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. 1999 Sep 6;146(5):941–954. doi: 10.1083/jcb.146.5.941

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9904020.f3a.jpg — hBUBR1 is essential for cells to proceed normally through mitosis. (A) Synchronized Hela cells released from the G1/S boundary were injected with nonimmune or hBUBR1 antibodies and sampled at 12 or 16 h later. (rows 1–3) 12 h after release from the G1/S boundary, injected cells entered mitosis. (row 1) Metaphase cell injected with nonimmune IgG (middle) stained for endogenous hBUBR1 (right). (rows 2 and 3) Prometaphase and anaphase cells injected with hBUBR1 antibodies (middle) and stained for CENP-E (right). (row 4) 16 h after release from the G1/S boundary, hBUBR1 injected cells (middle) divided with lagging chromosomes trapped in the cleavage furrow. Centromeres (arrowheads) stained with ACA and visualized with Texas red anti-human secondary antibodies. Insets in rows 3 and 4 are longer exposures and enlargements of boxed areas. Injected antibodies were visualized with Cy5 anti-rabbit secondary antibody (middle). Endogenous hBUBR1 was stained with rat anti–hBUBR1 and detected with Cy2 anti-rat (row 1, right). CENP-E was stained with mouse monoclonal anti-CENP-E (mAb177) and detected with Cy2 anti-mouse secondary antibodies (rows 2 and 3, right). Chromosomes and nuclei were stained with DAPI (left). (B) 16 h after release from the G1/S boundary, nonimmune IgG– and hBUBR1 antibody–injected cells were scored for mitotic (open bar), normal telophase (dotted bar) or aberrantly divided cut (checkered bar) based on a combination of DAPI staining and phase-contrast images. Histogram compares relative percentages of each of the three phenotypes. The plotted values are the means plus SD from at least three experiments. (C) Cells expressing gfp:hBUBR1Δkinase exit mitosis with prematurely. Synchronized Hela cells transfected with gfp:hBUBR1Δkinase enter mitosis (top row) 12 h after release from the G1/S boundary. By 16 h, most cells expressing the kinase mutant produced cells that divided with lagging chromosomes trapped in the cleavage furrow (bottom). Bars, 10 μm.

graphic file with name JCB9904020.f3b.jpg — hBUBR1 is essential for cells to proceed normally through mitosis. (A) Synchronized Hela cells released from the G1/S boundary were injected with nonimmune or hBUBR1 antibodies and sampled at 12 or 16 h later. (rows 1–3) 12 h after release from the G1/S boundary, injected cells entered mitosis. (row 1) Metaphase cell injected with nonimmune IgG (middle) stained for endogenous hBUBR1 (right). (rows 2 and 3) Prometaphase and anaphase cells injected with hBUBR1 antibodies (middle) and stained for CENP-E (right). (row 4) 16 h after release from the G1/S boundary, hBUBR1 injected cells (middle) divided with lagging chromosomes trapped in the cleavage furrow. Centromeres (arrowheads) stained with ACA and visualized with Texas red anti-human secondary antibodies. Insets in rows 3 and 4 are longer exposures and enlargements of boxed areas. Injected antibodies were visualized with Cy5 anti-rabbit secondary antibody (middle). Endogenous hBUBR1 was stained with rat anti–hBUBR1 and detected with Cy2 anti-rat (row 1, right). CENP-E was stained with mouse monoclonal anti-CENP-E (mAb177) and detected with Cy2 anti-mouse secondary antibodies (rows 2 and 3, right). Chromosomes and nuclei were stained with DAPI (left). (B) 16 h after release from the G1/S boundary, nonimmune IgG– and hBUBR1 antibody–injected cells were scored for mitotic (open bar), normal telophase (dotted bar) or aberrantly divided cut (checkered bar) based on a combination of DAPI staining and phase-contrast images. Histogram compares relative percentages of each of the three phenotypes. The plotted values are the means plus SD from at least three experiments. (C) Cells expressing gfp:hBUBR1Δkinase exit mitosis with prematurely. Synchronized Hela cells transfected with gfp:hBUBR1Δkinase enter mitosis (top row) 12 h after release from the G1/S boundary. By 16 h, most cells expressing the kinase mutant produced cells that divided with lagging chromosomes trapped in the cleavage furrow (bottom). Bars, 10 μm.