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• W2126460280 abstract "Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans. Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans. Complete 3D kinetochore tracking throughout meiosis I in mouse oocytes Chromosome congression precedes biorientation, forming the prometaphase belt Initial kinetochore-microtubule attachments are predominantly incorrect Quantitative analysis revealed that bivalent biorientation is highly error prone Proper segregation of homologous chromosomes during the first meiotic division in female oocytes is essential to prevent generation of aneuploid eggs. Fertilization of aneuploid eggs in humans is a leading cause of pregnancy loss and, if survived to term, results in developmental disabilities (Hassold and Hunt, 2001Hassold T. Hunt P. To err (meiotically) is human: the genesis of human aneuploidy.Nat. Rev. Genet. 2001; 2: 280-291Crossref PubMed Scopus (1618) Google Scholar). To segregate homologous chromosomes faithfully, it is essential that all bivalents, the paired homologous chromosomes, achieve biorientation, i.e., that their homologous kinetochores are attached to microtubules from the opposite poles of the meiotic spindle. Although the mechanism of sister chromatid biorientation has been well studied in somatic mitotic cells (Kops et al., 2010Kops G.J.P.L. Saurin A.T. Meraldi P. Finding the middle ground: how kinetochores power chromosome congression.Cell. Mol. Life Sci. 2010; 67: 2145-2161Crossref PubMed Scopus (43) Google Scholar, Maiato et al., 2004Maiato H. DeLuca J. Salmon E.D. Earnshaw W.C. The dynamic kinetochore-microtubule interface.J. Cell Sci. 2004; 117: 5461-5477Crossref PubMed Scopus (313) Google Scholar, Walczak and Heald, 2008Walczak C.E. Heald R. Mechanisms of mitotic spindle assembly and function.Int. Rev. Cytol. 2008; 265: 111-158Crossref PubMed Scopus (273) Google Scholar), homologous chromosome biorientation in animal oocytes is poorly understood. Somatic animal cells contain two centrosomes that predefine the poles of the mitotic spindle. One of the most widely used models to explain biorientation is the so-called “search-and-capture” mechanism, in which sister kinetochores become attached to microtubules emanating from the spindle poles. If both kinetochores are attached in an end-on manner from opposite poles (amphitelic), the force balance leads to stable biorientation. If the attachment is incorrect, for example if one kinetochore is attached to two poles (merotelic) or both are attached to the same pole (syntelic), the force balance cannot be achieved. Such erroneous attachments are sensed by Aurora B kinase, which phosphorylates a set of kinetochore substrates to detach the incorrect microtubules. The detached kinetochores activate the spindle assembly checkpoint, giving the cell time for a new round of biorientation (Nezi and Musacchio, 2009Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar). Merotelic and syntelic attachments are regarded as errors that must be corrected because they would cause chromosome missegregation if they persisted until anaphase (Cimini et al., 2003Cimini D. Moree B. Canman J.C. Salmon E.D. Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms.J. Cell Sci. 2003; 116: 4213-4225Crossref PubMed Scopus (186) Google Scholar). Although no comprehensive quantitative analysis has been carried out, the available data (Cimini et al., 2003Cimini D. Moree B. Canman J.C. Salmon E.D. Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms.J. Cell Sci. 2003; 116: 4213-4225Crossref PubMed Scopus (186) Google Scholar) allow estimation of the number of erroneous attachments during prometaphase in somatic mitotic cells to less than 10%, although this may be an underestimate (Salmon et al., 2005Salmon E.D. Cimini D. Cameron L.A. DeLuca J.G. Merotelic kinetochores in mammalian tissue cells.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2005; 360: 553-568Crossref PubMed Scopus (90) Google Scholar). The classical search-and-capture model explains chromosome congression as a result of the biorientation process because the balance of the pulling forces on the sister kinetochores positions the chromosomes at the spindle equator. Although congression mechanisms that do not require biorientation have been described (Cai et al., 2009Cai S. O'Connell C.B. Khodjakov A. Walczak C.E. Chromosome congression in the absence of kinetochore fibres.Nat. Cell Biol. 2009; 11: 832-838Crossref PubMed Scopus (119) Google Scholar, Kapoor et al., 2006Kapoor T.M. Lampson M.A. Hergert P. Cameron L. Cimini D. Salmon E.D. McEwen B.F. Khodjakov A. Chromosomes can congress to the metaphase plate before biorientation.Science. 