Normally, kMTs are stable to cold- or calcium-induced depolymerization because of their kinetochore plus-end attach- ments; in contrast, most spindle MTs with free plus-ends are not stable before anaphase [43,58]. The phenotypes of collapsed spindles with chromosomes excluded from the spindle structure or arranged in a dome-like configuration surrounding the spindle in MK2-depleted oocytes strongly suggest that down- regulation of MK2 results in failure of proper chromosome alignment because of defects in spindles and/or kinetochore– microtubule attachment [42]. Moreover, cold treatment resulted in a highly unstable and much disintegrated spindle in MK2- deficient oocytes. In taxol treated MK2-deficient oocytes, it is clearly seen that chromosome attachment is either amphitelic, syntelic/monotelic, or that chromosomes are detached from spindle asters. These results further suggest that the spindle microtubules are not properly attached to kinetochores in MK2- deficient cells. In order for homologous chromosomes to align properly at the metaphase plate and segregate equally to daughter cells through bipolar spindle dynamics, correct microtubule–kinetochore inter- actions must be established to direct sister kinetochores to opposite poles (amphitelic kinetochore orientation). However, we found that various incorrect kinetochore–microtubule attachments occur in MK2 deficient oocytes. The kinetochores were either not bound to microtubules (detached), or one kinetochore was bound to microtubules while the other one was unbound (monotelic kinetochore orientation), or both kinetochores were bound to microtubules from the same pole (syntelic kinetochore orientation), and one kinetochore was bound to microtubules from both spindle poles (merotelic kinetochore orientation) (Fig. S4). If errors are not corrected before the onset of anaphase, meiosis progression is arrested. Therefore, additional regulation is needed to eliminate improper microtubule–kinetochore associations to allow for correct cell division [59]. As previously reported, the spindle assembly checkpoint Bub3 [60,61], BubR1 [62,63] and Bub1 [60,64] localized to the kinetochores and is required for the establishment of efficient K-MT attachments. Moreover, histone deacetylase 3 localized to the mitotic spindle and is required for kinetochore-microtubule attachment [42]. Importantly, numerous studies revealed that Aurora B is concentrated at the centromere in prometaphase and metaphase [65] and interaction with mitotic centromere-associated kinesin (MCAK) is important for proper chromosome biorientation and for correcting or preventing improper microtubule-kinetochore attachments in mammalian cells [66,67,68,69]. Our study supports the idea that MK2 may play a critical role in the establishment of correct kinetochore- microtubule attachments by phosphorylating some unknown key substrates. In conclusion, in this study we report for the first time that MK2 localizes at the meiotic bipolar spindle microtubule minus ends and cohesion of chromosomes, and we provide evidence that MK2 is required for bipolar spindle formation, homologous chromosome segregation and proper kinetochore–microtubule attachment. Materials and Methods All chemicals and media were purchased from Sigma Chemical Company (St. Louis, MO) except for those specifically mentioned. Ethics Statement Animal care and use were conducted in accordance with the Animal Research Committee guidelines of the Institute of Zoology, Chinese Academy of Sciences. Roles of MK2 in Oocyte Meiosis Mouse oocyte collection and culture Immature oocytes were collected from ovaries of 6-week-old Kunming White mice in M2 medium. Only those immature oocytes displaying a germinal vesicle (GV) were cultured further in M16 medium under liquid paraffin oil at 37uC in an atmosphere of 5% CO2 in air. At different times of culture, oocytes were collected for immunostaining, drug treatment, or microinjection. Taxol and nocodazole treatment of oocytes Oocytes were treated at various stages with taxol or nocodazole. For taxol treatment, 5mM taxol in DMSO stock was diluted in M16 medium to give a final concentration of 10 mM and oocytes were incubated for 45 min; for nocodazole treatment, 10 mg/ml nocodazole in DMSO stock was diluted in M16 medium to give a final concentration of 20 mg/ml and oocytes were incubated for 10 min. After treatment, oocytes were washed thoroughly and used for immunofluorescence. Control oocytes were treated with the same concentration of DMSO in the medium before examination. CMPD1 treatment and Cold treatment For MK2 inhibitor CMPD1 (Calbiochem) treatment, 30 mM CMPD1 in DMSO stock was diluted in M16 medium to give a final concentration of 30 mM. The oocytes were incubated in M16 medium containing 30 mM CMPD1 for different times, and then washed thoroughly for subsequent experiments. Control oocytes were treated with the same concentration of DMSO in the medium before examination. For cold treatment, oocytes at the appropriate stages were transferred to M16 medium which was pre-cooled to 4uC and cultured for 20 minutes at this temperature, followed by immunofluorescent staining. Morpholinos microinjection and RNA Interference Oocytes were arrested at the GV stage in M2-containing 2.5 mM milrinone, 2 mM MK2 morpholino antisense oligos (GENE TOOLS, LLC, TCTGGCCCGGAGAGCCCGACAG- CAT) microinjected into the cytoplasm to deplete MK2. The same amount of negative control morpholino (GENE TOOLS, LLC) was injected as control. The GV-intact oocytes were microinjected in M2 medium containing 2.5 mM Milrinone with the negative control siRNA(- Qiagen) and MK2 siRNAs (Ambion) (MK2-1 siRNA, GAAC- GAUGGGAGGAUGUCAtt) or (MK2-2 siRNA, ACAGAAUU- CAUGAACCACCtt). The final concentration of the control or MK2 siRNA was 25 mM. After microinjection, the oocytes were arrested at the GV stage for 24 h in M2-containing 2.5 mM milrinone to knock down MK2, and then transferred to Milrinone-free M16 medium to resume meiosis. Oocytes were incubated for different times and then collected for the subsequent experiments. Immunofluorescence and confocal microscopy For staining of proteins, oocytes were fixed in 4% paraformal- dehyde in PBS (pH 7.4) for at least 30 min at room temperature and processed for indirect immunofluorescence microscopy as described previously [70]. The immunostained cells were mounted on glass slides and examined with a Confocal Laser-Scanning Microscope (Zeiss LSM 510 META, Germany). The following primary antibodies were used, respectively: rabbit anti-phospho-MAPKAPK-2 (Thr334; Cell Signaling; 1:100), mouse anti-Plk1 antibody (Cell Signaling; 1:50), mouse anti-c- tubulin antibody (Sigma; 1:200), mouse anti-a-tubulin-FITC PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11247