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

Figure 6. Cac delivery to active zones (AZs) correlates with AZ size and is regulated by α2δ.

(A) Representative images of FRAP experiments in CTRL (cacGFP) and α2δ mutant (cacGFP;α2δ2/k10814) neuromuscular junctions (NMJs). Fixed and Bruchpilot (BRP)-immunostained (magenta) NMJs are shown 24 hr post-bleaching, with unbleached NMJs as a control. Boutons inside white boxes are enlarged. (B) Percent of BRP-positive AZs with Cac-GFP max pixel intensity greater than 150% of average background fluorescence (pre: 95.56±0.9347; 0H: 90.37±2.347, p=0.0422). (C) Distribution of single AZ Cac-GFP intensity (sum Cac-GFP fluorescence within an ROI encompassing each BRP-positive AZ) in unbleached NMJs (UNBL) and 24 hr after bleaching (pre: 13,6052±3018, n=946 AZs; 24H: 72,261±1389, n=804 AZs). (D, E) Correlations of Cac-GFP and BRP at individual AZs in unbleached controls (D) and NMJs 24 hr post-bleach (E). (F) Pearson r value for the correlation of Cac-GFP and BRP intensity across the AZ population at each NMJ in unbleached control NMJs (UNBL) and 24 hr post-bleach (pre: 0.8801±0.01191; 24H: 0.7913±0.01348, p<0.001). (G) Average AZ Cac-GFP intensity in unbleached segments in cacGFP and α2δ2/k10814 NMJs (cacGFP: 136,462±6346, n=11 NMJs; α2δ2/k10814: 44,340±3252, n=12 NMJs, p<0.0001). (H) Average AZ Cac-GFP intensity 24 hr post-bleach (cacGFP: 70,528±3508, n=9 NMJs; α2δ2/k10814: 31,086±2326, n=8 NMJs, p<0.0001). (I) AZ number at NMJs (cacGFP: 88.26±4.13, n=19 NMJs; α2δ2/k10814: 92.45±3.689, n=20 NMJs). (J, K) Correlation of Cac-GFP and BRP at individual AZs in unbleached α2δ2/k10814 mutants (J) and α2δ2/k10814 NMJs 24 hr post-bleach (K). (L) Pearson r value for the correlation of Cac-GFP and BRP intensity across the AZ population in unbleached NMJs (UNBL) and NMJs 24 hr post-bleach in α2δ2/k10814 mutants (UNBL: 0.7952±0.01635; 24H: 0.7285±0.01955, p=0.018).

Figure 6—source data 1. Source data for Figure 6.
elife-78648-fig6-data1.xlsx (110.6KB, xlsx)

Figure 6.

Figure 6—figure supplement 1. Photobleaching validation and GluRIIA correlations.

Figure 6—figure supplement 1.

(A) Representative images of muscle 26 in intravitally imaged late 2nd instar larvae expressing endogenously tagged Cac-GFP (green) and GluRIIA-RFP (magenta) before (left column; pre-bleach) and immediately after (right column; 0 hr) Cac-GFP photobleaching. In the 0 hr timepoint, the neuromuscular junction (NMJ) is outlined with a white dotted line. (B) Representative images of the NMJ shown in panel A (left column) after fixation – NMJ area outlined in white. Right column is an NMJ from an unbleached segment of the same animal, used as a control. (C) Active zone (AZ) number (number of GluRIIA-RFP puncta) in pre-bleach and 24 hr post-bleach NMJs, with fold-increase in AZ number shown above (pre: 57.31±2.198, n=16 NMJs; 24H: 106±4.716, n=14 NMJs, p<0.0001). (D) Average AZ Cac fluorescence (intravital) for each NMJ pre-bleach and immediately after bleaching (0H), measured as sum pixel intensity of an ROI encompassing each GluRIIA-positive receptor field (pre: 158,514±8197; 0H: 7609±446.8, p<0.0001). (E, F) Correlations of Cac-GFP and GluRIIA-RFP at individual AZs in unbleached controls (E) and 24 hr post-bleach (F). (G) Pearson r value for the correlation of Cac-GFP and GluRIIA intensity across the AZ population at each NMJ in control segments (unbleached; UNBL) and 24 hr post-bleach (UNBL: 0.792±0.01618, n=6 NMJs; 24H: 0.7222±0.02212, n=12 NMJs).
Figure 6—figure supplement 1—source data 1. Source data for Figure 6—figure supplement 1.