RESEARCH ARTICLE

Anaphase asymmetry and dynamic repositioning of the divisionplane during maize meiosisNatalie J. Nannas1, David M. Higgins1 and R. Kelly Dawe1,2,*

ABSTRACTThe success of an organism is contingent upon its ability to transmitgenetic material through meiotic cell division. In plant meiosis I, theprocess begins in a large spherical cell without physical cues toguide the process. Yet, two microtubule-based structures, the spindleand phragmoplast, divide the chromosomes and the cell withextraordinary accuracy. Using a live-cell system and fluorescentlylabeled spindles and chromosomes, we found that the process self-corrects as meiosis proceeds. Metaphase spindles frequently initiatedivision off-center, and in these cases anaphase progression isasymmetric with the two masses of chromosomes traveling unequaldistances on the spindle. The asymmetry is compensatory, such thatthe chromosomes on the side of the spindle that is farthest from thecell cortex travel a longer distance at a faster rate. The phragmoplastforms at an equidistant point between the telophase nuclei rather thanat the original spindle mid-zone. This asymmetry in chromosomemovement implies a structural difference between the two halves of abipolar spindle and could allow meiotic cells to dynamically adapt toerrors in metaphase and accurately divide the cell volume.

KEY WORDS: Chromosome, Spindle, Phragmoplast, Meiosis

INTRODUCTIONOrganisms package their genetic material into chromosomes thatmust be faithfully separated into two new cells during division.Although this process is crucial in mitosis where mistakes can leadto tumorigenesis (Gordon et al., 2012; Stumpff et al., 2014),mistakes in meiosis result in congenital birth defects and disorders(Nagaoka et al., 2012), and embryo lethality (Hyde and Schust,2015). Despite being more error-prone than mitosis (Nagaokaet al., 2012), less is known about the meiotic regulation of spindleassembly and chromosome segregation. Female meiosis inmammals (Schatten and Sun, 2009), Caenorhabditis elegans(Sumiyoshi et al., 2002), Drosophila (Casal et al., 1990) andXenopus (Kalt, 1973) are different from their mitotic and malemeiotic divisions. They must assemble spindles, position themwithin the cell volume and segregate chromosomes, all without theorganizational support of centrosomes (Dumont and Desai, 2012).The lack of centrosomes could account for the higher rates of mis-segregation seen during female meiosis compared to male meiosis(Hassold and Hunt, 2001). A study on human oocytes has foundthat less than 20% of meiosis I spindles are stable. The majority ofthese spindles cannot focus their poles, form multi-polar spindles

or have a high frequency of lagging chromosomes (Holubcováet al., 2015).

Female meiosis in animals is also known for unequal cellulardivision (Brunet and Verlhac, 2011). In mammals, the meioticspindle is positioned near the cell periphery to produce one largeegg and three small polar bodies (Brunet and Verlhac, 2011).Mistakes in spindle positioning are characteristic of aging and low-quality oocytes that do not produce successful embryos (Brunet andVerlhac, 2011). Meiotic spindles are positioned at the cell cortex byactin microfilaments (Longo and Chen, 1985), their nucleatorFORMIN2 (Dumont et al., 2007; Leader et al., 2002) and myosins(Simerly et al., 1998; Weber et al., 2004). A dense actin networkinteracts directly with both chromosomes (Longo and Chen, 1985;Maro et al., 1986) and microtubules (Azoury et al., 2008) to positionthe spindle at the cell cortex for asymmetric division. In otherasymmetric cell divisions, such as the first zygotic division ofC. elegans (Cowan and Hyman, 2004) or Drosophila neuroblastdevelopment (Yu et al., 2006), spindles are positioned within thecell volume through cortical pulling forces (Kiyomitsu, 2015).Astral microtubules interact with the minus-end-directed motordynein, which is asymmetrically anchored on one side of the cellcortex through G-coupled protein receptors (Kotak et al., 2012), andthe spindle is pulled into position (Siller and Doe, 2009). Corticalpulling is also used to position spindles symmetrically, but in thesedivisions dynein localization is non-polarized. Accurate spindlepositioning within the cell volume is crucial because the spindlemid-zone has been shown to set the location of the cleavage furrowand, thus, the size and shape of resulting daughter cells (Burgess andChang, 2005).

Much less is known about spindle positioning and chromosomesegregation in plants, where all cell divisions occur in the absence ofcentrosomes (Schmit, 2002; Wasteneys, 2002; Zhang and Dawe,2011). Meiosis in plants such as maize have been heavily studied atthe level of fixed specimens (Dawe, 1998; Zamariola et al., 2014)but far less studied at the level of live cells (Yu et al., 1997, 1999),leaving gaps in our understanding of spindle and chromosomemovements.

In this study, we imaged live-cell division during male maizemeiosis I and meiosis II. Maize meiotic cells are easily collectedfrom immature tassels and provide an excellent system in which tostudy acentrosomal spindles. Early attempts to live-image maizemeiosis were limited to observing only chromosome movementswith a cell-permeant DNA stain (Yu et al., 1997), but a newlydeveloped fluorescent tubulin fusion (Mohanty et al., 2009) hasallowed this first study of live meiotic spindle dynamics. Imagingmeiosis I and meiosis II spindles revealed an unexpected andpreviously undocumented phenomenon – the separation ofchromosomes is not consistently symmetric. The observedasymmetry is limited to anaphase A and correlates with theposition of the spindle relative to the cell cortex. In cases where thespindle was positioned significantly off-center, the mass ofReceived 8 July 2016; Accepted 5 September 2016

1Department of Plant Biology, University of Georgia, Athens, GA 30605, USA.2Department of Genetics, University of Georgia, Athens, GA 30605, USA.

