Figure 4
From: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1
Localization of Epac1 at the plasma membrane is sufficient and required to inhibit neurite outgrowth.
(A) Representative images of N2A cells expressing Epac1-CAAX to tether Epac1 to the PM or control Epac1; scale bar corresponds to 25 μm. (B) N2A cells expressing Epac1 or Epac1-CAAX were grown in either SFM (solid bars) or RM (open bars) for 24 hrs. Data shown are the mean ± SEM neurite lengths for three independent experiments where at least 4 images of >40 cells each were analyzed for each condition. Comparison between Epac1 (SFM) and Epac1-CAAX (SFM) was performed by Student’s t-test; ***P < 0.0001. (C) Cells expressing YFP-Epac1-R82A mutant (deficient in binding phosphatidic acid at the PM) and treated with si-ctl or si-impβ1, were analyzed by live cell imaging. Representative images show that YFP-Epac1-R82A does not localize to the PM when impβ1 is knocked down; scale bar corresponds to 25 μm. Confirmation of the inability of Epac1-R82A to accumulate at the PM in response to 1 μM 8-pCPT-AM is shown in the lower left image. Western blot confirms impβ1 knockdown. (D) Cells expressing either YFP-Epac1 or YFP-Epac1-R82A mutant and treated with either si-ctl or si-impβ1 were analyzed for neurite outgrowth after culture in serum-free medium for 24 hrs. Data analyzes were performed by Two-Way ANOVA; ***P < 0.0001.
