Figure 2.

Fluorescence-based genetic screen for tethering partners of Brp. (A) Screening strategy. The density of SVs is low in the motoneuron axon (i) and increases upon tethering to membrane-anchored Brp (ii; CD8::EGFP::Brp^C-long). RNAi-mediated knockdown of factors mediating the SV–Brp interaction should prevent the axonal accumulation of SVs. (B) Confocal images of SVs (mRFP::Syt-1) in motoneuron axons in the absence (left, CD8::EGFP) and presence (right, CD8::EGFP::Brp^C-long) of membrane-bound Brp (ok6-GAL4 driver). Cpx^RNAi prevents Brp-dependent localization of SVs in the axon, whereas other RNAi lines, e.g., syn^RNAi and dysb^RNAi, have no discernible effect (Table S3). (C) Maximal projection of confocal images showing Cpx trapped in axons of ok6>CD8:EGFP::brp^C-long (experimental condition), but not ok6>CD8:EGFP (control condition), larvae (arrows). Cpx was detected by a rb-α-Cpx antibody (Huntwork and Littleton, 2007), and GFP signals were enhanced using a ms-α-GFP antibody. Asterisks indicate unspecifically labeled tracheae. (D) Cpx layout, N-terminal domain (NTD), accessory helix (AH), central helix (CH), and C-terminal domain (CTD). Deletion of the terminal glutamine disrupts the CAAX motif in the cpx¹²⁵⁷ mutant. (E) Maximal projections of confocal stacks show colocalization of Cpx (green, α-Cpx) with SVs (magenta, α-Csp) in WT motoneuron boutons (left). Cpx staining intensity is strongly reduced in cpx¹²⁵⁷ boutons (center) and no longer matches the SV distribution (right; image taken with sixfold increase in laser power). (F) Quantification of nerve/NMJ ratio of the mRFP::Syt signal. The BRP^C-long-dependent increase of SVs in the nerve is prevented by brp^C-tip and cpx^RNAi expression (ok6-GAL4) and in the cpx¹²⁵⁷ mutant. Data are presented as mean ± SEM (n ≥ 13; Table S4). *, P ≤ 0.05 (rank sum test). Scale bars: (B) 10 µm, 2 µm (inset); (C) 30 µm; and (E) 3 µm. ns, not significant.