*Author for correspondence (kdawe@uga.edu)

N.J.N., 0000-0002-2617-8822; R.K.D., 0000-0003-3407-4553

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chromosomes furthest from the edge of the cell moved faster andfarther, helping to adjust the position of the chromosomes and tocorrect the initial asymmetry. Additional data show that thephragmoplast, the plant equivalent of the cleavage furrow whichdetermines the plane of division (Otegui et al., 2005), forms midwaybetween the chromosome masses and not at the spindle mid-zone,helping to assure that cell division accurately divides the cell intoequal parts.

RESULTSChromosome segregation dynamics during male maizemeiosis I and meiosis IIChromosome segregation in male maize meiosis was studied byperforming live imaging. Microtubules were labeled with cyanfluorescent protein (CFP) fusion with β-tubulin (Mohanty et al.,2009), and chromosomes were labeled with SYTO12, a greennucleic acid stain that penetrates live cells (Yu et al., 1997). Meioticcells (meiocytes) were extruded from anthers into a culture mediumthat has been previously demonstrated to support cell growth (De LaPeña, 1986; Yu et al., 1997) and were imaged by using fluorescencemicroscopy. Spindle morphology and chromosome movementswere tracked over time, and movies were captured of both meiosis I(Fig. 1A;Movie 1) and meiosis II (Fig. 1B;Movie 2).We confirmedthat the CFP–tubulin tag did not affect spindle dynamics bycomparisons with unlabeled fixed cells (Fig. S1A,B). Allcharacterization and data analysis presented below is based on 41live cells.In both meiosis I and meiosis II, chromosomes align on the

spindle metaphase plate, separate into two masses in anaphase anddecondense into two new nuclei that are separated by a growingphragmoplast (n=41). Anaphase I lasted an average of 12.7±3.2 min(mean±s.d.), and anaphase II lasted an average of 11.0±3.7 min; thistiming was not statistically different (Student’s t-test, P=0.36).Anaphase was defined as the period of time from chromosomeseparation until spindle breakdown. Only one lagging chromosomewas observed out of 41 imaged cells (2%) (Fig. 1C); it remained inthe center of the spindle and was not pulled to one pole. Maizemeiotic chromosome segregation is thus more stable than humanfemale meiosis where these ‘persistent’ lagging chromosomes havebeen observed in 40% of cells (Holubcová et al., 2015). In 17% ofcells, at least one chromosome was observed to be trailing the mainmass after initial separation but always caught up to the mass within5–10 min (Fig. 1C). Because these slow-moving chromosomeshave not been previously reported in studies on fixed cells (Yu andDawe, 2000; Li and Dawe, 2009), we cannot rule out the possibilitythat culturing and imaging the meiocytes impact chromosomesegregation. We also observed astral-like microtubules in somespindles that appeared to contact and curl around the cell cortex(Fig. S2A).Spindle dynamics are similar in both meiosis I and meiosis II.

Metaphase spindle length in meiosis I is 34.2±3.6 μm and35.3±5.2 μm in meiosis II (mean±s.d.) (Fig. 2A). Eukaryoticchromosomes are separated in anaphase through two mechanisms –movement of chromosomes to the spindle poles (anaphase A) andelongation of the spindle with the poles pushing apart (anaphase B)(Fig. 2B). The usage of these two mechanisms and their degree ofcontribution to chromosome separation varies from species tospecies (Maiato and Lince-Faria, 2010). We found that spindles didnot elongate from their metaphase length during anaphase(Fig. 2A). In fact, spindles were significantly shorter 20 min afteranaphase onset during both meiosis I (24.9±7.0 μm, P<10−5) andmeiosis II (23.7±5.2 μm, P=0.01) (Fig. 2C). The segregation of

maize chromosomes appears to be exclusively achieved throughmovement in anaphase A, with no observed contribution duringanaphase B.

Chromosome movement to the poles (anaphase A) exhibitsdifferences between meiosis I and meiosis II. Although spindlelength was the same, chromosomes in meiosis II were pulledsignificantly further apart than in meiosis I. Homologouschromosomes were pulled an average of 5.6±2.1 μm (mean±s.d.)from the metaphase plate to the poles in meiosis I, and sisterchromatids were pulled 6.7±0.7 μm (Fig. 2D, P=0.001). If wenormalize by spindle length, where being pulled 50% of spindlelength means being pulled from the metaphase plate all the way tothe poles, meiosis I chromosomes were pulled an average of32±12% (mean±s.d.), and meiosis II chromosomes were pulled40±8% (Fig. S3A, P=0.009). Meiosis II chromosomes were pulledcloser to the poles of the spindle, both in terms of raw distance andas a percentage of spindle length.

Asymmetric chromosome segregation during anaphase AWe found that the standard deviation of chromosome segregationdistance (anaphase A distance) was larger during meiosis I thanduring meiosis II (Fig. 2D), suggesting more variation between thedistances traveled by each mass of chromosomes. We tracked themovement of the twomasses over time and found that chromosomesmoved asymmetrically on the spindle in anaphase (Fig. 3A). Fig. 3Bshows an example of an asymmetric division where thechromosome masses are outlined in white and tracked in threedimensions from metaphase (T=0 min) through anaphase (T=5–10 min). The asymmetry was quantified by designating onechromosome mass as ‘A’ and determining that it traveled distanceDA, and designating the other mass as ‘B’ and that it traveleddistance DB. The difference in segregation distance is thenΔDA−B=DA−DB (Fig. 3C). A small ΔDA−B value means that thetwo masses of chromosomes traveled approximately equal distancestoward their respective poles, whereas a large value means thechromosomes segregated asymmetrically.

