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

Figure 4. Bruchpilot (BRP) biosynthesis rate-limits active zone (AZ) BRP incorporation.

(A) Representative images of BRP and endogenously tagged Cac-GFP at AZs in controls, brpDf/+ heterozygotes and pan-neuronally (elav-GAL4) expressed BRP RNAi. (B) Quantification of single AZ BRP intensity, with average AZ BRP intensity listed as a percent of control above each genotype (control: 18,161±210.7, n=1779 AZs; brpDf/+: 11,534±176.7, n=1350 AZs, p<0.0001; BRP RNAi: 2806±79.91, n=1140 AZs, p<0.0001). (C) Representative image of Western blots of adult head extracts from control and brpDf/+ heterozygotes (left panel), and control and pan-neuronally expressed BRP RNAi animals (right panel) stained for syntaxin 1 (SYX1) (loading control), Tubulin, and BRP. (D) Quantification of BRP intensity in Western blots of the indicated genotypes. Each point represents BRP intensity in one lane, with BRP intensity normalized to the SYX1 loading control. Percent of protein abundance compared to control (100%) is shown above each genotype (control: 1.0±0.02912, n=9 lanes; brpDf/+: 0.6518±0.03862, n=10 lanes, p<0.0001; BRP RNAi: 0.1366±0.02117, n=5 lanes, p<0.0001). (E) Quantification of endogenously tagged Cac-GFP intensity at single AZs in controls, brpDf/+ heterozygotes, and pan-neuronally expressed BRP RNAi (control: 17,268±232.4, n=1757 AZs; brpDf/+: 16,788±217, n=1403 AZs; BRP RNAi: 11,959±259, n=1140 AZs, p<0.0001). (F, G) Average traces and quantified evoked peak currents (nA) in control and brpDf/+ heterozygotes at muscle 6 (control: 26.78±1.632, n=11 neuromuscular junctions (NMJs); brpDf/+: 28.71±3.105, n=13 NMJs).

Figure 4—source data 1. Source data for Figure 4.
elife-78648-fig4-data1.xlsx (465.2KB, xlsx)
Figure 4—source data 2. Western for Figure 4 panel C - BRP deficiency heterozygote.
Figure 4—source data 3. Western regions used for Figure 4 panel C - BRP RNAi.

Figure 4.

Figure 4—figure supplement 1. Single active zone (AZ) analysis of Cac and Bruchpilot (BRP) following BRP RNAi knockdown.

Figure 4—figure supplement 1.

(A) Representative images of endogenously tagged Cac-GFP (green) and BRP (magenta) in control (C155, cacGFP) and BRP RNAi (C155, cacGFP; UAS-BRP-RNAi) neuromuscular junctions (NMJs). (B) Quantification of single AZ BRP abundance (max pixel intensity per AZ) at control and BRP RNAi NMJs. AZs are defined by Cac-positive puncta. AZs below the red dotted line have zero measurable BRP (control: 23,623±230.1, n=3037 AZs; BRP RNAi: 2742±61.27, n=2363 AZs). (C) Quantification of single AZ Cac abundance (max pixel intensity per AZ) at all Cac-positive sites at control and BRP RNAi NMJs (control: 15,059±172.6; BRP RNAi: 10,592±165.9). (D, E) Quantification and histogram of single AZ BRP abundance (max pixel intensity) for all BRP-positive AZs (control: 23,691±229.7, n=3028 AZs; BRP RNAi: 5093±96.25, n=1055 AZs). (F,G) Quantification and histogram of single AZ Cac abundance (max pixel intensity) for all BRP-positive AZs (control: 15,090±172.8; BRP RNAi: 15,933±269.8).
Figure 4—figure supplement 1—source data 1. Source data for Figure 4—figure supplement 1.