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. 2018 Oct 1;29(20):2410–2432. doi: 10.1091/mbc.E18-05-0337

FIGURE 2:

FIGURE 2:

Exogenous Kv2 expression remodels ER–PM junctions in HEK293T cells and cultured rat hippocampal neurons. (A, B) TIRF images of live HEK293T cells expressing DsRed2-ER5 (magenta) either alone (A) or coexpressed with (in green) GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4, as indicated (B). Scale bar in A is 5 µm and holds for all panels. (C) Graph of mean ER–PM junction (EPJ) size per cell measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 or expressing DsRed2-ER5 alone (control). (D) Graph of percentage of the PM area per cell occupied by cortical ER measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 or expressing DsRed2-ER5 alone (control). (E) Graph of PCC between DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv constructs. The dashed line denotes a PCC of 0.5. Bars on all graphs are mean ± SD. See Supplemental Tables 1–3 for values and statistical analyses for C–E. (F) TIRF image of a live CHN expressing DsRed2-ER5 (magenta) alone. (G) TIRF image of a live CHN coexpressing DsRed2-ER5 (magenta) and GFP-Kv2.2 (green). Scale bar in DsRed2-ER5 panel is 10 µm and holds for all panels in that row. Magnified images are shown in bottom row. Scale bar in DsRed2-ER5 magnified inset panel is 2.5 µm and holds for all panels in that row. (H) Scatterplot shows sizes of Kv2.2 clusters and associated ER–PM junctions (EPJs, as indicated by DsRed2-ER5 in TIRF) in HEK293T cells (red points) and CHNs (black points). n = 3 cells each.