2006; 311: 388-391Crossref PubMed Scopus (312) Google Scholar, Wignall and Villeneuve, 2009Wignall S.M. Villeneuve A.M. Lateral microtubule bundles promote chromosome alignment during acentrosomal oocyte meiosis.Nat. Cell Biol. 2009; 11: 839-844Crossref PubMed Scopus (95) Google Scholar), it remains unknown what fraction of chromosomes normally congress without biorientation in mammalian cells (Foley and Kapoor, 2009Foley E.A. Kapoor T.M. Chromosome congression: on the bi-orient express.Nat. Cell Biol. 2009; 11: 787-789Crossref PubMed Scopus (4) Google Scholar). By contrast, oocytes in humans and all other mammals analyzed so far (and also in Gallus, Xenopus, Drosophila, and C. elegans) lack centrosomes (Manandhar et al., 2005Manandhar G. Schatten H. Sutovsky P. Centrosome reduction during gametogenesis and its significance.Biol. Reprod. 2005; 72: 2-13Crossref PubMed Scopus (202) Google Scholar), and the search-and-capture mechanism should become insufficient in their large cytoplasm (Wollman et al., 2005Wollman R. Cytrynbaum E.N. Jones J.T. Meyer T. Scholey J.M. Mogilner A. Efficient chromosome capture requires a bias in the ‘search-and-capture’ process during mitotic-spindle assembly.Curr. Biol. 2005; 15: 828-832Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Oocytes must therefore achieve spindle bipolarization and chromosome biorientation through a different process (Walczak and Heald, 2008Walczak C.E. Heald R. Mechanisms of mitotic spindle assembly and function.Int. Rev. Cytol. 2008; 265: 111-158Crossref PubMed Scopus (273) Google Scholar). Because mice represent the closest experimentally tractable model to humans, we have focused our analysis on their oocytes. We previously showed that the acentrosomal spindle in mouse oocytes is assembled by self-organization of many microtubule-organizing centers (MTOCs) (Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Upon nuclear envelope breakdown (NEBD), over 80 MTOCs scattered in the cytoplasm attract each other and form a cluster in the center of the oocyte that radiates microtubules to the outside, the apolar microtubule ball. This ball then bipolarizes through a several hour tug of war of attractive and repulsive forces between the MTOCs, giving rise to multipolar intermediates and ultimately a barrel-shaped spindle with poorly focused poles. How homologous chromosome biorientation is achieved during this lengthy and complex spindle self-assembly process is not known to date. In this study, we have addressed this issue, using 3D confocal fluorescence microscopy at high spatial and temporal resolution in live mouse oocytes. We succeeded to track all homologous kinetochores during the approximately 8 hr from NEBD to the onset of chromosome segregation in anaphase. These datasets allowed a systematic quantitative analysis of kinetochore and chromosome dynamics during the first meiotic division. We show that chromosome congression precedes biorientation, forming an intermediate chromosome configuration, the prometaphase belt. Furthermore we show that two-thirds of all biorientation attempts are erroneous, and that 86% of all homologous chromosomes undergo error corrections of their kinetochore-microtubule attachments before they establish stable biorientation. These results show that homologous chromosome biorientation is a highly error-prone process in acentrosomal mammalian oocytes, which may explain the high incidence of segregation errors in meiosis I observed in vivo (Hassold and Hunt, 2001Hassold T. Hunt P. To err (meiotically) is human: the genesis of human aneuploidy.Nat. Rev. Genet. 2001; 2: 280-291Crossref PubMed Scopus (1618) Google Scholar). To analyze spatiotemporal dynamics of kinetochores and chromosomes during the first meiotic division of live mouse oocytes, we recorded four-dimensional (4D) datasets of kinetochores and chromosomes labeled with EGFP-CENP-C and Histone 2B (H2B)-mCherry, respectively, expressed after quantitative mRNA microinjection (Jaffe and Terasaki, 2004Jaffe L.A. Terasaki M. Quantitative microinjection of oocytes, eggs, and embryos.Methods Cell Biol. 2004; 74: 219-242Crossref PubMed Scopus (55) Google Scholar) (Figure 1A ; Movie S1 available online). We used an automated confocal microscope that can focus and track all chromosomes within the oocyte to record 3D stacks of optical sections for 9 hr after induction of maturation (Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, Rabut and Ellenberg, 2004Rabut G. Ellenberg J. Automatic real-time three-dimensional cell tracking by fluorescence microscopy.J. Microsc. 