°C (n = 77/8), and in control without UAS-shits1 in LL at 29°C (n = 39/4). (J–M) Brp-short-cherry puncta (green in left panels, gray in right panels) in a single R8 of hiscl1134, ort1 double mutant flies maintained in LD (J) or LL (K), or upon expression of lexAop-TrpA1 with ort-LexA in LL at 25°C (L) or 29°C (M). (N) Boxplot comparing the total number of Brp-short-cherry puncta: hiscl1134, ort1 double mutant in LD (n = 19/3) and LL (n = 63/9); lexAop-TrpA1 with ort-LexA in LL at 25°C (n = 104/6), 29°C (n = 127/7), without lexAop-TrpA1 in LL at 29°C (n = 90/4), or without ort-LexA in LL at 29°C (n = 105/6). Scale bar, 5 μm. See also Figure S4. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Suppression of the Divergent Canonical Wnt Pathway Contributes to the Relocalization of Brp after Prolonged Light Exposure (A) Scheme of the divergent canonical Wnt pathway. (B–E, G, and H) Brp-GFP BAC (green in left panels, gray in right panels) in R8 in control (B), in dsh1 mutant (C), upon overexpression of UAS-sgg with Rh6-Gal4 (D), in futschN94 mutant (E) in LD, or upon overexpression of UAS-arr with Rh6-Gal4 (H), without UAS-arr as a control (G) in LL. Scale bar, 5 μm. (F and I) Boxplots comparing the total number of Brp-GFP BAC puncta: (F) same background control (n = 57 axons/8 brains), dsh1 mutants (n = 56/8), futschN94 mutants (n = 70/8), overexpression of UAS-sgg (n = 47/7), and control minus UAS-sgg (n = 31/4) in LD; (I) boxplots comparing the total number of Brp-GFP BAC puncta: UAS-arr (LD, n = 52/7; LL, n = 48/7) and control minus UAS-arr (LD, n = 31/4; LL, n = 39/4). (J–L) Confocal image of the medulla of animals carrying Ort-LexA and lexAop-TrpA1 at 25°C (J) or 29°C (K), immunolabeled with anti-Wg antibodies (green in the top panels, gray in the bottom ones) and anti-CadN to highlight the medulla (blue, top panels). Endogenous Wg was upregulated in Ort-LexA > lexAop-TrpA1 at 29°C (65.2%, 15 brains/23 brains analyzed), but hardly in Ort-LexA > lexAop-TrpA1 at 25°C (8.3%, 1/12), Ort-LeXA at 29°C (7.1%, 1/14), or in lexAop-TrpA1 at 29°C (12.5%, 2/16). Scale bar, 20 μm. (L) Medulla of animals carrying Rh6-GFP with Ort-LexA and lexAop-TrpA1 at 29°C, immunolabeled with anti-Wg antibodies (green in the left and central panels) and highlighted R8 (magenta in left and right panels). Scale bar, 5 μm. See also Figure S5. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Exposure to Light Induces Microtubule Disorganization (A–F) GFP-αTub84B (gray) in one R8 axon in LD (A) and LL (B), in control futschN94/+ (C), futschN94 mutant (D), control Rh6-Gal4 (E), or Rh6-Gal4 UAS-spas (F). futschN94/+ (C) and control Rh6-Gal4 (E) display tubulin threads. futschN94 mutants (D) or Rh6-Gal4 UAS-spas (F) display disorganized tubulin. White arrows point to loops (microtubule threads), and yellow arrows point to diffuse tubulin signal (disorganized tubulin). Scale bar, 5 μm. (G) Quantification of the GFP-αTub84B phenotypes. Number of R8s examined for the control in LD (n = 126 axons/9 brains), control in LL (n = 95/6), futschN94/+ (n = 95/6), futschN94 (n = 112/5), Rh6-Gal4 control (n = 253/17), or Rh6-Gal4 UAS-spas (n = 145/9). (H and I) Brp-GFP BAC (green, left panels; gray, right panels) and mAb24B10 highlighting R7 and R8 (red, left panels) in one R8 axon in Rh6-Gal4 control (H) or Rh6-Gal4 UAS-spas (I) in LD at 29°C. (J) Boxplot comparing the total number of Brp-GFP BAC puncta: control Rh6-Gal4 (n = 31/4) and Rh6-Gal4 UAS-spas (n = 117/13). (K) Brp-short-cherry (green, left and middle panels) and Imac-GFP (magenta, left and right panels) in one R8 axon. Brp-short-cherry signals delocalized after overexpression of Imac-GFP. (L and M) Brp-short-cherry (green, left panels; gray, right panels) and mAb24B10 highlighting R7 and R8 (red in the left panels) in one R8 axon in control Rh6-Gal4 (L) or Rh6-Gal4 UAS-imac RNAi (M) in LL at 29°C. (N) Boxplot comparing the total number of Brp-short-cherry puncta: control Rh6-Gal4 (n = 169/10) and Rh6-Gal4 UAS-imac-GFP (n = 95/5) in LD; control Rh6-Gal4 (n = 98/7) and Rh6-Gal4 UAS-imac RNAi in LL (n = 66/6). See also Figure S6. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 A Model of the AZ Remodeling upon Prolonged Light Exposure (A) In DD or LD, photoreceptors are not activated or activated for a restricted period of time. While photoreceptors are not activated, postsynaptic histamine chloride channel-positive cells are not hyperpolarized. In these conditions, a feedback signal mediated by the divergent canonical Wnt pathway promotes microtubule stability in presynaptic photoreceptors to maintain the basic AZ structure. (B) In LL, photoreceptors are active for an extended period of time, and the postsynaptic neurons become hyperpolarized. This condition suppresses the Arrow-mediated activation of the divergent canonical Wnt pathway, leading to microtubule disorganization and consequently to AZ remodeling, including delocalization of Brp, DLiprin-α, and DRBP. Neuron 2015 86, 711-725DOI: (10.1016/j.neuron.2015.03.046) Copyright © 2015 Elsevier Inc. Terms and Conditions