FIGURE 6.

NL2 can undergo dynamin-dependent regulated endocytosis in neurons. Cultured hippocampal neurons were transfected to express HA-NL2 at 9 DIV and assayed at 12 DIV. Live neurons were incubated with FITC-conjugated rat anti-HA Fab fragment for 10 min, then for 1 h with soluble NL2 ligand neurexin Nrx-AP or AP control in the presence of the dynamin inhibitor dynasore or vehicle DMSO control. Cells were fixed without permeabilization and incubated with Alexa 568-conjugated anti-rat antibody. Thus, green FITC indicates surface plus internalized HA-NL2, whereas red Alexa 568 indicates surface HA-NL2 remaining. This HA-NL2 was expressed at a higher level than the HFY-NL2 in Fig. 1, hence there is a higher level at extrasynaptic sites along dendrites, but this high expression was needed for sufficient sensitivity for the internalization assay. A, Nrxn1β-AP relative to AP control induced an obvious increase in HA-NL2 internalization. Scale bar, 20 μm. B, the fraction of initial surface HA-NL2 remaining, measured as the ratio of Alexa 568 to FITC signal normalized to the AP + DMSO control condition, differed significantly among groups (analysis of variance, p = 0.0006, *, p < 0.05; **, p < 0.001 by Tukey's multiple comparison test, n = 34–37 from two independent experiments).