Figure 4

Synaptic vesicle protein accumulation occurs at clustered neurexin and correlates with levels of clustered neurexin. (A) Time-lapse images of an axon transfected with neurexin-1β-GFP (green) contacting a neuroligin-1-expressing HEK293 cell (white outline). Neurexin is recruited to contact sites within minutes and enhanced at these sites as imaging progresses. (B) Images of two axons that are transfected with neurexin-1β-GFP (green) and contact cells expressing neuroligin-1 (left, white outline) or neuroligin-1 + S-SCAM (right, white outline). At neuroligin-1-only contacts, neurexin appears both punctate (arrows) and diffuse (bars). Addition of S-SCAM enhanced neurexin-1β-GFP clustering and reduced diffuse neurexin-1β-GFP labeling at contacts (arrow). (C) Persistent sites of synaptophysin-RFP (magenta) recruitment appeared at sites of punctate neurexin-1β-GFP (green) recruitment (arrows). t = time after contact established. (D) Kymographs of neurexin-1β-GFP and synaptophysin-RFP, starting immediately after contact with a neuroligin-1-expressing cell was established. Neurexin and synaptophysin clustered simultaneously at the same sites in the axon (arrows). (E) At an individual recruitment site, synaptophysin-RFP and neurexin-1β-GFP levels were positively (r = 0.98) and significantly (P = 2.0 × 10⁻⁵) correlated over several hours of imaging. This correlation was significant for 11 of 14 co-recruitment sites that were present for at least 5 hours. (F) When combining data from all recruitment sites in the same axon, synaptophysin and neurexin integrated densities were also correlated (r = 0.91, P = 6.0 × 10⁻²⁰). Similar correlation was observed in five of seven axons. (G) When the data from all axons were pooled, neurexin and synaptophysin were positively and significantly correlated (P = 1.0 × 10⁻⁷⁴, r = 0.82). (A-C) scale bars = 10 μm, (D) scale bar = 5 μm, (E-G) correlations made from non-transformed data and plotted in log-log form for display purposes.