Figure 6.
Inducible relocalization of ER in mitotic cells. (A) Schematic diagram of the ER clearance procedure. Rapamycin induces the heterodimerization of the ER-resident FKBP-GFP-Sec61β and the plasma-membrane localized Stargazin-mCherry-FRB. (B) Cumulative histogram showing the time to detection of ER clearance. An automated segmentation procedure was used to monitor ER localization in mitotic cells. The time at which the largest decrease in ER localization occurred was taken (n = 35−37, see Materials and methods). Random occurrence is shown for comparison. The median (IQR) ER clearance time in rapamycin-treated cells was 15 (12–24) min; rapamycin is applied after the first frame (T = 0). (C) Induced relocalization of FKBP-GFP-Sec61β to the plasma membrane causes ER clearance. Typical immunofluorescence micrographs of mitotic HCT116 cells pretreated with GSK923295, expressing FKBP-GFP-Sec61β (green) and Stargazin-mCherry-FRB (blue), treated or not with rapamycin (200 nM). Cells were stained for ER markers KDEL or Calnexin as indicated (red), DNA was stained with DAPI (gray). Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). (D) SBF-SEM imaging of control or ER-cleared (rapamycin) mitotic HCT116 cells. A single slice is shown with segmentation of ER (green), plasma membrane (yellow), mitochondria (blue), and chromosomes (red). Scale bars, 5 µm; 1 µm (insets). Insets are 2× expansions of the indicated ROI shown without segmentation; green arrowheads indicate ER attachment to the plasma membrane.

Inducible relocalization of ER in mitotic cells. (A) Schematic diagram of the ER clearance procedure. Rapamycin induces the heterodimerization of the ER-resident FKBP-GFP-Sec61β and the plasma-membrane localized Stargazin-mCherry-FRB. (B) Cumulative histogram showing the time to detection of ER clearance. An automated segmentation procedure was used to monitor ER localization in mitotic cells. The time at which the largest decrease in ER localization occurred was taken (n = 35−37, see Materials and methods). Random occurrence is shown for comparison. The median (IQR) ER clearance time in rapamycin-treated cells was 15 (12–24) min; rapamycin is applied after the first frame (T = 0). (C) Induced relocalization of FKBP-GFP-Sec61β to the plasma membrane causes ER clearance. Typical immunofluorescence micrographs of mitotic HCT116 cells pretreated with GSK923295, expressing FKBP-GFP-Sec61β (green) and Stargazin-mCherry-FRB (blue), treated or not with rapamycin (200 nM). Cells were stained for ER markers KDEL or Calnexin as indicated (red), DNA was stained with DAPI (gray). Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). (D) SBF-SEM imaging of control or ER-cleared (rapamycin) mitotic HCT116 cells. A single slice is shown with segmentation of ER (green), plasma membrane (yellow), mitochondria (blue), and chromosomes (red). Scale bars, 5 µm; 1 µm (insets). Insets are 2× expansions of the indicated ROI shown without segmentation; green arrowheads indicate ER attachment to the plasma membrane.

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