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. 2013 Jul 22;202(2):221-9.
doi: 10.1083/jcb.201303019. Epub 2013 Jul 15.

Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I

Olga Davydenko  1 , Richard M Schultz, Michael A Lampson
Affiliations

Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I

Olga Davydenko et al. J Cell Biol. .

Abstract

Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore-microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3-4 h, and stabilization of K-MT attachments is delayed an additional 3-4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.

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Figures

Figure 1.
Figure 1.
Stable K-MT attachment is delayed until late in MI. Oocytes were cultured for 2.5 h after GVBD to prometaphase I or 6.5 h to MI, then analyzed for cold-stable MTs. (A) Images are projections of a confocal z series showing MTs (green), kinetochores (CREST, red), and DNA (blue). Individual kinetochores were classified as end-on attached (1), unattached (2), or lateral (3); insets are optical sections showing examples of each (bar, 0.5 µm). (B) The percent of kinetochores in each category was averaged over multiple cells (n ≥ 17, 15 kinetochores per cell) at each time point (*, P < 0.001).
Figure 2.
Figure 2.
Aurora B/C kinase activity and tension regulate K-MT attachments during meiosis I. (A and B) Oocytes were cultured for 5.5 h after GVBD, then treated with Aurora B/C inhibitor ZM447439 or DMSO (control) for 1 h before analysis of cold-stable MTs. (C and D) Oocytes injected with AA-separase cRNA together with securin MO, or GFP cRNA as a control, were matured for 6.5 h after GVBD, then analyzed for cold-stable MTs. (E and F) Oocytes injected with AA-separase cRNA and securin MO were cultured for 5.5 h after GVBD, then treated with ZM447439 (or DMSO) for 1 h before analyzing cold-stable MTs. Only oocytes with complete chromatid separation were analyzed, indicating loss of cohesion. Images (A, C, and E) are projections of a confocal z series showing MTs (green), kinetochores (CREST, red), and DNA (blue). Insets in C are optical sections showing lateral interactions (bar, 0.5 µm). Percentages of end-on attached, unattached, and lateral kinetochores were averaged over multiple cells (n ≥ 13, ≥15 kinetochores per cell; *, P < 0.001).
Figure 3.
Figure 3.
Partial CDK1 inhibition slows stabilization of attachments. (A) Histone-H1 kinase activity was measured in oocytes at the indicated time points relative to GVBD. Each data point represents H1 phosphorylation averaged over three lysates, each with three oocytes. Arrows indicate time of GVBD, anaphase I (AI), and first polar body extrusion (PBE). (B) Oocytes were matured with low concentrations of CDK1 inhibitor RO-3306 (0.5 and 2 µM) or DMSO (control). Meiosis I progression from GV to AI was followed live by DIC microscopy. Times from milrinone washout to GVBD, GVBD to chromosome alignment, and alignment to anaphase I were averaged over multiple cells (n ≥ 18). (C–F) Oocytes were cultured with 0.5 µM RO-3306 or DMSO for 4.5, 6.5, or 11 h after GVBD, then analyzed for cold-stable MTs. Images in C are projections of a confocal z series or optical sections showing MTs (green) and kinetochores (CREST, red). Insets show individual kinetochores classified as attached (1), unattached (2), or lateral (3); bar, 0.5 µm. Percentages of each attachment state were averaged over multiple cells (n ≥ 23, 15 kinetochores per cell; *, P < 0.001) at each time point.
Figure 4.
Figure 4.
Prematurely increasing CDK1 activity stabilizes K-MT attachments. (A and B) Oocytes injected with GFP cRNA or cyclin B–GFP cRNA (700 ng/µl) were analyzed for histone-H1 kinase activity (A) or cold-stable MTs (B) at the indicated time points. H1 kinase activity (A) was averaged over three lysates, each with one oocyte. Images in B are projections of a confocal z series showing MTs (green) and kinetochores (CREST, red). Insets show individual kinetochores classified as attached (1), unattached (2), or lateral (3); bar, 0.5 µm. Injecting lower levels of cyclin B–GFP cRNA (200 ng/µl) together with CDK1 cRNA gave similar results in both the H1 kinase and cold-stable MT assays. (C and D) Percentages of each attachment state were averaged over multiple cells (n ≥ 22, 15 kinetochores per cell; *, P < 0.001) at each time point. Results of the cold-stable MT assay were combined for oocytes injected with cyclin B–GFP cRNA (700 ng/µl) or CDK1 cRNA together with lower levels cyclin B-GFP cRNA (200 ng/µl).
Figure 5.
Figure 5.
Prematurely increasing CDK1 activity leads to lagging chromosomes at anaphase I. (A and B) Oocytes microinjected with H2B-mCherry cRNA, with or without cyclin B–GFP cRNA, were cultured for 6.5 h after GVBD, incubated with 10 µM cycloheximide to allow normal anaphase I progression, and imaged live from MI through telophase I (TI). Images in A are maximal intensity projections of a confocal z series showing all the chromosomes (time stamps relative to anaphase onset). The inset shows lagging chromosomes at anaphase (brightness increased for clarity); bar, 2 µm. The percentage of anaphases with lagging chromosomes was averaged over three independent experiments (n ≥ 5 cells per experiment; *, P < 0.001). (C) Model schematic depicts the timing of K-MT stabilization when CDK1 activity rises normally (left) vs. prematurely (right) during MI. Normally, kinetochores interact with MTs laterally to achieve chromosome congression, and attachments are stabilized ∼7 h after GVBD when CDK1 activity is maximal (left). If K-MT attachments are stabilized too early, in the presence of multipolar spindle intermediates and multiple MTOCs close to the chromosomes, incorrect attachments can lead to lagging chromosomes at anaphase I (right).
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