Figure 8: GluA2–PlexA interaction in the somatodendritic region is required for Sema3A-induced dendritic localization of GluA2.
(a) Overexpression of PlexA1-DN (A1-DN) attenuated Sema3A-enhanced immunostaining of both GluA2 and PlexA4 in dendrites. Overexpression of PlexA1 IPT domain (A1-IPT) attenuated Sema3A-enhanced immunostaining of GluA2 without modifying that of PlexA4 (n=33–104 neurons from more than three independent cultures). To discriminate between endogenous PlexA4 and exogenous PlexA mutants, we utilized mutant forms of PlexA1 instead of PlexA4 that is recognized by the anti-PlexA4 antibody (see text). (b) Overexpression of A1-IPT suppressed the number of PlexA4–GluA2 interaction signals in the soma of the neurons without Sema3A. The A1-IPT also suppressed the number of signals in the soma and dendrites treated with Sema3A for 30 min. The mean number of PlexA4–GluA2 interacting signals per 10 μm2 (soma) or 10 μm (dendrite) is shown in c (n=30 neurons from three independent cultures). (d) Knockdown of GRIP1 suppressed Sema3A-induced increase in the number of PlexA4-GluA2 interacting signals in the dendrites but not soma. The mean number of PlexA4–GluA2 interacting signals per 10 μm2 (soma) or 10 μm (dendrite) is shown in e (n=44–45 neurons from three independent cultures). (f) Pretreatment with sPlexA1-IPT, using the microfluidic culture system, in the somatodendritic region, but not the axonal region, attenuated the enhanced immunostaining of GluA2 by Sema3A stimulation in the axonal region. Magnified images of boxed areas in upper panels are shown in lower panels. Arrowheads indicate axon. Normalized mean immunostaining intensity of dendrite per neuron is shown in g (n=15–38 neurons from more than three independent cultures). Scale bars in b, d, 20 μm; f, 20 μm (upper panel); 10 μm (lower panel). The representative images of a are shown in Supplementary Fig. 8c,d. *P<0.05, **P<0.01 by ANOVA compared with vehicle control. ##P<0.01 by ANOVA compared with EGFP only. Data are presented as mean±s.e.m.
