Journal: British Journal of Cancer

Article Title: Loss of Coxsackie and adenovirus receptor downregulates α -catenin expression

doi: 10.1038/sj.bjc.6605331

Figure Lengend Snippet: Impact of CAR and α -catenin on cellular morphology. Cells after CAR knockdown appear round and smaller compared with controls (DLD1) or display less intercellular attachment sites (IEC-6). In matrigel, cells after CAR knockdown form amorphous clusters in contrast to the organised-appearing formations of matching controls. Ectopic ‘re’-expression of α -catenin partially reverses the effects seen after CAR knockdown when grown as a monolayer, and particularly results in organised cell formations similar to those of vector controls when cultured in matrigel. Images show representative results for DLD1 ( A ) and IEC-6 ( B ).

Article Snippet: The human colon cancer cell line DLD1 and the rat small intestine cell line IEC-6 were obtained from the American Type Culture Collection (Rockville, MD, USA) and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively, and were cultured in recommended growth media.

Techniques: Knockdown, Expressing, Plasmid Preparation, Cell Culture

Journal: British Journal of Cancer

Article Title: Loss of Coxsackie and adenovirus receptor downregulates α -catenin expression

doi: 10.1038/sj.bjc.6605331

Figure Lengend Snippet: Functional impact of CAR downregulation and α -catenin ‘re’-expression in DLD1 and IEC-6 cell lines. Proliferation ( A ), migration ( B ), and invasion ( C ) in DLD1 and IEC-6 were determined after CAR knockdown and ‘re’-expression of α -catenin in comparison with controls.

Article Snippet: The human colon cancer cell line DLD1 and the rat small intestine cell line IEC-6 were obtained from the American Type Culture Collection (Rockville, MD, USA) and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively, and were cultured in recommended growth media.

