Dynamic nanoscale-sized holes in ER sheets. (A–D) Overview and magnified confocal and STED images of COS-7 cell expressing Halo-KDEL. White arrowhead in right panel of B shows uniform sheet region near holes (magenta box; magnified in C). Green boxed regions show clustered tubules. Boxed regions in subsequent magnified live images may be slightly shifted because of ER movements. (E) STED images of COS-7 cells expressing SNAP-KDEL acquired every 250 nm along the z axis near the NE. Magnified STED images show nanoscale-sized holes in the NE (red box) and ER sheet adjacent to the NE (orange box). (F) STED image of fixed U2OS cells expressing SNAP-Sec61Bβ (magenta) and immunolabeled for endogenous Rtn4B/D (green). Arrow in magnified image points to a nanohole that contains Rtn4, and arrowhead points to a uniform sheet region devoid of Rtn4. (G) STED image acquisition schematic. (H) Confocal image and magnified STED image of COS-7 cell expressing SNAP-KDEL. Blue rectangular region in magnified image outlines the 4.8 × 1.2-µm region imaged live with smaller magenta boxed region of the video shown in H. (I) Select STED images from time-lapse series paired with images pseudocolored to highlight manually tracked nanoholes during time course of video. n = 24 holes of n = 1 cell. Time shown in seconds.