Membrane-bound extracellular molecules of acetylcholinesterase (AChE) are clustered and co-localized with clustered neurexins in primary neurons. A-1. Confocal microscopic images illustrating the intracellular localization of immunofluorescence of neurexins (Nrxn, left column) and membrane-bound extracellular AChE (middle column) in cultured hippocampal neurons under control conditions (top panel) and in neurons treated with the AChE inhibitor BW284c51 (lower panel). The right column illustrates the immunofluorescence overlay of the two proteins. Insets under each panel illustrate the subcellular distribution of the two proteins. Note co-localization of neurexin and AChE immunofluorescent clusters in neurites. A-2a. Total area (pixels) of AChE-immunoreactive clusters in control and BW284c51-treated neurons (control: 356 ± 100, n = 10 image fields; BW284c51: 682 ± 78, n = 10 image fields; P < 0.05). A-2b. Fraction of colocalization of AChE clusters with Nrxn clusters (control: 0.6 ± 0.08, n = 12 image fields; BW284c51: 0.82 ± 0.07, n = 12 image fields; P < 0.05). B-1. Immunoblots showing increase of AChE and decrease of neurexin in BW284c51-treated neurons. B-2. BW284c51treatment increased the total quantity of AChE (140% ± 11% of control) but decreased the total amount of neurexin (59% ± 10% of control). C. Co-immunoprecipitation assay of lysates of control neurons and neurons treated with BW284c51. The immunoprecipitating AChE co-precipitates with neurexin (Nrxn, middle lane), and BW284c51 enhances this co-precipitation (right lane). D. Assay for co-immunoprecipitating AChE using a neurexin antibody from lysates of control neurons under various control conditions. From left to right, lane 1: control neuron lysate without anti-neurexin antibody (anti-Nrxn); lane 2: control neuron lysate with IgG; lane 3: control neuron lysate with anti-neurexin; lane 4: BW284c51-treated neuron lysate with anti-neurexin. Note co-precipitation of AChE in lane 3 and increase in co-precipitation of AChE in BW284c51-treated neurons.