The asymmetry in chromosome movement was statisticallysignificant. The average total distance traveled by mass A (DA) wasstatistically greater than that of mass B (DB) (Fig. 3D; 6.6±1.9 μmvs4.9±1.7 μm, P<10−4; mean±s.d.). Mass A also traveled further thanmass B over multiple time points throughout anaphase, beginning10 min after the metaphase–anaphase transition (P<0.004)(Fig. 3E). The average difference in chromosome segregationdistance (ΔDA−B) was twice as large in meiosis I than in meiosis II(Fig. 3F; 1.8±1.5 μm vs 0.9±0.6 μm, P=0.02), meaningchromosomes segregated more asymmetrically during meiosis I(Fig. 3F). Approximately half of meiosis I cells segregated in asimilar manner to meiosis II cells with less than 2-μm difference,whereas the other half traveled quite asymmetrically with a range of2- to 7-μm difference in chromosome paths (Fig. 3G).

To better compare chromosome movements between cells, DA

and DB were normalized to total movement (DA+DB). Insymmetrically segregating cells, DA and DB were nearly equal,thus each contributed 50% to chromosome movement. Thepercentage of total chromosome movement attributed to eachchromosome mass was plotted and ordered by symmetry (Fig. 3H).Plotting this data shows that there were not two discrete populationsof symmetric and asymmetric cells; instead, we observed acontinuum of asymmetry from 50–50% to 80–20% at theextremes. A plot of all cells and their non-normalizedchromosome segregation distances, as well as their spindlelengths can be found in Fig. S3B. The asymmetry in chromosome

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segregation distance (ΔDA−B) was not correlated with spindle length(Fig. S3C), total distance traveled during anaphase A (Fig. S3D) orthe presence of slow-moving chromosomes (Fig. S1C).The timing of anaphase was not correlated with asymmetry

(Fig. S3E), and chromosomes that traveled further did not spend alonger time moving (P=0.4), suggesting that the two masses ofchromosomes move at different rates. The data show that in 95% ofthe observed cells (39/41), the chromosome mass that traveledfurther (mass A) did so at a faster rate than the chromosome massthat moved the shorter distances (mass B) (P<0.0001, Fisher’s exacttest). Both the average rate of segregation and the maximum speedwas greater for chromosome mass A than that for mass B (Fig. 3I;P=0.003 and P=0.05, respectively; see Fig. S3G,H for plot of allcells). Because cells showed a continuum of symmetry phenotypesrather than two discrete populations (Fig. 3H), we compared the top25% most asymmetric divisions with the 25% least asymmetricdivisions (symmetric divisions). By comparing top and bottomquartiles for the phenotype, we found that in symmetric divisionsthere was no difference in mass A and mass B speeds at any pointduring anaphase (Fig. S3F; P>0.7), but in asymmetric divisionsmass A was faster than mass B at both 5 and 10 min post-transition

to anaphase (Fig. 3J; P≤0.005; see Fig. S3I for plot of all cells).There was also a significant increase in the speed of chromosomemass A from the 5-min time point (0.58 μm min−1) to that at the10-min time point (0.90 μm min−1, P=0.01). The difference in theinitial rate of movement between masses A and B suggests that thechromosome masses could be under different force conditions atmetaphase.

Asymmetry is a corrective mechanism resulting from offsetspindlesAs they segregate at different rates, the two masses of chromosomescould be experiencing different forces at the metaphase–anaphasetransition. Forces are generated on chromosomes during metaphaseas microtubules depolymerize and pull the paired chromosomestoward the poles, but these forces are resisted by chiasma duringmeiosis I and cohesin during meiosis II. Microtubulepolymerization, chromosome elasticity and viscous drag alsogenerate force, which all balance to align chromosomes atmetaphase (Dumont and Mitchison, 2009). It is possible that thechromosomes in asymmetrically dividing cells were not aligned inthe center of the spindle at the start of anaphase, and thus

Fig. 1. Live-imaging of maize male meiosis. Meiosiswas imaged in live male maize cells using CFP–tubulin(β-TUB1) to label spindles (green) and SYTO12 DNAstain to label chromosomes (magenta). The cell volumecould be determined by using the visible diffuseunincorporated CFP–tubulin monomers in the cytoplasm.Timing of anaphase in meiosis I (A) and meiosis II (B) wasnot statistically different (Student’s t-test, P=0.36), lastingapproximately 12 min from chromosome separation tospindle breakdown. During both meiosis I and meiosis II,chromosomes were pulled towards the poles (anaphaseA) but there was no visible elongation of the spindle(anaphase B). The phragmoplast, a microtubule-basedstructure that directs cell division, appeared between thenew nuclei after spindle breakdown during both meiosis Iand meiosis II. Images in panels A and B are frames fromMovies 1 and 2, respectively. (C) Of the 41 live-meiosisimages, only one cell showed a lagging chromosome thatremained behind on the spindle and was not pulled to apole (white arrow). In approximately 20% of cells, a slow-moving chromosome initially trailed behind the mainchromosome mass, but rejoined the main mass duringanaphase (white arrows). Scale bars: 10 μm. n=41 cells.(D) Most live divisions had uniform separation ofchromosomes (80%, 33). As shown in C, ~20% ofdivisions had a slow-moving chromosome (7) and 2% hada lagging chromosome (1).