2004; 216: 131-137Crossref PubMed Scopus (120) Google Scholar). Live imaging did not perturb oocyte maturation, as the rate of polar body extrusion and average time from NEBD to polar body extrusion were indistinguishable from normal in vitro culture conditions (83% [n = 12 with imaging] versus 78% [n = 61 w/o imaging], p = 1.0; 8.8 ± 0.6 hr [n = 10] versus 8.6 ± 0.8 hr [n = 7], p = 0.7). In these 4D datasets, we could detect 99.6% of all kinetochores (n = 200 from 5 oocytes) at all time points from NEBD to anaphase onset with high spatial (xy 0.3 μm, z 3.0 μm) and temporal (90 s) resolution. The 4D datasets were processed by an in-house-developed computational pipeline, which detects kinetochore positions and tracks them in 3D after registration of global cellular movements. The resulting kinetochore tracks were interactively validated and rare errors corrected. In this manner we could obtain complete 3D tracking datasets of all kinetochores during the entire process of the first meiotic division (Figures 1B and 1E; Movie S2). We make these datasets available online as a resource for the analysis of kinetochore and chromosome dynamics (http://www.ellenberg.embl.de/apps/KTTracking/). To gain insight into the processes that govern kinetochore dynamics, we analyzed different parameters for reproducible global changes during the first meiotic division. The average kinetochore speed changed in a canonical and stepwise fashion after NEBD (Figures 1C and 1D; Movie S2), suggesting changes in the interactions of kinetochores and/or chromosome arms with microtubules. Thus we can define five kinetic phases of chromosome dynamics during the first meiotic division: phase 1 (∼0–1 hr), in which kinetochores move relatively rapidly (0.19 ± 0.04 μm/min, up to 0.31 μm/min) outwards; phase 2 (∼1–2 hr), in which kinetochores move more slowly (0.13 ± 0.03 μm/min, up to 0.25 μm/min) with no obvious radial directionality; phase 3 (∼2–4 hr), in which chromosomes exhibit rapid oscillations (0.27 ± 0.11 μm/min, up to 0.59 μm/min) along the spindle axis; phase 4 (from ∼4 hr to anaphase onset), in which chromosomes oscillate more slowly (0.19 ± 0.05 μm/min, up to 0.34 μm/min); and finally anaphase, during which chromosomes very rapidly move to the opposite spindle poles (0.80 ± 0.23 μm/min, up to 1.2 μm/min). Anaphase is triggered after completion of biorientation, but how chromosome biorientation is achieved during the preceding four phases is poorly understood in mammalian meiosis, and we therefore decided to analyze them in more detail. We previously reported that clustered chromosomes are individualized and moved onto the forming microtubule ball ∼0–1 hr after NEBD (Schuh and Ellenberg, 2007Schuh M. Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes.Cell. 2007; 130: 484-498Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), which corresponds to phase 1. By analyzing kinetochore tracks during phase 1, we found that not only chromosomes but also kinetochores were individualized from their clusters during the chromosome-sorting process (Figures S1A–S1D), resulting in a spherical shell-like chromosome configuration on the surface of the microtubule ball (see also Figure 4). To understand how the individualized chromosomes congress to the metaphase plate, we carefully analyzed their spatial distribution in three dimensions over time. At the beginning of phase 2, chromosomes and kinetochores were distributed randomly (Figures 2A and 2B , 0:49) on the surface of the microtubule ball (see also Figure 4B). With the onset of chromosome congression, this distribution became progressively ordered as shown by a decrease in aspect ratio of the ellipsoid fitted to all chromosome positions (Figure 2C). At the end of phase 2, all chromosomes had relocated close to the equator of the overall chromosome distribution (Figures 2A–2C, 1:59; Movie S3). Viewing chromosome positions from the top onto the equator revealed that they formed a belt-like arrangement with a chromosome-free region inside of the equator (Figure 2B, top view, 1:59). Thus, chromosome congression results in a belt-like chromosome configuration, which we refer to hereafter as the “prometaphase belt.” To investigate how chromosomes move to the prometaphase belt, we analyzed their