Figure 5.

VAP coexpression triggers Nir2 recruitment to ER–PM junctions mediated by Kv2.1 in live cultured rat hippocampal neurons. A, SDC optical sections taken at the basal surface of a cultured rat hippocampal neuron transfected with mCherry–Nir2. The scale bar is 5 μm and holds for all images. B, SDC optical sections taken at the basal surface of a cultured rat hippocampal neuron cotransfected with mCherry–Nir2 (red) and GFP–VAPA (green). Note the robust colocalization of GFP–VAPA with mCherry–Nir2. Line scan analysis of mCherry–Nir2 and GFP–VAPA intensity from selection indicated in merged image of B is shown to the right of B. C, SDC optical sections taken at the basal surface of a cultured rat hippocampal neuron cotransfected with mCherry–Nir2 (red) and CFP–Kv2.1 (blue). Note the lack of minimal colocalization of mCherry–Nir2 with CFP–Kv2.1. Line scan analysis of mCherry–Nir2 and CFP–Kv2.1 intensity from selection indicated in merged image of C is shown to the right of C. D, SDC optical sections taken at the basal surface of a cultured rat hippocampal neuron cotransfected with mCherry–Nir2 (red), GFP–VAPA (green), and CFP–Kv2.1 (blue). Note the robust colocalization of mCherry–Nir2 with GFP–VAPA and CFP–Kv2.1. Line scan analysis of mCherry–Nir2, GFP–VAPA, and CFP–Kv2.1 intensity from selection indicated in merged image of D is shown to the right of D. E, enlarged images of mCherry–Nir2 (red), GFP–VAPA (green), and CFP–Kv2.1 (blue) from selection indicated in merged image of D. F, summary graph of Pearson's correlation coefficient values between mCherry–Nir2 and GFP–VAPA or CFP–Kv2.1, measured from cultured rat hippocampal neurons transfected with mCherry–Nir2 and CFP–Kv2.1 (circles) or mCherry–Nir2, CFP–Kv2.1, and GFP–VAPA (triangles). Bars are mean ± S.D. (VAPA: ****, p value = 0.0001, n = 16 cells; Kv2.1: ****, p value = 0.0001, n = 17 cells; ordinary one-way ANOVA followed by Dunnett's multiple comparisons test).