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experienced unequal forces, driving them apart (Fig. 4A). Thethree-dimensional location of the spindle center was measured andsubtracted from the three-dimensional location of the metaphasechromosomes to determine the chromosome–spindle offset(ΔChrom-Spindle). If the asymmetric movement is caused bymisalignment of the chromosomes, we would expect the offsetdistance to be greater in the top quartile of asymmetric divisions, asone mass must travel further to approach its pole (DA>DB; Fig. 4A).Chromosomes were offset from spindle center by 0.7±0.4 μm(mean±s.d.) in asymmetric divisions and by 0.7±0.3 μm insymmetric divisions (Fig. 4B), with no significant difference inoffset. Thus, the asymmetric movement was not due to a failure toalign chromosomes in a central position on the spindle.Positioning of the spindle within the cell volume can define the

size of daughter cells, as the site of cell division is often specified bythe spindle mid-zone in both animals and plants (Kiyomitsu, 2015).Given that four equally sized products is the optimal outcome inmaize male meiosis, it is possible that the asymmetric segregation isa post-metaphase correction mechanism to pull chromosomes intoequal volumes when the spindle is not centered in the cell (Fig. 4C).If this were true, we would expect spindles in asymmetric divisionsto be more offset from the cell center (ΔSpindle-Cell) thanin symmetric divisions. Spindles in asymmetric divisions wereindeed more offset (3.3±2.2 μm) than those in symmetric divisions(1.5±0.9 μm, P=0.04) (Fig. 4D).We also expect that the further-moving chromosome mass (mass

A) should travel in the direction of larger cell volume (Fig. 5A). Wemeasured the distance from spindle pole to cell cortex (distances Xand Y) and found our expectation to be true. In the top quartile of

asymmetric divisions, all cells had greater chromosome movementon the side of the spindle furthest from the cell cortex (distance X),which was significant compared to randomly oriented movement(Fisher’s exact test, P=0.01) (Fig. 5B). The difference in distancebetween the pole and cell wall (X−Y) was greater in asymmetricdivisions, both in terms of absolute distance (X−Y, P=0.003) andwhen normalized for cell size (X−Y/X+Y, P=0.002) (Fig. 5C). Incells in which spindles were offset by more than a standarddeviation, asymmetry of chromosome movement was significantlygreater than the average of all observed cells (P=0.01; Fig. 5D).

Most convincingly, the extent to which spindles were offsetcorrelated with asymmetric movement such that the greater thespindle was shifted towards one wall, the greater the difference inchromosome movement. The position of the spindle in the cell canexplain ∼59% of the observed anaphase asymmetry (R2=0.588;Fig. 5E). Two cells were excluded from this analysis owing toconfounding features that could have increased asymmetry in waysthat were unrelated to spindle position (red data points in Fig. 5E).One had a persistent lagging chromosome with possible merotelicattachments, which impede chromosome movement towards onepole, and the other had a large spindle morphology defect. Images ofthese divisions can be found in Fig. S2B,C. No other cells showedsignificant aberrations in chromosome alignment or spindle shape.

Asymmetry is often dictated by spindle position (Siller and Doe,2009). In some systems, such as the first zygotic division inC. elegans, the whole spindle is shifted during anaphase to achievethe desired location within the cell volume (Li, 2013). To determineif offset spindles shift toward the cell center, we measured the pole-to-wall distance (X) throughout anaphase (T=0–20 min) in the top

Fig. 2. Characterization of spindle and chromosome dynamics in meiosis I versusmeiosis II. (A) Maize meiotic spindles do not elongate during anaphase.Themean length of meiosis II spindles did not statistically change frommetaphase to anaphase (P=0.37), whereasmeiosis I spindles shortened slightly (P=0.02).(B) Chromosomes that aligned on the metaphase spindle were segregated as a result of two types of movement – movement of chromosomes to the poles(anaphase A) and elongation of the spindle that drives the poles apart (anaphase B). Organisms utilize one or both of these mechanisms with varying levels ofcontribution to separate their chromosomes. (C) Spindle length tracked over time in meiosis I (black circles) and meiosis II (gray boxes) showed a shorteningrather than elongation of spindles (meiosis I, P<10−5; and meiosis II, P=0.01; Student’s t-test), demonstrating that there is no anaphase B in male maize meiosis.The blue box marks anaphase. (D) Chromosomes were separated entirely by movements during anaphase A, which on average, comprised longer distances inmeiosis II than in meiosis I (P=0.001). All error bars represent standard deviation from the mean. n=35 for meiosis I and n=6 for meiosis II. Unless otherwiseindicated, all P-values were calculated using Student’s t-test. *P<0.05.

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quartile of asymmetric divisions. Distance at each time point (XT)was normalized to the initial pole–wall distance (X0), such that avalue less than 1 indicates a decrease in distance and shifting of thespindle towards cell center (Fig. 5F). The data show that the averagepole–wall distance was not significantly different at the end ofanaphase than at the beginning. Because the spindles in our assaydid not shift (Fig. 5F) or elongate (Fig. 2A,C), the observedasymmetric movement is due solely to unequal movement of

chromosomes toward their poles. This represents a new mechanismfor positioning chromosomes within a cell volume.

The phragmoplast is established at a site equidistant fromchromosomes, not at the spindle mid-zoneFollowing chromosome segregation during anaphase, cytokinesisstructures form at the spindle mid-zone to split the cell in two(Otegui et al., 2005). These include the cleavage furrow in animals

