PLK1 activates the APC/C through multiple pathways.

<p>(A) Imaging of oocytes expressing securin-EGFP (green) and H2B-mCherry (chromosomes, red) in the presence of DMSO (control), 100 nM BI2536 and/or 1 μM reversine. Maximum intensity z-projection images are shown. Time after NEBD (h). Scale bar = 10 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s012" target="_blank">S5 Movie</a>. (B) Normalized intensities of cytoplasmic securin-EGFP signals (I_norm) were plotted. Average and s.d. are shown (n = 6, 4, 12, 15). (C-E) A line was fitted to the decrease of I_norm (C). Time for the start of securin-EGFP degradation (T_start) was defined as the time when the fitted line reaches the I_norm value 1.0. The degradation rate (D_rate) was defined as the negative value of the slope of the fitted line. Averages with s.d. of T_start and D_rate are shown in (D) and (E), respectively (n = 6, 12, 15. **p < 0.01).</p

PLK1 is required for chromosome segregation, first polar body extrusion, and maintenance of the condensed state of chromosomes.

<p>(A) Experimental scheme. Oocytes were cultured for 6 hours in control medium, and then MG132 was added. The oocytes were incubated for 4 hours to arrest oocytes at the late metaphase I. After release from MG132, 100 nM BI2536 was added and oocytes were imaged. (B) Anaphase phenotypes after MG132 release in control and BI2536-treated oocytes. PB = the first polar body. (C) Imaging of securin-EGFP (green) and H2B-mCherry (red) after DMSO (control, top) or 100 nM BI2536 (lower panels) was added at the time of the MG132 washout. Each phenotype from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g006" target="_blank">Fig. 6B</a> is shown on a representative image sequence. Arrows indicate lagging chromosomes. Note that none of the BI2536-treated oocytes undergoing abnormal chromosome segregation extruded the first polar body. Time after MG132 washout (h:mm). Scale bar = 30 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s014" target="_blank">S7 Movie</a>. (D) Quantification of securin-EGFP destruction. Values were normalized to 1 when the imaging was started. Time relative to MG132 washout (h). The ‘BI2536 with anaphase’ curve represents BI2536-treated oocytes that underwent abnormal chromosome segregation either with or without DNA decondensation (3^rd and 4^th rows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). The ‘BI2536 w/o anaphase’ curve represents BI2536-treated oocytes that did not undergo chromosome segregation (2^nd row in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). Average and s.d. are shown (n = 23, 40). (E) Degradation rate of securin-EGFP calculated from (D). Average and s.d. are shown. ***p < 0.001.</p

PLK1 is required for chromosome alignment.

<p>(A) Imaging of meiosis I in oocytes expressing EGFP-CENP-C (kinetochores, green) and H2B-mCherry (chromosomes, red) in the presence of DMSO (control) or 100 nM BI2536. Maximum intensity z-projection images are shown. White lines indicate kinetochore tracks over 5 timepoints. Time after NEBD (h:mm). Scale bar = 10 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s011" target="_blank">S4 Movie</a>. (B) Kinetochore positions were determined from (A) and shown in the 3D plot as green spheres. Red bars connect homologous kinetochores. The view along the chromosome distribution equator (side view) is shown. Black arrowheads indicate misaligned chromosomes. Time after prometaphase belt formation (h:mm). The unit of the grid is 5 μm. (C) Chromosome positions along the estimated spindle axis were plotted for all twenty chromosomes of a single oocyte cultured in the presence of DMSO (control, black) or 100 nM BI2536 (red). (D) Distances between chromosomes and the equator at 0 and 2 hours after the prometaphase belt formation were potted. The box indicates 10–90 percentile (n = 60, 60, 60, 60 from three oocytes for each condition). ***p < 0.0001. (E) Oocytes 4 hours after NEBD were briefly treated with a cold buffer and fixed for immunostaining of microtubules (blue) and kinetochores (red). 100 nM BI2536 was added at 2 hours after NEBD. DNA was stained with Hoechst33342 (blue). Insets show magnified images of kinetochore-microtubule attachments. Scale bar = 10 μm. Average and s.d. of the population of unattached kinetochores are shown (n = 5, 5. **p < 0.01).</p

PLK1 localizes to MTOCs and kinetochores and becomes activated around NEBD.

<p>(A) Imaging of meiosis I in oocytes expressing EGFP-PLK1 (green) and 3mCherry-CENP-C (kinetochores, red). Maximum intensity z-projection images are shown. At 7:50, the saturated signal locates at the spindle midzone, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s001" target="_blank">S1A Fig.</a> Time after induction of meiotic resumption (h:mm). Scale bar = 10 μm. Insets show magnified images on kinetochores. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s008" target="_blank">S1 Movie</a>. (B) Immunostaining of active PLK1 phosphorylated on T210 (pPLK1) during meiosis I. Pictures represent single selected confocal sections through MTOCs for pPLK1 (fire-pseudocolored or green) and pericentrin (MTOCs, red) and maximum intensity z-projection for DAPI (DNA, blue). Arrowheads indicate pPLK1 signals on MTOCs. Time after induction of meiotic resumption (h:mm). Quantification of MTOC-associated pPLK1 signals (n = 11, 5, 7, 5, 5, 9, 17 oocytes). Averages with the 95% confidence intervals are shown. Scale bar = 20 μm. Insets show magnified images on MTOCs. (C) Localization of pPLK1 on kinetochores in metaphase I oocytes. Oocytes stained for pPLK1 (green), kinetochores (CREST, red), acetylated α-tubulin, and DAPI (DNA). Scale bar = 10 μm.</p

PLK1 is required for EMI1 destruction and full APC/C activation.

<p>(A) Imaging of oocytes expressing EGFP-EMI1 or EGFP-EMI1–2A (green) and H2B-mCherry (red) in the presence of DMSO (control) or 100 nM BI2536. Scale bar = 10 μm. Time after NEBD (h). (B) Cytoplasmic EGFP-EMI1 signals were measured and normalized. Average and s.d. are shown (n = 7, 5, 3). Time after NEBD (h). (C) Imaging of securin-EGFP (green) and H2B-mCherry (red) after DMSO (control, top) or 100 nM BI2536 (bottom) was added at the time of metaphase I (6.5 hours after the induction of meiotic resumption). Note that in the control oocyte at 0:30, the metaphase plate is viewed from the top of the spindle. Time after BI2536 addition (h:mm). Scale bar = 20 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s013" target="_blank">S6 Movie</a>. (D) Quantification of securin-EGFP destruction. Values were normalized to 1 at the time when imaging was started (n = 11, 18). Time is relative to BI2536 addition (h). (E) Degradation rate of securin-EGFP in oocytes with BI2536 addition during metaphase I. Average and s.d. are shown.***p < 0.001.</p

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

<div><p>Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.</p></div