Fig 2: R1 and R2 tubules differentially accommodate ER proteins. (A) Representative two-color STORM image of the immunolabeled endogenous Rtn4 (green) and calreticulin (magenta) in an untransfected COS-7 cell. (B and C) Representative two-color STORM images of the immunolabeled endogenous Rtn4 (green) and expressed ER-luminal protein mEmerald-ER-3 (magenta in B) and ER-membrane protein HaloTag-sec61b (magenta in C) in COS-7 cells. (D) Representative two-color STORM image of the immunolabeled endogenous Rtn4 (green) and calnexin (magenta) in an untransfected COS-7 cell. Filled and hollow arrowheads point to examples of R2 and R1 tubules, respectively. (Scale bars, 1 µm.)

Fig 3: STORM SRM of endogenous Rtn4 unveils a structural dichotomy for ER tubules. (A) Representative STORM image of immunolabeled Rtn4 in the untransfected COS-7 cell, compared with a diffraction-limited fluorescence image (Upper Left). Filled and hollow arrowheads point to examples of R2 (Rtn double-line) and R1 (Rtn single-line) tubules, respectively. (B) Result from another COS-7 cell, highlighting a long R1 tubule at the extremity of the ER network. (C) Representative STORM image intensity (as single-molecule counts) across an R2 tubule. (D) Distribution of the STORM-determined center-to-center separations between opposing Rtn4 lines in the R2 tubules in the native COS-7 cells, U2OS cells, and cultured rat astrocytes. Whiskers and boxes show 10, 25, 50, 75, and 90% quantiles. For each data point, n = 200 local distances were pooled from five cells. (E) Representative STORM image intensity across an R1 tubule. Blue curve: fit to a Gaussian distribution of 50-nm FWHM. (F) The fractions of R1 and R2 tubules by length, observed in different cell types. Blue, R1; red, R2; gray, ambiguous. Error bars indicate SDs between n = 5 cells of each type. (G–M) Representative STORM images of immunolabeled endogenous Rtn4 in different cell lines: BS-C-1 (monkey epithelial; G), U2OS (human epithelial; H), A7r5 (rat fibroblast; J), NIH 3T3 (mouse fibroblast; K), and C2C12 (mouse myoblast; L), as well as in cultured primary astrocytes (I) and neurons (M) from rat hippocampus. (Scale bars, 1 µm.)

Fig 4: Live-cell imaging unveils fast R1–R2 remodeling and associated cotraveling of ER proteins. (A) Two-color fluorescence micrographs of Rtn4b-GFP (green) and mCherry-Climp63 (magenta) coexpressed in a COS-7 cell, shown as separate channels (Left) and an overlay (Right). (B) STORM of immunolabeled Rtn4 of the same view (cyan), overlayed with the mCherry-Climp63 micrograph (magenta). Filled and hollow arrowheads point to R2 and R1 examples, respectively. (C) Two-color micrographs of Rtn4b-GFP (green) and mCherry-Climp63 (magenta) for a small region in a living COS-7 cell at selected time points, highlighting ER-tubule outgrowth with a Climp63-free R1 tip (o1) and the ensuing extension of Climp63 into the tubule in segments (e1 to e3). The two separated color channels are shown for the final image. See also separated color channels in SI Appendix, Fig. S10 and a full series in Movie S1. (D) Another two-color live-cell dataset. (E) Selected time points for the boxed region in D. Arrowheads point to major structural changes vs. the preceding image. o1 to o4: tubule outgrowths with Climp63-free R1 tips; e1 to e4: extension of Climp63 into R1 tubules; s1 to s2: the sequential splitting of a Climp63-positive R2 segment into three R2 segments connected by Climp63-negative R1 segments; f1 and f2: fusion of an R2 tubule with two small R2 segments; t1 and t2: translational motions of two R2 segments along tubules; b1 to b3: Climp63 entering new tubule branches; w1 and w2: withdrawal of newly extended tubules. See separated color channels in SI Appendix, Fig. S11 and a full series in Movies S3 and S4. (F) Live-cell three-color imaging of Rtn4b-GFP (green), mCherry-Climp63 (magenta), and JF635-labeled HaloTag-ER-3 (yellow) in a COS-7 cell, shown as merged and separated channels. (G and H) The same region after 7.5 s (G) and 16.5 s (H), shown as merged Rtn4b-GFP/mCherry-Climp63 (Left) and HaloTag-ER-3 alone (Right). See the full series in Movie S5. (I) Live-cell three-color imaging of JF635-labeled Rtn4b-HaloTag (green), mCherry-Climp63 (magenta), and mEmerald-sec61b (yellow) in a COS-7 cell, shown as merged and separated channels. (J and K) The same region after 50 s (J) and 154 s (K), shown as merged Rtn4b-HaloTag/mCherry-Climp63 (Left) and mEmerald-sec61b alone (Right). See the full series in Movie S6. Filled and hollow arrowheads in F and I point to R2 and R1 examples, respectively. Arrowheads in G, H, J, and K point to major structural changes annotated similar to E. (Scale bars, 1 µm.)

Fig 5: Rtn4 and Climp63 jointly define the R2 tubule width. (A) A simple model of the R2 tubule based on an elliptical cross-section, in which the luminal height h is varied as the membrane curvature at the two edges is held at 1/re = 0.08 nm−1. Color represents the local curvature. (B) The expected R2 tubule width w as a function of h according to this simple model (line). Colored squares mark the experimental w for cells expressing the different Climp63 mutants in C, calculated as the STORM-determined Rtn4–Rtn4 separations minus 5-nm lipid-bilayer thickness. (C) Distribution of center-to-center separations between opposing Rtn4 lines in R2 tubules, for COS-7 cells expressing mCherry-tagged mouse Climp63 mutants of different intraluminal sizes. Whiskers and boxes show 10, 25, 50, 75, and 90% quantiles. For each data point, n = 200 local distances were pooled from five cells. (C, Inset) Schematics of the full-length (FL) and truncated Climp63 versions examined. CC1 to CC3, intraluminal coiled-coil domains; Cyto, cytosolic domain; FP, mCherry fluorescent protein; TM, transmembrane domain. (D–I) Representative STORM images of the immunolabeled endogenous Rtn4 in COS-7 cells expressing mCherry-tagged full-length (1–575) (D), 2xlumen (E), 1–437 (F), 1–336 (G), 1–301 (H), and 1–192 (I) mutants of mouse Climp63. (D–I, Insets) Epifluorescence images of the mCherry channel. Filled and hollow arrowheads point to examples of R2 and R1 tubules, respectively. In H, the arrow points to an untransfected cell in the same view, which facilitates a direct comparison. (Scale bars, 1 µm.)