Fig. 3. Asymmetric anaphase-A chromosome segregation. (A) Chromosomes (magenta) travel unequal distances on the spindle (green) from their startingposition on themetaphase plate. Example stills from amovie are shown in Bwhere onemass of chromosomes, whose path is denoted by the orange line, travels ashorter distance than the mass of chromosomes marked with the blue path. Scale bars: 10 μm. (C) A schematic of the asymmetric division; the further-movingchromosome mass A is in blue and moves distance DA, the shorter-moving chromosome mass B is in orange and moves distance DB. The asymmetry iscalculated as DA−DB and represented as ΔDA−B. (D) Themean distance (dist) traveled by chromosomemass A is statistically greater than that of mass B (P<10−4,n=41). (E) Chromosome asymmetry was statistically significant within 10 min of the metaphase–anaphase (meta-anaphase) transition (P<0.004, n=28). Thegray box represents anaphase, which starts at the metaphase–anaphase transition (T=0 min). (F) The average asymmetry is greater in meiosis I than meiosis II(P=0.02). (G) A histogram of asymmetry values shows that cells in meiosis I exhibit a wide range of variation in this asymmetry phenotype, n=41. (H) The range ofasymmetry is displayed by normalizing the distance traveled during anaphase A to 100%. In symmetric divisions, each chromosomemass (mass A, blue; mass B,orange) traveled 50% of the total distance in anaphase; during asymmetric division, a greater percentage of the distance in anaphase A was attributed tochromosomemass A. The data is sorted by increasing asymmetry, n=41. The most asymmetric division is represented by the left-most bar, wheremass A travels80% of distance during anaphase A, whereas mass B traveled only 20%. (I) On average, chromosome mass A moved statistically faster than mass B, both inaverage speed (P=0.003) and maximum speed (P=0.05), n=41. See Fig. S3G,H for individual cell data. (J) In the top quartile of asymmetric divisions (n=10),chromosome mass A moved faster than mass B at both 5 and 10 min post-metaphase–anaphase transition (T=5 min, P=0.037; T=10 min, P=0.019), andchromosomemass A sped-up over time (5 vs 10 min, P=0.038). All error bars represent standard deviation from the mean and P-values were calculated by usingStudent’s t-test, *P<0.05. See Fig. S3I for individual cell data.

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(Gould, 2016) and the phragmoplast in plants (Müller and Jürgens,2016). Our observations indicated that chromosomes areasymmetrically pulled away from the spindle mid-zone,suggesting that the distance from the phragmoplast to thechromosomes might also be asymmetric (Fig. 6A, option 1).However, anaphase asymmetry (ΔDA−B) clearly did not correlatewith the cytokinesis asymmetry (ΔPA−B) (Fig. 6B; R2=0.0007) and,instead, the phragmoplast formed at a site that was equidistant fromthe chromosome masses (Fig. 6A, option 2, 6C). The distance fromphragmoplast to chromosome mass strongly correlated with themidpoint distance between chromosomes, calculated by averagingDA and DB (Fig. 6C, R2=0.8424). By establishing itself relative tothe position of the chromosomes, the phragmoplast might be able toprovide a back-up mechanism to segregate chromosomes andensure that each daughter cell receives at least some geneticmaterial. We observed two instances where the spindle failed tosegregate chromosomes, in which the phragmoplast was able topush chromosomes apart into two masses (Fig. 6D).

DISCUSSIONWe investigated the dynamics of chromosome segregation in malemaize meiosis; a system that segregates chromosomes with anacentrosomal spindle (Zhang and Dawe, 2011). We found that thetwo masses of chromosomes did not segregate consistently equaldistances (Fig. 3D). Instead, we saw a large variation in anaphase Adistance, with masses of chromosomes traveling asymmetricdistances on the spindle. In the most extreme case, one mass ofchromosomes traveled 80% of the total anaphase A distance,whereas the other mass traveled only 20% (Fig. 3H). Theasymmetry observed here is different from the high incidence of

lagging chromosomes seen in human (Holubcová et al., 2015) andmouse (Yun et al., 2014) oocytes, as well as in the perturbed meiosisin other species (Dumont et al., 2010). In these previous studies, thetwo masses of chromosomes moved apart equally, with individualchromosomes lagging behind; here, we saw that all ten of thechromosomes on one side of the spindle frequently moved atdifferent speeds than those of the chromosomes on the other side(Fig. 3I).

To our knowledge, asymmetrical segregation during anaphase Ahas not been previously described. Previous live-imaging studies ofmeiosis in human (Holubcová et al., 2015), mouse (Lane et al.,2012), C. elegans (Dumont et al., 2010; Segbert et al., 2003) andDrosophila (Gilliland et al., 2007) showed no evidence ofasymmetry, and the previous data on mitosis also suggest thatanaphase A is predictably symmetric. There are, however, a fewdocumented examples of unequal chromosome movement duringanaphase B. InDrosophila (Kaltschmidt et al., 2000) and C.elegans(Ou et al., 2010) neurogenesis, chromosomes are segregated by acentrally positioned spindle, and in anaphase B, one side of thespindles elongates further than the other, creating a large neuroblastand a small ganglion mother cell (Kaltschmidt et al., 2000; Li, 2013;Ou et al., 2010). In Drosophila, the asymmetry in spindlemorphology is created by a larger centrosome that nucleates moremicrotubules to pull the chromosomes further in one direction (Caiet al., 2003), and in C. elegans, polarization of myosin II squeezesone side of the spindle, allowing the other side to preferentiallyelongate (Ou et al., 2010). Additionally, in a study on mitotictobacco culture cells, unequal elongation during anaphase B pullschromosomes further on one side (Hayashi et al., 2007). Althoughthere is evidence for anaphase B in mitotic maize cells (Duncan and

Fig. 4. Asymmetry is related to spindle position, not chromosome congression. (A) A model showing chromosome offset within the spindle (ΔChrom-Spindle), which could explain asymmetry. Asymmetry might occur when the center of the chromosome mass (white circle) is offset from the center of the spindle(black circle), and chromosomes move further (blue arrow) to compensate for this offset. (B) There was no difference in chromosome offset between asymmetricand symmetric divisions. (C) A model showing how spindle offset within the cell (ΔSpindle-Cell) might explain anaphase asymmetry. Offset of the spindle center(white circle) from cell center (black circle) could promote chromosomes to move further on one side (blue arrow) to correct for initial spindle position.(D) Asymmetric divisions had significantly larger spindle offsets than symmetric divisions (P=0.04). Error bars represent standard deviation from the mean,P-values were calculated by using Student’s t-test, *P<0.05, n=10 for both asymmetric and symmetric divisions.

