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. 2022 Jul 14;11:e78648. doi: 10.7554/eLife.78648

Figure 3. Active zone (AZ) Cac accumulation is regulated downstream of Cac biosynthesis.

(A) Representative images of Cac-GFP (green) and Bruchpilot (BRP) (magenta) at muscle 4 for endogenously C-terminally tagged Cac-GFP controls (cacGFP(C),C155/cacGFP(C)) and Cac overexpression animals (cacGFP(C),C155/cacGFP(C),UAS-Cac-GFP). (B) Quantification of AZ Cac intensity in control versus Cac overexpression neuromuscular junctions (NMJs). Each point represents the maximum Cac-GFP pixel intensity of a single AZ (control: 14617±130.8, n=3663 AZs from 10 NMJs from 6 animals; Cac OE: 14,383±148.2, n=3145 AZs from 10 NMJs from 6 animals). (C) Distribution of AZ Cac-GFP abundance across the AZ population in control versus Cac overexpression NMJs. (D) Representative images of endogenously tagged Cac-GFP (green) and overexpressed Cac-tdTomato (magenta) at AZs in control (cacGFP,C155) versus Cac-tdTomato overexpression (cacGFP,C155;UAS-Cac-tdT). (E) Quantification of AZ Cac-GFP intensity with and without Cac-tdTomato overexpression (control: 15,339±232, n=1613 AZs, Cac-tdT: 4477±123.9, n=850 AZs, p<0.0001). (F, G) Average traces and evoked peak currents (nA) at muscle 6 in Cac overexpression and control animals (control: 24.45±2.433, n=9 NMJs from 5 animals; Cac OE: 30.11±3.046, n=9 NMJs from 5 animals). (H, I) Quantifications and representative images of Western blots of adult head extracts from cacGFP/Df heterozygotes and cacGFP controls (n=11 samples, extracts from 5 adult heads per sample). Each point in H represents Cac intensity in one lane (cacGFP: 25,092±2146; cacGFP/Df: 13,055±1403, p<0.001). Each point in I represents syntaxin 1 (SYX1) intensity in one lane (cacGFP: 224,900±11,239; cacGFP/Df: 214,889±86,96). Representative images for Cac and SYX are shown above each quantification. (J) Representative images of endogenous Cac-GFP (green) and BRP (magenta) at muscle 4 AZs in cacGFP/Df heterozygotes and cacGFP controls. Ib and Is terminals are labeled in white. (K) Quantification of average Cac-GFP AZ abundance per MN4-Ib NMJs (cacGFP: 15,105±486.3, n=19 NMJs from 7 animals; cacGFP/Df: 13,617±365.9, n=21 NMJs from 7 animals, p<0.05). (L) Histogram of Cac-GFP intensity across the AZ population in cacGFP/Df heterozygotes and cacGFP controls at MN4-Ib NMJs. (M) BRP-positive AZ number per MN4-Ib NMJ (cacGFP: 243.4±17.05; cacGFP/Df: 260.3±9.963). (N) Quantification of average AZ Cac-GFP intensity per Is innervation of muscle 4 NMJs (cacGFP: 14,621±747.5, n=11 NMJs from 7 animals; cacGFP/Df: 10,219±601 n=12 NMJs from 7 animals, p<0.001). (O, P) Average traces and evoked peak currents (nA) at muscle 6 in cacGFP/Df heterozygotes and cacGFP controls (cacGFP: 41.96±3.879, n=9 NMJs from 5 animals; cacGFP/Df: 16.7±1.984, n=8 NMJs from 7 animals, p<0.0001).

Figure 3—source data 1. Source data for Figure 3.
Figure 3—source data 2. Western regions used for Figure 3.
Figure 3—source data 3. Western for Figure 3.

Figure 3.

Figure 3—figure supplement 1. Synaptic morphology of Cac-GFP C-terminal CRISPR-tagged larvae.

Figure 3—figure supplement 1.

(A) Representative images of muscle 4 neuromuscular junctions (NMJs) stained for Bruchpilot (BRP) and HRP in control animals and animals expressing endogenously C-terminally tagged Cac-GFP (cacGFP(C)). (B) Active zone (AZ) number in control and cacGFP(C) muscle 4 NMJs (control: 365.8±20.17, n=13 NMJs; cacGFP(C): 301.6±12.72, n=13 NMJs, p=0.0127). (B) NMJ area at control and cacGFP(C) muscle 4 NMJs (control: 207.1±12.54; cacGFP(C): 189.2±6.94).
Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1.
Figure 3—figure supplement 2. Quantitative imaging control.

Figure 3—figure supplement 2.

(A) Representative images of Cac-GFP at active zones (AZs) in female larvae homozygous for CRISPR-tagged Cac-GFP (cacGFP, left) and heterozygous for CRISPR-tagged Cac-GFP (cacGFP/+, right). Bruchpilot (BRP) immunostaining was used to identify AZs (magenta). (B) Quantification of single AZ Cac abundance using max pixel intensity per AZ (cacGFP: 21,181±218.3, n=3452 AZs; cacGFP/Df: 10,505±111.5, n=3,976 AZs, p<0.0001). (C) Sum AZ Cac abundance, with each point representing the sum of Cac-GFP fluorescence in an ROI encompassing a single AZ (cacGFP: 127,105±172, n=3157 AZs; cacGFP/Df: 60,865±859.1, n=3818 AZs, p<0.0001). (D) Average AZ Cac abundance per NMJ, with each point representing the average of max pixel intensities of the population of AZs at one NMJ (cacGFP: 21,434±1366, n=14 NMJs; cacGFP/Df: 10,552±686.2, n=14 NMJs, p<0.0001). (E) Average AZ Cac per NMJ, with each point representing the average of sum pixel intensities of the population of AZs at a single NMJ (cacGFP: 129,806±9724; cacGFP/Df: 60,724±4723, p<0.0001). (F) Total Cac at each NMJ (cacGFP: 36,003,272±2,818,383; cacGFP/Df: 20,063,946±2175784, p<0.001). (G) AZ number at each NMJ (cacGFP: 246.6±14.82; cacGFP/Df: 309.8±15.02, p<0.006).
Figure 3—figure supplement 2—source data 1. Source data for Figure 3—figure supplement 2.