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. 2006 Oct 9;175(1):17–23. doi: 10.1083/jcb.200606021

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Copyright © 2006, 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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Figure 4. — Chromosome capture and biorientation are inhibited in tub2-V169A and -C354A cells. (A–C) Cells contain YFP-Tub1, P_GAL1-CEN3-GFP, and P_MET3-CDC20 (T3531, CUY1858, and CUY1859). (A) Cells were treated with α factor for 3 h in medium containing glucose and lacking methionine and were shifted to medium lacking α factor and containing glucose and 2 mM methionine. Aliquots of cells were fixed in 3.7% formaldehyde at 10-min time intervals for imaging. Images show cells with unseparated and separated CENs associated with preanaphase spindles. (B) Cells were treated with α factor for 3 h in medium containing raffinose and lacking methionine and were shifted to medium containing galactose and 2 mM methionine. After 3 h, cells were transferred to medium containing glucose and 2 mM methionine (defined as t = 0). Aliquots of cells were fixed at the indicated times. The percentage of cells with free CEN3, CEN3 on a captured microtubule, CEN3 on the spindle but not separated, and CEN3 on the spindle and separated was determined. The ratio of separated to unseparated CENs on the spindle versus time is shown in the bottom graph. (C) Images of live cells subjected to the procedure described in B were captured at 30-s intervals. The zero time point is defined as the time when the captured CEN reaches the spindle. Top panel shows a TUB2 cell in which the CENs separated 210 s after reaching the SPB. Middle panel shows a tub2-V169A cell in which CENs separated 540 s after reaching the SPB. Bottom panel shows a tub2-C354A cell in which the CENs did not separate within 900 s after reaching the SPB. (A and C) Arrowheads indicate CEN-GFP dots. Videos 4–6 (available at http://www.jcb.org/ cgi/content/full/jcb.200606021/DC1) show the entire time course for these cells. Bars, 1 μm.