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Persidsky, 1958), we have found no evidence for anaphase B inmeiosis because the spindle does not elongate (Fig. 2A,C). Ourobservations indicate that all of the asymmetry occurs duringmovement during anaphase A (Figs 2E–I and 5F). Maize does nothave centrosomes to create an imbalance in microtubule nucleation,and there is no evidence of polarized actin in maize meiosis (Staigerand Cande, 1991), so neither of these established mechanisms canexplain the asymmetry seen here.The function of asymmetric chromosome segregation is typically

to move chromosomes into unequal cell volumes to producedaughter cells of different sizes and/or cell fates (Li, 2013).Asymmetry generally arises from the orientation and positioning ofthe spindle (Siller and Doe, 2009). In mouse meiosis, the spindle ispositioned near the cell periphery to create one large egg and threesmall polar bodies (Brunet and Verlhac, 2011). In the first zygoticdivision of C. elegans, the spindle is offset from cell center,producing a small posterior cell that gives rise to the germline andmuscle, and a larger anterior cell that becomes all other somatictissue (Gilbert, 2006; Li, 2013). Asymmetry and orientation of the

spindle is also used in stem cells to produce one self-renewing andone differentiating daughter (Knoblich, 2008). Maize male meiosispresents the opposite problem, where the spindle is frequently offsetfrom the cell center but the final products of meiosis are highlyuniform in size. If the purpose of anaphase asymmetry in maize is toplace the new nuclei in equal volumes, it should be correlated withhow offset the spindle is within the cell (Fig. 5A). We find that thereis indeed such a correlation, as the greater the spindle was shiftedtowards the edge of the cell, the greater the asymmetry (Fig. 5E).When the spindle was offset to a large extent (the top quartile ofasymmetric divisions), chromosomes always moved further on theside of the spindle with greater pole–wall spacing (Fig. 5B). Thespindle itself did not move (Fig. 5F), but the rate and distance ofchromosome movement within the spindle served to correct forirregularities in spindle position and to help to place chromosomesinto opposite hemispheres of the cell.

The phragmoplast forms the new cell wall after cell division inplants. In mitotic cells, the position of both the spindle andphragmoplast is dictated by the pre-prophase band, a microtubule

Fig. 5. Asymmetric chromosome movement determined by distance from cell cortex. (A) A model showing how chromosomes on the side of thespindle furthest from the cell cortex (X) might move further at anaphase (chromosome mass A, blue). Chromosomes on the side closest to the cortex (Y) wouldthen move the shorter distance (chromosome mass B, orange). The pole–wall offset was normalized to the combined distance between poles and cell cortex.(B) Histogram showing all divisions in order of asymmetry (ΔDA−B), which is the difference in movement of the two chromosome masses (see Fig. 3C for ΔDA−B

diagram, n=41). Cases that matched the prediction in A, where the further-moving chromosome mass Awas most distant from the cell cortex, are colored black.The trend was most apparent for highly asymmetric divisions. (C) The normalized pole–wall offset was statistically greater in asymmetric divisions than insymmetric divisions (P=0.002, n=10 for each). (D) In cells that had spindles offset from the cortex bymore than a standard deviation (Offset, n=5), asymmetry wasstatistically larger when compared to that of all observed cells (All, n=41) (P=0.01). (E) Asymmetry correlated with normalized pole–wall offset. Two data points(red) were excluded from the analysis owing to spindle errors (see text for discussion, n=39). (F) The distance from pole to wall did not significantly change fromthe beginning of anaphase (T=0 min) to the end of anaphase (T=20 min). The pole–wall distance (X) was normalized to the initial distance (ΔX0−T) such that avalue less than 1 meant the distance had decreased (n=10). Error bars represent standard deviation from the mean, and P-values were calculated by using aStudent’s t-test, *P<0.05.

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array around the cell periphery (Mineyuki et al., 1991), and itsassociated proteins, TONNEAU (Azimzadeh et al., 2008; Camilleriet al., 2002; Traas et al., 1987) and TANGLED (Cleary and Smith,1998; Smith et al., 1996). Meiotic plant cells lack a pre-prophase

band (Chan and Cande, 1998), and analysis of fixed specimenssuggests that phragmoplasts are formed from the central fibers of thespindle that remain after chromosomes segregation (Otegui andStaehelin, 2000; Shamina et al., 2007; Staehelin and Hepler, 1996).

Fig. 6. The phragmoplast is established at a site that is equidistant from chromosomes. (A) The separation of chromosomes in anaphase is oftenasymmetric. In these instances, the phragmoplast could either form (1) at the position of the original metaphase plate (spindle mid-zone) or (2) midway betweenthe chromosomes. If the phragmoplast is established at the mid-zone (option 1), the asymmetry seen in anaphase (ΔDA−B) should be roughly equal to theasymmetry in cytokinesis (ΔPA−B). If the phragmoplast appears midway between the chromosomes (option 2), the distance from phragmoplast to chromosome(PA) should be approximately equal to the mean of DA and DB. (B) Plot to test option 1. The scatter plot shows that phragmoplast position (cytokinesis asymmetry,ΔPA−B) was not correlated with anaphase asymmetry (ΔDA−B), thus it was not located at the spindle midzone. Red dots indicate cells excluded from correlationanalysis, see text and Fig. S2B,C for explanation of excluded cells (n=39). (C) Plot to test option 2. The scatter plot shows that phragmoplast position (distancefrom chromosome mass A to the phragmoplast, PA) was strongly correlated with the midpoint between chromosomes [(DA+DB)/2]. Red dots indicate cellsexcluded from correlation analysis (n=39). (D) The phragmoplast was capable of pushing chromosomes apart when the spindle failed to separate chromosomes.FITC, DNA; CFP, tubulin. Scale bars: 10 μm.

