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Science 339 (6123): 1071-1074

Copyright © 2013 by the American Association for the Advancement of Science

Mps1 and Ipl1/Aurora B Act Sequentially to Correctly Orient Chromosomes on the Meiotic Spindle of Budding Yeast

Régis E. Meyer1, Seoyoung Kim1,2,*, David Obeso1,2,{dagger}, Paul D. Straight3,{ddagger}, Mark Winey3, and Dean S. Dawson1,2,§

1 Department of Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
2 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309–0347, USA.


Figure 1
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Fig. 1. Ipl1 is necessary to release centromeres from the SPB in meiotic prophase. (A) Dispersion of kinetochores was monitored in wild-type (square), ipl1-mn (circle), and mps1-R170S/mps1-mn (triangle) cells carrying SPB (Spc42-DsRed) and kinetochore (Mtw1-GFP) markers. Cells with one SPB and dispersed kinetochores were counted (n ≥ 100 cells). T = 0 represents the time at which cells were switched to sporulation medium. (B) As in (A), ipl1-mn diploid cells with clustered (light gray) or dispersed kinetochores (dark gray) were analyzed (n ≥ 100 cells) with or without addition of benomyl. (C) Separation of CEN1–green fluorescent protein (GFP) from the SPBs (Spc42-DsRed) was monitored in ipl1-mn cells with or without inactivation of CEN1 (materials and methods, supplementary materials). Separation was scored as either close to (<0.75 μm, light gray) or far away from the SPB (≥0.75 μm, dark gray) (n ≥ 30 cells). (D) Cells with Zip1 staining were scored for having clustered (light gray) or dispersed (dark gray) kinetochores 3 hours after meiotic induction (n ≥ 38 cells). Cells were stained with antibodies to GFP (MTW1-GFP; green) and Zip1 (red). Scale bar, 2 μm.

 

Figure 2
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Fig. 2. Chromosomes begin pro-metaphase with monopolar spindle attachments. (A) NDJ of CEN1 toward the new (light gray) or old SPB (dark gray) was determined in anaphase I cells (n ≥ 60 cells) homozygous for CEN1-GFP and expressing Spc42-RedStar, which differentiates old from new SPBs (fig. S5). An example of NDJ to the old SPB is shown. (B) The frequency of mono- or bipolar orientation of CEN1-GFP on newly formed spindles (Spc42-DsRed) was observed by means of time-lapse imaging (5-min intervals). The first frame with a bipolar spindle is t = 0. Cells with CEN1-GFP overlapping one pole were scored as mono-oriented (dark gray) and between poles as bi-oriented (light gray) (n = 29 cells). (C) Association of mono-oriented CEN1-GFP with the old (dark gray) or new SPB (light gray) measured in diploid cells carrying GAL4-ER, GAL-NDT80, homozygous CEN1-GFP, and SPC42-RedStar after release from prophase arrest (β-estradiol addition) (fig. S5). Cells with short spindles were scored (0.75 to 1.5 μm) (n = 104 cells). (D) Non-disjunction after inactivation of Ipl1-as5 or Mps1-as1. Cells were released from prophase in the presence or absence of inhibitor. Disjunction (light gray) or NDJ (dark gray) of CEN1-GFP was scored at anaphase I. Scale bar, 2 μm.

 

Figure 3
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Fig. 3. Ipl1 and Mps1 have different roles in re-orientation. (A to F) spo11{triangleup} [wild-type(WT)], spo11{triangleup} mps1-as1/mps1-mn (mps1) or spo11{triangleup} ipl1-as5/ipl1-mn (ipl1) cells with one CEN1-GFP–tagged chromosome and expressing Spc42-DsRed were observed by means of time-lapse imaging during meiosis at 2-min [(A), (B), and (E)] or 45-s intervals [(C), (D), and (F)] after release from prophase arrest, with or without inhibitor (fig. S7). (A) Images from representative cells (t = 0 represents first frame with SPB separation). (B) Cells in which CEN1 migrated to SPBs before spindle formation. (C) After release from the SPB, complete migrations of CEN1 to the opposite SPB were quantified as traverses. (D) The speed for crossing the spindle was evaluated for each individual traverse (n = 55 cells for WT, n = 19 cells for mps1-as1). (Bottom) An mps1-as1 mutant showing back-and-forth movements. Scale bar, 2 μm. (E) Dwell-time of CEN1 at SPBs. (F) Migrations away from and then back to the same SPB (trials).

 

Figure 4
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Fig. 4. Mps1 is necessary to generate end-on attachment during meiosis I. (A) Colocalization of Mtw1-RFP and Dam1-GFP were observed in spread nuclei from WT or mps1-as1/mps1-mn (mps1-as1) chromosome spreads after release from prophase arrest with addition of inhibitor (fig. S7). The proportion of chromosomes (Mtw1) located between the SPBs with Dam1 overlapping staining (white arrow) was determined. The difference between WT and mps1 mutant is significant (Fischer's test, P < 0.001). Scale bar, 2 μm. (B) Model showing kinetochore behaviors in meiosis and the roles of Ipl1 and Mps1.

 


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