tracks during congression. Chromosomes that were located far from the equator at the beginning of phase 2 congressed toward it, whereas chromosomes already located near the equator remained stationary until the end of phase 2 (Figure 2D). The paths of the congressing chromosomes defined arcs around a 7.5 μm radius sphere, consistent with the size of the microtubule ball (see also Figure 4). Collectively, chromosomes therefore congress to form the prometaphase belt by sliding along the surface of the microtubule ball during phase 2 (Figure 3E , “phase 2”). We reasoned that plus-end-directed forces generated by chromokinesins might be responsible for chromosome individualization in phase 1 and/or prometaphase belt formation in phase 2. The chromokinesin Kid is a prime candidate because it has been shown to be required for expulsion of chromosomes on a monopolar spindle in mitotic cells (Levesque and Compton, 2001Levesque A.A. Compton D.A. The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles.J. Cell Biol. 2001; 154: 1135-1146Crossref PubMed Scopus (170) Google Scholar) and congression in Xenopus egg extracts (Antonio et al., 2000Antonio C. Ferby I. Wilhelm H. Jones M. Karsenti E. Nebreda A.R. Vernos I. Xkid, a chromokinesin required for chromosome alignment on the metaphase plate.Cell. 2000; 102: 425-435Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, Funabiki and Murray, 2000Funabiki H. Murray A.W. The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement.Cell. 2000; 102: 411-424Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). We therefore imaged chromosome and kinetochore dynamics in oocytes from Kid knockout mice (Ohsugi et al., 2008Ohsugi M. Adachi K. Horai R. Kakuta S. Sudo K. Kotaki H. Tokai-Nishizumi N. Sagara H. Iwakura Y. Yamamoto T. Kid-mediated chromosome compaction ensures proper nuclear envelope formation.Cell. 2008; 132: 771-782Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) (Figure S2A ). Quantitative analysis of these data revealed that neither chromosome individualization nor congression was significantly affected by the absence of Kid (Figures S2B–S2D), demonstrating that Kid is dispensable for these processes in mouse oocytes. Although the prometaphase belt formed during phase 2 represents a congressed chromosome configuration, in that all paired kinetochores are found around one plane, it is not yet the final arrangement in which chromosomes are stably bioriented. Therefore, we next addressed how the prometaphase belt with its chromosome-free interior transforms into the metaphase plate. During phase 3, chromosomes moved from the belt toward the center of the plane (Figures 3A–3D; Movie S3). This centripetal movement resulted in an even distribution of the chromosomes across the equatorial plane by the end of phase 3 (Figure S3A ), thereby defining the metaphase plate. Thus, the metaphase plate is formed by chromosomes invading the spindle from the peripheral prometaphase belt (Figure 3E, “phase 3”). Both the change in direction and the significant increase in speed of the invading chromosomes compared to phase 2 (average speed of congressing chromosomes = 0.13 ± 0.02 [n = 10]; average speed of invading chromosomes = 0.17 ± 0.04 [n = 6]; p = 0.01) indicated a different mechanism of this motion. Furthermore, the inward movements were concomitant with the onset of kinetochore oscillations perpendicular to the equator (Figure 3D), suggesting that kinetochore-microtubule attachments are dynamic during the invasion of chromosomes. Next, we wanted to determine when bivalents are stretched by directional microtubule forces. By measuring interkinetochore distances, we found that most bivalents were not stretched in phase 2, although they had already congressed, forming the prometaphase belt (Figures 4A and 4D and Movie S4, from −130 to −70 min). During the invasion in phase 3, however, the fraction of stretched bivalents increased progressively, reaching 89% ± 9% (Figures 4A and 4D and Movie S4, from −70 to 50 min). Thus, the majority of bivalents became stretched only after the formation of the prometaphase belt, as indicated by the significantly different half-time of chromosome congression and fraction of stretched bivalents (p = 0.0001, Figure 4E). To examine the relationship of spindle elongation with chromosome biorientation, we imaged kinetochores and microtubules by 3mCherry-CENP-C and EGFP-MAP4, respectively (Figure 4B). During phase 3 the spindle elongated, increasing its aspect ratio almost exactly