Techniques: Functional Assay, Expressing, Migration, Knockdown, Comparison

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Misaligned chromosomes outside the exclusion zone are ensheathed in endomembranes. (A) Confocal image of a mitotic RPE-1 cell stably coexpressing GFP-Sec61β (green) Histone H3.2-mCherry (DNA, red) and stained with SiR-Tubulin (gray). Scale bar, 10 µm. (B) SBF-SEM imaging of mitotic cells and subsequent segmentation reveals the endomembranes (ER, blue) and mitochondria (Mito, orange) beyond the exclusion zone boundary (EZ, pink), with the chromosomes (DNA, gray) within. Angle of rotation about y axis is shown. Scale bar, 2 µm. (C) Confocal image of an untreated HeLa cell coexpressing GFP-Sec61β (green) and Histone H2B-mCherry (magenta) with a spontaneously occurring ensheathed chromosome. (D) SBF-SEM imaging of an untreated HeLa cell with a spontaneously occurring ensheathed chromosome. Model shows the position of two ensheathed chromosomes (red) away from the metaphase plate; height of slice 232 is indicated. Scale bar, 2 µm. Segmentation shows endomembranes (green and lilac surrounding the chromosome marked with a star), rendered in 3D (reconstruction). Scale bars, 1 µm. See and .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Induction of misaligned chromosomes in stably diploid RPE1 cells by pretreatment with a CENP-E inhibitor. (A) Polar, misaligned chromosomes can be induced by treatment with CENP-E inhibitor GSK923295 (150 nM, 3 h) and subsequent washout (1 h). (B) Confocal micrographs to show that these misaligned chromosomes (SiR-DNA, red) are either outside the exclusion zone delineated by GFP-Sec61β (green), termed ensheathed, or at the boundary and inside the exclusion zone, termed free. Scale bars, 10 µm; 1 µm (inset). (C) Spatially averaged 3D view of all CENP-C–positive kinetochores in the dataset; see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (free, blue). Spindle poles are shown in black. (D) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Chromosome misalignment was induced by pretreatment with GSK923295 (150 nM). Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log2 scale. Dots represent kinetochore ratios from 31 RPE-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Inset: Schematic diagram to show how the position of kinetochores relative to the exclusion zone boundary was calculated. C is the centroid of aligned kinetochores, P is a kinetochore, and Q is the point along the 3D path ( CP ) that intersects the exclusion zone boundary. The ratio of CP to CQ is taken for each kinetochore (aligned kinetochores, gray; free, blue; and ensheathed, orange). (E) Single SBF-SEM image showing an ensheathed chromosome. Boxed region is shown expanded and modeled (zoom). Single slice and a 3D model (bottom right) of slices 87–126 are shown. Scale bar, 2 µm (black) and 500 nm (white). (F) Modeled substacks from SBF-SEM images showing a chromosome outside the exclusion zone, ensheathed in ER. Slices shown and angles and axes of rotation are indicated (see ). Scale bar, 2 µm.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes in DLD-1 cells after targeted missegregation of Y-chromosome. (A) Schematic diagram after , showing how reexpression of a CENP-A mutant (C-H3) in DLD-1 cells where CENP-A is degraded causes selective misalignment of the Y-chromosome. WT and C-H3 lines were further modified to express mCherry-Sec61β. (B) Western blot of lysates from WT or C-H3 DLD-1 cells treated with doxycycline (Dox) and/or indole-3-acetic acid (IAA) as indicated. Upper blot shows anti-CENP-A detection of endogenous CENP-A fused to EYFP-AID tag (66 kD) and expression of untagged CENP-A (either WT or C-H3). Lower blot shows GAPDH loading control. (C) Typical FISH images locating the Y-chromosome in the main nucleus in control cells and in a micronucleus in cells expressing C-H3 CENP-A. Scale bar, 10 µm. (D) Western blot of lysates from stable cell lines expressing mCherry-Sec61β derived from WT (G2) or C-H3 (G11). Detection of Sec61β or mCherry is shown as indicated with actin loading controls. Migration of Sec61β and mCherry-Sec61β is indicated by black and red arrowheads, respectively. Note that the expression of mCherry-Sec61β downregulates endogenous Sec61β. (E) Deconvolved wide-field microscopy images showing an ensheathed chromosome in G11 cells but not in G2 cells treated with Dox/IAA. Scale bars, 10 µm; 2 µm (insets). (F) Spatially averaged view of all kinetochores in the G11 DLD-1 Dox/IAA dataset (see Materials and methods). Small gray points represent kinetochores at the metaphase plate. Colored points represent misaligned chromosomes that were ensheathed (orange) and those that were not (blue). Spindle poles are shown in black. (G) Box plot to show the relative position of each kinetochore relative to the exclusion zone boundary. Ratio of kinetochores within the exclusion zone are <0 and those within the ER are >0 on a log 2 scale. Dots represent kinetochore ratios from 50 DLD-1 cells at metaphase. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. Source data are available for this figure: .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Mutagenesis, Modification, Western Blot, Expressing, Stable Transfection, Derivative Assay, Migration, Microscopy