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Although, in most cell types, the position of the metaphase platemarks the location of the spindle mid-zone, owing to anaphaseasymmetry, these positions often differed in our assay. We foundthat the phragmoplast appeared halfway between the twochromosome masses after anaphase rather than at the originalspindle mid-zone (Fig. 6A–C). Dynamic establishment of thephragmoplast relative to chromosomes allowed cells to correct thedivision plane if the spindle was improperly positioned (Fig. 5B,E)and provided a back-up mechanism for segregation when thespindle failed to fully separate chromosomes (Fig. 6D).We found that chromosomes moved farther and faster on the side

of the spindle that was most distant from the cell cortex (Fig. 3I,J,Fig. 5A,B). Maize meiotic cells must have a sensing mechanism inorder to integrate spindle position with chromosome segregationdistances. One possibility is astral microtubules that reach from thespindle poles towards the cortex and relay positional information.Higher plants lack centrosomes (Schmit, 2002; Wasteneys, 2002)and were thought to lack astral microtubules as well (Lloyd andHussey, 2001; Smirnova and Bajer, 1992), but recent microscopyanalyses have revealed astral-like microtubules that reach from theacentrosomal pole towards the cell cortex in Arabidopsis (Chanet al., 2005) and tobacco (Dhonukshe et al., 2005). We observedsimilar astral connections to the cortex in our time-lapse data(Fig. S1A). In animals, astral microtubules work in concert withcortical dynein to move spindles into position (Siller and Doe,2009). However, higher plants lack dynein (Lawrence et al., 2001).It is possible that astral-like microtubules act as messengers,relaying positional information to the spindle and modulatingmicrotubule dynamics. Astral-like microtubules could bind toregulatory molecules such as KLP10 or CLASP, which regulatemicrotubule flux rates in Drosophila and produce a similarasymmetry phenotype with unequal movement of chromosomestowards poles when depleted (Matos et al., 2009). Although verylittle is known about microtubule flux regulation in plants(Dhonukshe et al., 2006), our data suggest that such regulatorscould exist and are sensitive to spindle position within the cell.Overall, our findings demonstrate that positional signals aretransduced from the cortex to modulate chromosome dynamics aslate as anaphase to correct errors in spindle placement.

MATERIALS AND METHODSMaize lines and genotypingAmaize line (Zea mays ssp.mays) containing CFP fused to the N-terminusof β-tubulin (β-TUB1) was generated by the laboratory of Anne Sylvester(University of Wyoming, WY). Plants were genotyped for the CFP–tubulin transgene using a CTAB DNA extraction protocol (Clarke, 2009)on leaf tissue and primers that annealed within the CFP (5′-GGAGTAC-AACTACATCAGCCACAACGTC) and tubulin (5′-CCGGACTGACC-GAAGACGAAGTTGT) sequences. The maize line containing dv1 wasobtained from the Maize Genetics Cooperation Stock Center (Universityof Illinois) and genotyped as previously described (Higgins et al., 2016).All chemicals and reagents, unless otherwise stated, were purchased fromSigma Aldrich.

Live imagingMale meiotic cells were harvested from immature tassels as previouslydescribed (Yu et al., 1997). Meiocytes were extruded from anthers into live-cell imaging medium, pH 5.8–5.9 (De La Peña, 1986; Yu et al., 1997), thatcontained a final concentration of 2 μMSYTO12GreenDNAdye (InvitrogenMolecular Probes). Cells were staged for meiosis I andmeiosis II, loaded ontopoly-L-lysine-coated coverslips (Corning) and sealed onto microscope slides(Fisher Scientific). Cells were imaged on a Zeiss Axio Imager.M1fluorescence microscope with a 63× Plan-APO Chromat oil objective.Images were collected every 3–7 min in three dimensions using a 20-μm

Z-range and 1-μm step sizewith 50-ms exposure for CFP and 30-ms exposurefor SYTO12 and 2×2 binning. Three-dimensional volume renderings of anexample meiosis I cell (Movie 3) and meiosis II cell (Movie 4) demonstratethat cellswere not compressed between the coverslip and slide during imaging.The sample size was 41 cells, and the asymmetric and symmetric categorieswere defined as the top and bottom quartile of the asymmetric phenotype.Sample size was sufficient for statistical power in both Student’s t-test andFisher’s exact test, used as described in the text.

Image analysisImages were analyzed using Slidebook software (Intelligent ImagingInnovations, Denver, CO, USA). Cells, spindles, chromosomes andphragmoplasts were identified as objects by thresholding. Cells, labeledby diffuse cytoplasmic CFP–tubulin monomers, were thresholded atapproximately 35% above CFP background, spindle and phragmoplastsignals at approximately 50% above background, and chromosomes atapproximately 5% above FITC background. Object statistics were extractedincluding center of volume (x, y, z coordinates of the center of the object)and longest chord (distance between the two furthest pixels within theobject). Spindle length was measured as the longest chord within the spindleobject after constrained iterative deconvolution using a calculated point-spread function. Chromosome movements were calculated as the three-dimensional distance between the center of volume at different time points,and chromosome speed was calculated as this distance divided by time. Thetotal distance traveled in anaphase (DA or DB) was calculated as the distancebetween the initial position on the metaphase plate and the final positionnear the spindle pole. The asymmetry value (ΔDA−B) is the difference inthese distances: ΔDA−B=DA−DB. Congression of chromosomes on themetaphase plate (ΔChromosome-Spindle) was calculated as the differencebetween the chromosome center of volume and the spindle center of volumeat metaphase. The offset of the spindle from the cell center (ΔSpindle-Cell)was calculated as the difference between the spindle center of volume andthe cell center of volume. Cytokinesis ΔPA−B is the difference in distancefrom each chromosome mass to the phragmoplast center of volume. The xyzposition of the spindle pole was determined by the edge of the thresholdedspindle outline (50% above deconvolved background CFP signal) as thefurthest point from the center of the spindle, measured using Slidebook rulerfunction. The distance from pole to cell cortex was measured by determiningthe shortest xy distance from the pole to the thresholded outline of the cell(35% above non-deconvolved background CFP signal) within the same zplane as the pole using Slidebook ruler function.

