amphitelic; some chromosomes localized to the periphery of the aster and the attachments were syntelic/monotelic; and some chromosomes were scattered or detached from the spindle (Fig. 6Db,c). In control oocytes (n = 112), most chromosomes (95%) exhibited a clear amphitelic configuration, whereas in MK2-depleted oocytes (n = 217), only few chromosomes (11%) were localized between the periphery of the two asters with a clear amphitelic configuration. The collapsed and cold sensitive spindles with the scattered chromosome configurations in MK2-deficient oocytes suggests that kinetochores were not properly captured by microtubules. To determine whether the lack of microtubule–kinetochore attach- ment is due to defective kinetochore structure, we next examined the localization of kinetochore-associated protein kinase Plk1 to assess the integrity of the kinetochore structure in MK2-deficient oocytes. We found that Plk1 was localized properly at the kinetochores in MK2-deficient oocytes (Fig. 3D, Fig. 4D, Fig. 5C). Taken together, these results indicate that knockdown of MK2 impairs correct microtubule-kinetochore attachments but does not affect kinetochore integrity. Discussion The formation of a stable bipolar meiotic spindle is crucial for chromosome disjunction in which chromosome attachment, alignment and pulling toward spindle poles are critical steps for accurate segregation. Although chromatin interaction with microtubules appears to influence microtubule dynamics, a bipolar spindle can still form in the absence of chromatin, as is the case in enucleated mouse oocytes [45,46]. Our studies for the first time showed that phosphorylated MAPKAPK 2 (p-MK2) is localized to bipolar spindle minus ends and chromosomes during metaphase I and metaphase II in meiosis. In addition, in the Pro-MI oocytes, in which the connection between microtubules and chromosomes had not yet been completely established and the bipolar spindle had not yet formed, p-MK2 was only localized along the interstitial axes of homologous chromosomes. Furthermore, at anaphase I, p-MK2 was localized in the region of the equatorial plate between the separating chromosomes. MK2-deficient oocytes after inhibitor treatment and morpholino injection both resulted in collapsed spindles with no centrosomes apparent at the poles. These data suggest that MK2 most likely contributes to meiotic bipolar spindle assembly, similar to mitosis where MK2 functions in bipolar spindle formation shown by MK2 depletion which induced mitotic defects in bipolar spindle formation and subsequent activation of the spindle checkpoint [47]. However, in mitosis, p-MK2 localization to spindle poles during prophase and metaphase is different from our findings showing p-MK2 localization to both microtubule minus ends and chromosomes, perhaps because our p-MK2 antibody may have been more suitable for immunofluorescence. To further clarify the correlation between p-MK2 and microtubule dynamics, we destructed bipolar spindle structures, and found that all cytoplasmic p-MK2 emerged around the chromosomes at the MI stage, but the cytoplasmic p-MK2 signals disappeared completely at the MII stage. We speculate that p- MK2 accumulated at the minus ends of microtubules and was retained at the kinetochores of homologous chromosomes and sister chromatids. Another explanation might be that p-MK2 may have different roles in first meiotic bipolar spindle and second meiotic bipolar spindle formation. There are some proteins regulating meiosis I spindle formation differently from meiosis II spindle formation, such as ATRX [48] and Ran [49]. However, we find that inhibition of MK2 at first meiotic and second meiotic Roles of MK2 in Oocyte Meiosis division both results in collapsed spindles and unaligned chromosomes. The third explanation might be that different mechanisms are employed for meiosis I and II spindle formation [49]. In mouse oocytes, the first meiotic M phase is very long, lasting from 6 to 11 h, depending on the genetic background [50,51]. However, the MII spindle assembles rapidly, and 1 h after first polar body emission in mouse oocytes a stable bipolar spindle has already formed. We further observed that p-MK2 was localized to the spindle minus ends of bipolar microtubule fibers and not to cytoplasmic asters after taxol treatment. This unexpected localization of cytoplasmic MK2 suggests that it serves functions in the establishment or stabilization of the bipolar spindle, but not in microtubule nucleation or organization, which is different from functions of other microtubule nucleation-related protein kinases such as MEK and Polo-like kinase 1 [5,52]. To our knowledge, we are the first to show that p-MK2 signals are localized along the interstitial axes of homologous chromo- somes in meiosis extending over centromere and arm regions at the proMI and MI stages. Moreover, at the MII stage, p-MK2 signals were detected as centromeric dots of sister chromatids. Clearly, similar to the REC8 signals in meiosis which distributes along the entire axis of chromosomes during meiosis I, p-MK2 localization on the cohesion domains of meiotic homologous chromosomes and sister chromatids, disappears from chromo- some arms at the onset of anaphase I, but persists at centromeres until the onset of anaphase II[53]. However, at the AI or TI stage, p-MK2 was no longer associated with the centromeric domains of meiotic chromosomes; therefore, we suggest that MK2 is not part of the cohesin complex and is not directly responsible for holding sister chromatids together, but it may be essential for cohesin degradation or activation, which needs further investigation. The cohesin family of proteins play a well-established role in maintaining sister chromatid cohesion in mitosis and meiosis in a number of species [12,54]. In contrast, meiotic sister chromatids do not lose cohesin from their arms until the anaphase I onset, and this is mediated solely by REC8 degradation through separase activity rather than dissociation [13,14]. Recently, several proteins were reported to be involved in the regulation of REC8 degradation; for example, Aurora B kinase is localized to the chromosomal arms distal to chiasmata and leads to phosphorylation and localized destruction of the meiotic cohesin REC8 [17,55]. Moreover, cohesin phosphorylation depends on Polo-like kinase (Plk1) and reduces the ability of cohesin to bind to chromatin; therefore, Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes [16,56]. Furthermore, MK2 has been proved to directly phosphorylate Ser326 of Plk1 [47]. We found that knock-down of MK2 by inhibitor treatment and morpholino injection both lead to homologous chromosome segregation failure and meiosis progression arrest. However, inhibition release of phosphorylated MK2 could break through the metaphase I arrest and allow extrusion of the polar body. The explanation is that MK2- deficiency reduces the level of Plk1 phosphorylation which may downregulate cohesin phosphorylation and prevent homologous chromosome separation. Because of the lack of phospho-Plk1 and REC8 antibodies, this assumption could not be confirmed further in our study. Kinetochore microtubules (kMTs) are spindle microtubule whose minus ends are typically anchored at the spindle poles and whose plus ends terminate end-on at the kinetochore. Chromosome movement depends on the assembly dynamics of kMT plus ends within their kinetochore attachment sites [57]. PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11247