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Impact of ensheathed chromosomes on cell division. (A) Mitotic timing of RPE-1 cells. Cumulative frequencies for NEB to metaphase (NEB-Meta) and metaphase to anaphase (Meta-Ana) are shown. RPE-1 stably expressing GFP-Sec61β were treated with 150 nM GSK923295 for 3 h before washout. Three classes of metaphase were seen: all chromosomes aligned (Aligned, n = 29), cells with one or more free chromosomes (Free, n = 11), and cells with one or more ensheathed chromosome (Ensheathed, n = 107). Timing of untreated parental (Parental, n = 69) and stable RPE-1 (Control, n = 52) cells is also shown. Inset in Meta-Ana shows same data on an expanded time scale. Comparison of NEB-Meta and Meta-Ana timing distributions for ensheathed vs. control, P = 1.9 × 10 −57 and 7.8 × 10 −23 , Kolmogorov–Smirnov test. (B) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, red) at kinetochores (CENP-C, blue) in cells stably expressing GFP-Sec61β (green); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (C) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the GFP-Sec61β signal. Dots represent kinetochores, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: n A = 132, n F = 30, n E = 37; (Mad2: n A = 103, n F = 20, n E = 31). (D) Stills from live-cell imaging experiments to track Mad2 levels at kinetochores of ensheathed chromosomes. A GSK923295-pretreated RPE-1 cell is shown, stably coexpressing GFP-Mad2 (green) and mCherry-Sec61β (red); DNA is stained using SiR-DNA (blue). Time relative to anaphase is shown in minutes. Insets show 2× zoom of the indicated ROI. Scale bars, 10 µm; 2 µm (insets). (E) Quantification of live Mad2 imaging experiments. Kaplan–Meier plot to show congression times of the last misaligned chromosome to align. Measurement of mCherry-Sec61β (mean ± SD) and GFP-Mad2 is shown for the misaligned that congressed and those that were missegregated (misseg). A linear regression fit with 95% confidence intervals is shown for GFP-Mad2. All plots are shown in time (minutes) relative to anaphase onset. Total cells with misaligned chromosomes, n = 72; cells where all chromosomes congressed, n = 56; and where there was missegregation, n = 16.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Expressing, Immunofluorescence, Staining, Live Cell Imaging, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Spindle assembly checkpoint and micronucleus formation in DLD-1 cells. (A) Micrographs of immunofluorescence experiments to detect Bub1 or Mad2 (SAC, green) at kinetochores (CENP-C, blue) in cells stably expressing mCherry-Sec61β (red); DAPI-stained DNA is shown in gray. Scale bars, 10 µm; 2 µm (insets). (B) Quantification of Bub1 and Mad2 immunofluorescence at kinetochores marked by CENP-C. Ensheathed chromosomes were classified using the mCherry-Sec61β signal. Dots show kinetochore measurements, boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles (Bub1: n A = 52, n F = 49, n E = 52; (Mad2: n A = 55, n F = 57, n E = 55). (C) Stills from a video showing an example of ensheathed chromosomes in G11 DLD-1 cells forming micronuclei following Dox/IAA treatment. Scale, 10 µm.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes do not have stable microtubule-kinetochore attachment. (A) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for tubulin (red) and CENP-C (green); DNA stained with DAPI. Examples show end-on attachments at aligned kinetochores and potential lateral kinetochore-MT contacts for ensheathed chromosomes. (B) Micrographs of RPE-1 cells stably expressing GFP-Sec61β (gray) pretreated with GSK923295 immunostained for kinastrin (red) and CENP-C (green); DNA stained by DAPI. Scale bars, 10 µm; 2 µm (insets). (C) Frequency distributions of the proximity of the nearest kinastrin punctum to each kinetochore (CENP-C punctum). Kinetochores (n, % with kinastrin <600 nm): aligned (3,124, 26.8%); free (74, 4.1%); ensheathed (227, 6.2%). ( D and E ) Still images from live-cell imaging experiments of RPE-1 cells stably expressing GFP-Sec61β (green) and Histone H3.2-mCherry (gray), pretreated with 150 nM GSK923295 and stained with SiR-Tubulin (red). Similar results were recorded in 25 cells with free chromosomes and 16 cells with ensheathed chromosomes. Scale bars, 10 µm; 2 µm (insets).

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Expressing, Staining, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Ensheathed chromosomes promote formation of micronuclei. (A) Stills from live-cell imaging experiments to track the fate of ensheathed chromosomes. A control or GSK923295-pretreated GFP-Sec61β RPE-1 cell is shown; DNA is stained using SiR-DNA (red). Scale bars, 10 µm; 2 µm (insets). Shown in and . (B) Sankey diagram to show the fate (right) of cells in each of the three metaphase classes (left). Fates include normal division, micronucleus formation, death, and other defects (lagging chromosome, cytokinesis failure). Note that the fate of cells (and not chromosomes) is tracked. A cell with three misaligned chromosomes, only one of which is ensheathed, is classified as ensheathed. Parental RPE-1 cells (Parental, n = 92) and untreated RPE-1 stably expressing GFP-Sec61β (Control, n = 69) are from two and three independent overnight experiments, respectively. Fates of GSK923295-pretreated GFP-Sec61β cells ( n = 186) were compiled from seven experiments. Fates of individual chromosomes are shown in .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Live Cell Imaging, Staining, Stable Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Missegregation of an ensheathed chromosome results in a micronucleus with a disrupted NE. (A) Confocal images showing examples of an intact or a disrupted micronucleus as indicated. Images show mCherry-BAF or LBR-mCherry (red) stably coexpressed with GFP-Sec61β (green) in RPE-1 cells; H3K27ac was detected by immunofluorescence (blue), and DNA was stained with DAPI. XY view is through the center of the micronucleus; YZ (right) and XZ (below) are orthogonal views at the positions indicated. Scale bar, 10 µm. (B) Scatter plots to show the fluorescence intensity of H3K27ac (blue) and either mCherry-BAF or LBR-mCherry (red) vs. GFP-Sec61β intensity. Data are plotted as the log 2 ratio of intensity at the micronucleus vs. main nucleus. For RPE1 GFP-Sec61β mCherry-BAF, n = 71 cells, and LBR-mCherry, n = 73 cells, from three independent experiments in each cell type.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Stable Transfection, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: 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.

