Fig. 3: mScarlet3-S2 enables dual-color 3D STED.
a, A 3D STED reconstruction of the nuclear lamina (mScarlet3-S2, left) and ER (HaloTag-conjugated BD626HTL, right) in fixed U-2 OS cells. Cells were cotransfected with HaloTag-Sec61β and mScarlet3-S2-laminA/C. After 24 h, cells were incubated with 10 nM BD626HTL for 30 min at 37 °C, washed twice with PBS and fixed with 4% paraformaldehyde. Scale bar, 5 μm. b, Imaris-reconstructed dual-color 3D image of the ER (pink) and nuclear membrane with NR channels (cyan). Scale bar, 5 μm. c, Enlarged view of the blue rectangle in b showing simultaneous invagination of the ER and nuclear lamina. Scale bar, 5 μm. d, Enlarged view of the rectangle in b revealing NR channels of varying diameters and morphologies. Scale bar, 5 μm. e, XY, XZ and YZ cross-sections of U-2 OS cells co-expressing HaloTag-Sec61β and mScarlet3-S2-laminA/C, with arrows marking ER-only protrusions. Scale bar, 5 μm. The experiment was independently repeated twice, with three technical replicates each, yielding similar results. f, Imaris-reconstructed ER–NE junctions at the top and bottom of the nucleus (left). Scale bar, 5 μm. Magnified views of ER–NE junctions (yellow boxes) displaying ribbon-like (1), branched ribbon (2) and punctate (3) morphologies (right). Area ratios of ER–NE junctions to nuclear membranes (top versus bottom) are shown in the lower right corner. Surface plots of ER and lamina were generated using Imaris software, and the top and bottom views of the rendered images were captured using the snapshot tool. The areas of ER and lamina were measured in ImageJ, and their area ratio was subsequently calculated. Significance was calculated by two-tailed Student’s t-test; *P < 0.05 (P = 0.0438). Scale bar, 1 μm. g, Imaris-reconstructed of planar three-way ER junctions (top) and nonplanar three-way/four-way ER junctions (bottom). Scale bar, 1 μm. The excitation and emission wavelengths are 561 nm (excitation) and 571–630 nm (emission) for red, and 640 nm (excitation) and 650–755 nm (emission) for far red, respectively.
