Roles of MK2 in Oocyte Meiosis this hypothesis, we tested the stability of the spindle microtubules in response to cold treatment [43]. Although cold treatment moderately affected spindle morphology in control cells, the overall structure of the kinetochore attached microtubules remained robust in control cells (Fig. 6A,B). In contrast, cold treatment resulted in highly unstable and much disintegrated microtubules in MK2 MO-injected or CMPD1-treated oocytes (Fig. 6A,B,C). This result suggests that spindle microtubules are not properly attached to kinetochores in MK2-deficient oocytes. To further confirm this speculation, we performed an assay designed to quantify microtubule–kinetochore interactions [44]. For this assay, GV oocytes were cultured in M16 medium with DMSO or CMPD1 for 8.5 h and subjected to taxol for 45 min, which causes the collapse of the bipolar spindle into a monopolar configuration. The microtubule–kinetochore interactions could be scored easily in this structure. We found that in control MI oocytes, most of the chromosomes aligned at the equatorial plate displayed both kinetochores attached to microtubules in an amphitelic configuration (Fig. 6Da). However, in the MK2- deficient oocytes, bundles of abnormal spindle microtubules were excessively polymerized, and it was clearly observed that some chromosomes localized between asters and the attachments were Figure 6. MK2 depletion interferes with correct microtubule–kinetochore attachment. (A) After culture for 12 h in M16 medium at 37uC, oocytes injected with control or MK2 morpholino were quickly transferred to ice-cold medium at 4uC for 20 min. These oocytes were fixed and stained for a-tubulin (green) and DNA (blue). Scale bar, 20 mm. (B) After 8.5 h of culture in M16 medium with DMSO or CMPD1 at 37uC, oocytes were quickly transferred to ice-cold medium at 4uC for 20 min. These oocytes were fixed and stained for a-tubulin (green) and DNA (blue). Scale bar, 10 mm. (C) Percentages of oocytes with stable K-MTs after cold treatment as in (A,B). (D) Oocytes cultured in medium with DMSO or CMPD1 for 8.5 h were incubated in M16 medium containing 10 mM taxol for 45 min and then double stained with antibodies against p-MK2 (red), a-tubulin (green), and DNA label (blue). Bar 20 mm. White arrowhead indicates homologous chromosome attachment to microtubules. doi:10.1371/journal.pone.0011247.g006 PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11247