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Immunofluorescence, Expressing, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Rescue of ensheathed chromosomes by the induced relocalization of ER. (A) Stills from live-cell imaging of ER clearance experiments. FKBP-GFP-Sec61β (green), Stargazin-mCherry-FRB (red), and SiR-DNA (gray) are shown. Insets are 2× expansions of the ROI shown. Scale bars, 10 µm; 1 µm (insets). See . (B) Semiautomated 4D tracking of misaligned chromosome location is used to monitor congression. Two tracks from the cells in A are shown. The shortest Euclidean distance from the centroid of the misaligned chromosome to the edge of the main chromosome plate is plotted as a function of time. (C) Fate of misaligned chromosomes in control or rapamycin-treated cells. Rescue of misaligned chromosomes was detected in 26 of 30 rapamycin-treated cells. Coloring in B and C is with the color scale shown. Tracks terminate at 90 min or when the chromosome merges with the plate. Median termination time was 93 min (control, n = 36) and 45 min (rapamycin, n = 30); P = 7.1 × 10 −9 , Wilcoxon rank test. Rapamycin is applied after the first frame ( T = 0).

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

doi: 10.1083/jcb.202203021

Figure Lengend Snippet: Stable transgene expression in RPE1 cells and fate of misaligned chromosomes in RPE1 cells stably expressing GFP-Sec61β. (A–D) Western blots to examine expression of proteins in parental RPE1 cells or clonal cells stably expressing GFP-Sec61β alone or with Histone3.2-mCherry, LBR-mCherry, or mCherry-BAF, as indicated. Membranes were probed for GFP, Sec61β, mCherry, LBR, BAF. Actin or tubulin is shown as a loading control. Green or red arrowheads indicate the expected position of GFP- or mCherry-tagged protein; black arrowheads indicate the untagged protein. (E) Mitotic timing of RPE1 cells stably expressing transgenes. Cumulative frequencies for NEB to metaphase, metaphase to anaphase, and NEB to anaphase are shown. Parental, n = 69; GFP-Sec61β alone, n = 52; GFP-Sec61β and LBR-mCherry, n = 66; GFP-Sec61β and mCherry-BAF, n = 51. (F) Sankey diagram to show the fate (right) of RPE1 cells in each of the three metaphase classes (left). Fates include normal division, micronuclei formation, death, and other defects (lagging chromosome, cytokinesis failure). Note that the fate of cells (and not chromosomes) is tracked. LBR-mCherry/GFP-Sec61β, n = 51; mCherry-BAF/GFP-Sec61β, n = 67; pooled from three experiments. (G) Sankey diagram to show the fate (right) of chromosomes in each of the three metaphase classes (left) after GSK923295 pretreatment. Fates include rescue, micronuclei formation, death, and other defects (lagging chromosome, cytokinesis failure). Number of chromosomes: free, 146; ensheathed, 207; lagging, 9. The same dataset was analyzed for the outcome of cells (classified by the final misaligned chromosome) in . Note that ensheathed chromosomes at metaphase that were rescued all became “free” chromosomes before rescue. Source data are available for this figure: .

Article Snippet: DLD-1-WT mCherry-Sec61β and DLD-1-C-H3 mCherry-Sec61β stable cell lines were generated by GeneJuice (Merck Millipore) transfection of mCherry-Sec61β into the respective parental lines.

Techniques: Expressing, Stable Transfection, Western Blot