concomitantly with the increase of stretched bivalents (Figures 4C and 4D, from −70 to 50 min), indicated by indistinguishable half-times of the increases (p = 1.0, Figure 4E). This precise kinetic correlation suggests that spindle elongation contributes to chromosome biorientation. To understand how homologous chromosomes achieve biorientation, we analyzed dynamics of bivalent stretching and alignment by measuring changes in interkinetochore distances and chromosome angles with the spindle axis, respectively. Average bivalent stretching (Figure 5B ) and alignment (Figure 5E) increased progressively with linear kinetics during phase 3 (from −70 to 50 min), confirming that the majority of chromosomes becomes bioriented during spindle elongation (compare Figure 4D). To analyze the kinetics of individual chromosome biorientation attempts (Figure 5A), we counted the number of significant bivalent stretching events (see Experimental Procedures and Figure S4A ) over time. Biorientation attempts occurred most frequently in the first half of phase 3 (Figure 5D, from −50 to 0 min), when the spindle starts to elongate (compare Figure 4D), and oriented the chromosomes parallel to the spindle axis (Figure 5G). By contrast, biorientation attempts that occurred prior to spindle elongation in phase 2 (compare Figure 4D) failed to orient chromosomes along the spindle axis (Figures 5D and 5G, from −150 to −50 min). These results indicate that spindle elongation promotes biorientation and enhances its fidelity. The complete kinetochore-tracking datasets allowed us to investigate biorientation systematically for each individual chromosome (Figure 5A; Movie S5 and Movie S6). Analyzing bivalent stretching and alignment at the single-chromosome level revealed that only very few chromosomes achieved biorientation at the first attempt, and the kinetic path taken was very different between individual chromosomes (Figure 5A). The “Red” chromosome, for example, started biorientation and alignment early and gradually as revealed by its slow stretching and early alignment (Figures 5A, 5C, and 5F, from −127 to −28 min, red brackets), eventually leading to stable biorientation in a continuous process over ∼1.5 hr. By contrast, the “Black” chromosome started biorientation only very late (−5 min), stretching abruptly and snapping into a parallel position with the spindle axis (Figures 5A, 5C, and 5F, 4 min, white and black arrowheads), leading to biorientation within less than 20 min. We found that most chromosomes underwent multiple biorientation attempts, indicated by repetitive bivalent stretching/relaxation (Figures 5A and 5C; and Movie S6) and reorientation along the spindle axis (Figure 5F). For example, the “Blue” chromosome showed three biorientation attempts. The first occurred at −45 min, but the bioriented state was lost at 4 min. The second biorientation attempt occurred at 15 min, which however halted transiently at 22 min, and was followed by the third attempt at 35 min (Figures 5A, 5C, and 5F, blue arrowheads), leading to stable biorientation. Overall, 86% of all chromosomes underwent two or more biorientation attempts with a maximum of six and an average of 3.1 ± 1.5 biorientation events per chromosome (Figure 5H, n = 200 from 5 oocytes). Sixty-seven percent of bivalent stretching events collapsed or stalled and were thus not able to establish stable chromosome biorientation. This is most likely due to frequent improper kinetochore-microtubule attachments that would require to be dissolved to be corrected (Nezi and Musacchio, 2009Nezi L. Musacchio A. Sister chromatid tension and the spindle assembly checkpoint.Curr. Opin. Cell Biol. 2009; 21: 785-795Crossref PubMed Scopus (117) Google Scholar). Indeed, we found that some chromosomes rotated between biorientation events, reverting their orientation between spindle poles (Figure S4C), demonstrating that kinetochore-microtubule attachments were lost and reformed. The dynamic nature of kinetochore-microtubule attachments was additionally indicated by the fact that the amplitude of chromosome oscillations along the spindle axis was maximal in phase 3 (Figures S4D–S4F). Together our results demonstrate that chromosome biorientation is unexpectedly error prone in meiosis I. Most chromosomes underwent multiple kinetochore-microtubule attachments that presumably had to be actively corrected before reaching stable biorientation. The frequently observed collapse or stalling of bivalent stretching suggested that kinetochore-microtubule