Figure 5.

MINKΔCNH-mediated reduction of dendritic complexity requires Rap2 activation. A, Representative images of hippocampal neurons transfected with GFP to visualize transfected cell morphology along with Flag-tagged constructs (indicated left). Scale bar, 50 μm. B, MINK ΔCNH compromises dendritic complexity. Neurons were transfected as indicated (right) and subjected to Sholl analysis (n ≥ 12 neurons for each). A significant difference between transfected groups was confirmed by a 7 × 5 mixed-effect ANOVA, with transfected group as the seven-level between-group effect and distance from the soma as the five-level within-group effect (F_(6,574) = 54.051; p < 0.0001). Post hoc tests (PLSD) determined MINK ΔCNH alone, and Rap2(ca) either alone or with MINK or MINK KD each displayed significantly fewer dendritic branch point crossings relative to GFP control (****p < 0.0001). C, Rap2 activation is required for MINK ΔCNH to reduce dendritic arborization. Neurons were transfected as indicated (right) and subjected to Sholl analysis (n ≥ 12 neurons for each). A significant difference between transfected groups was confirmed by a 6 × 5 mixed-effect ANOVA, with transfected group as the six-level between-group effect and distance from the soma as the five-level within-group effect (F_(5,504) = 31.438; p < 0.0001). Post hoc tests (PLSD) revealed expression of MINK ΔCNH alone, or MINK ΔCNH coexpressed with HA-Rap1(dn) both significantly reduced dendritic complexity relative to empty vector control (****p < 0.0001 for each relative to control). Dominant-negative Rap2 subtly decreased complexity relative to control (**p < 0.01 for each relative to control). However, when MINK ΔCNH was coexpressed with HA-Rap2(dn) the number of dendritic branch point crossings failed to decrease below the level of HA-Rap2(dn) expressed alone [p = 0.5673 for HA-Rap2(dn) expressed with MINK ΔCNH relative to HA-Rap2(dn) alone].