Fig. 5: 4Pi-SIM revealed the 3D remodeling of the ER.

a, Left: 4Pi-SIM image (maximum intensity projection) of the ER (oxStayGold-Sec61β and oxStayGold-KDEL) in a live COS-7 cell (Supplementary Video 12). Right: magnified views of the yellow boxed region at 8,462 s and 15,824 s, respectively. b–d, Top: time-lapse magnified views of the boxed regions denoted in a, showing that a small hole appeared and spread within the ER sheet (b), that an ER sheet transformed into tubules (c) and that an ER nanohole formed (d). Bottom: the x–z cross-sections at positions denoted by the arrowheads. e, Left: 4Pi-SIM image (maximum intensity projection) of ER in a live COS-7 cell. Right: magnified views of the yellow boxed region at 0 s and 9,084 s (Supplementary Video 13). f,g, Top: time-lapse magnified views of the boxed regions denoted in e, showing the formation of ER sheets either extending from pre-existing small sheets (f) or individual tubules (g). Bottom: the x–z cross-sections at positions denoted by the arrowheads. h, 4Pi-SIM image (maximum intensity projection) of ER at a volume rate of 0.7 Hz (Supplementary Video 14). i, Left: time-lapse magnified views of the boxed regions denoted in h, showing the retraction of an ER tubule. Right: the y–z cross-sections at positions denoted by the arrowheads. j,k, Top: time-lapse magnified views of the boxed regions denoted in h, showing the growth of ER tubules as they form stable connections (j) or tri-junctions (k) with other tubules. Bottom: the x–z cross-sections at positions denoted by the arrowheads. Color represents depth denoted by the color bars. Scale bars: 2 μm (a,e,h); 1 μm (b–d,f,g,j,k); and 500 nm (i). The thickness of all cross-section slices is 30 nm. Representative results are shown from four to six independent experiments.