attachments are erroneous. Observation of single kinetochore fibers was not possible in live oocytes due to the very high density of non-kinetochore microtubules in the meiotic spindle. To directly investigate kinetochore-microtubule attachments, we therefore immunostained kinetochores and microtubules after destabilizing dynamic non-kinetochore microtubules with calcium in a fixed cell time course (Figure S5G ). This allowed us to systematically analyze the nature of the kinetochore-microtubule attachment throughout meiosis (Figures 6A and 6B ; and Figure S5H).Figure 6Predominant Improper Kinetochore-Microtubule Attachments in Phase 3Show full caption(A) Oocytes were stained with anti-Tubulin (microtubules, green), CREST (kinetochores, red), and Hoechst 33342 (chromosomes, blue) after brief treatment with a 0.1 mM Ca2+-containing buffer (see Extended Experimental Procedures). Maximum intensity z projection images across the whole spindle are shown in the top panel. Z projection images of four selected sections are shown in the bottom panel. Circles and numbers indicate the kinetochores magnified in (B). Time after NEBD (hr). Scale bar is 5 μm.(B) Magnified views for the kinetochore-microtubule attachments in the oocytes shown in (A). Kinetochore-microtubule attachments are classified into three categories: “Mixed/Undefined” (green), “Merotelic/Lateral” (red), and “Amphitelic” (blue). The images are enwrapped with squares colored according to the categories. Scale bar is 1 μm. The full list of the magnified views for the kinetochore-microtubule attachments are in Figure S5H.(C) All kinetochores (n = 40) in an oocyte are classified according to their kinetochore-microtubule attachments. The fraction of each category is shown over time, relative to NEBD. Averages and standard deviations from 3 oocytes at each time point are shown. Note that phase 3 is prolonged until 5 hr in this particular experiment, which is evident from quantitative analysis of the chromosome distribution and the spindle (Figures S5A–S5F), presumably because of a temperature control problem during oocyte collection every hour.(D) Biorientation losses (>20% consecutive decrease of normalized interkinetochore distance) are counted over time from the live imaging data. Averages and standard deviations from 5 oocytes are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Oocytes were stained with anti-Tubulin (microtubules, green), CREST (kinetochores, red), and Hoechst 33342 (chromosomes, blue) after brief treatment with a 0.1 mM Ca2+-containing buffer (see Extended Experimental Procedures). Maximum intensity z projection images across the whole spindle are shown in the top panel. Z projection images of four selected sections are shown in the bottom panel. Circles and numbers indicate the kinetochores magnified in (B). Time after NEBD (hr). Scale bar is 5 μm. (B) Magnified views for the kinetochore-microtubule attachments in the oocytes shown in (A). Kinetochore-microtubule attachments are classified into three categories: “Mixed/Undefined” (green), “Merotelic/Lateral” (red), and “Amphitelic” (blue). The images are enwrapped with squares colored according to the categories. Scale bar is 1 μm. The full list of the magnified views for the kinetochore-microtubule attachments are in Figure S5H. (C) All kinetochores (n = 40) in an oocyte are classified according to their kinetochore-microtubule attachments. The fraction of each category is shown over time, relative to NEBD. Averages and standard deviations from 3 oocytes at each time point are shown. Note that phase 3 is prolonged until 5 hr in this particular experiment, which is evident from quantitative analysis of the chromosome distribution and the spindle (Figures S5A–S5F), presumably because of a temperature control problem during oocyte collection every hour. (D) Biorientation losses (>20% consecutive decrease of normalized interkinetochore distance) are counted over time from the live imaging data. Averages and standard deviations from 5 oocytes are shown. Up" @default.
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• W2126460280 date "2011-08-01" @default.
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• W2126460280 title "Complete Kinetochore Tracking Reveals Error-Prone Homologous Chromosome Biorientation in Mammalian Oocytes" @default.
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• W2126460280 doi "https://doi.org/10.1016/j.cell.2011.07.031" @default.
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