ImmunolocalizationImmunolocalization was performed as previously described (Higginset al., 2016). Anthers from immature tassels were fixed for 60 min in 4%paraformaldehyde-PHEMS buffer (60 mM PIPES, 25 mM Hepes, 10 mMEGTA, 2 mM MgCl2, 0.35 M Sorbitol, pH 6.8), washed three times in1× PBS and dissected for meiotic cells. Staged meiocytes were adhered topoly-L-lysine coverslips by centrifugation at 100 g for 1 min, thenpermeabilized for 1 h in a 1% Triton X-100, 1 mM EDTA, 1× PBSsolution. Coverslips with affixed cells were blocked in 10% goat serumfor 90 min, incubated with a monoclonal antibody against sea urchinα-tubulin (Asai et al., 1982) at 37°C overnight, blocked again with10% goat serum and then incubated with a Rhodamine-conjugatedAffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (JacksonImmunoResearch) for 150 min. Between each step, coverslips werewashed three times with 1× PBS solution. Coverslips were mounted withProLong Gold with DAPI (Thermo Fisher Scientific) and imaged asdescribed above, except there was no binning and exposure times wereoptimized for each channel and ranged from 0.1–1 s.

AcknowledgementsWe thank Caroline Jackson for genotyping the CFP–tubulin plants and Amy Hodgesfor genotyping the dv1 plants, as well as Jonathan Gent and other members of thePlant Biology Department for discussion of the data.

Competing interestsThe authors declare no competing or financial interests.

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Author contributionsConceptualization and methodology: N.J.N., D.M.H., R.K.D.; Investigation: N.J.N.,D.M.H.; Formal analysis: N.J.N.; Writing – original draft preparation: N.J.N.; Writing –

reviewand editing: N.J.N., D.M.H., R.K.D.; Visualization: N.J.N.; Funding acquisitionand resources: N.J.N., R.K.D.; Supervision: R.K.D.

FundingThis study was supported by a National Science Foundation fellowship [grantnumber IOS-1400616 to N.J.N.] and grants [grant numbers MCB-1412063 and IOS-0922703 to R.K.D.].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.194860.supplemental

ReferencesAsai, D. J., Thompson, W. C., Wilson, L. and Brokaw, C. J. (1982). Two differentmonoclonal antibodies to alpha-tubulin inhibit the bending of reactivated seaurchin spermatozoa. Cell Motil. 2, 599-614.

Azimzadeh, J., Nacry, P., Christodoulidou, A., Drevensek, S., Camilleri, C.,Amiour, N., Parcy, F., Pastuglia, M. and Bouchez, D. (2008). ArabidopsisTONNEAU1 proteins are essential for preprophase band formation and interactwith centrin. Plant Cell 20, 2146-2159.

Azoury, J., Lee, K. W., Georget, V., Rassinier, P., Leader, B. and Verlhac, M.-H.(2008). Spindle positioning in mouse oocytes relies on a dynamic meshwork ofactin filaments. Curr. Biol. 18, 1514-1519.

Brunet, S. and Verlhac, M. H. (2011). Positioning to get out of meiosis: theasymmetry of division. Hum. Reprod. Update 17, 68-75.

Burgess, D. R. and Chang, F. (2005). Site selection for the cleavage furrow atcytokinesis. Trends Cell Biol. 15, 156-162.

Cai, Y., Yu, F., Lin, S., Chia, W. and Yang, X. (2003). Apical complex genes controlmitotic spindle geometry and relative size of daughter cells in drosophilaneuroblast and pI asymmetric divisions. Cell 112, 51-62.

Camilleri, C., Azimzadeh, J., Pastuglia, M., Bellini, C., Grandjean, O. andBouchez, D. (2002). The arabidopsis TONNEAU2 gene encodes a putative novelprotein phosphatase 2A regulatory subunit essential for the control of the corticalcytoskeleton. Plant Cell 14, 833-845.

Casal, J., Gonzalez, C. and Ripoll, P. (1990). Spindles and centrosomes duringmale meiosis in drosophila melanogaster. Eur. J. Cell Biol. 51, 38-44.

Chan, A. and Cande, W. Z. (1998). Maize meiotic spindles assemble aroundchromatin and do not require paired chromosomes. J. Cell Sci. 111, 3507-3515.

Chan, J., Calder, G., Fox, S. and Lloyd, C. (2005). Localization of the microtubuleend binding protein EB1 reveals alternative pathways of spindle development inarabidopsis suspension cells. Plant Cell 17, 1737-1748.

Clarke, J. D. (2009). Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep forplant DNA isolation. Cold Spring Harb. Protoc. 2009, pdb.prot5177.

Cleary, A. L. and Smith, L. G. (1998). The Tangled1 gene is required for spatialcontrol of cytoskeletal arrays associated with cell division during maize leafdevelopment. Plant Cell 10, 1875-1888.

Cowan, C. R. and Hyman, A. A. (2004). Asymmetric cell division in C. elegans:cortical polarity and spindle positioning. Annu. Rev. Cell Dev. Biol. 20, 427-453.

Dawe, R. K. (1998). Meiotic chromosome organization and segregation in plants.Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 371-395.

De La Pen a, A. (1986). ‘In vitro’culture of isolatedmeiocytes of rye, secale cereale L.Environ. Exp. Bot. 26, 17-23.

Dhonukshe, P., Mathur, J., Hulskamp, M. and Gadella, T. W. (2005). Microtubuleplus-ends reveal essential links between intracellular polarization and localizedmodulation of endocytosis during division-plane establishment in plant cells. BMCBiology 3, 1.

Dhonukshe, P., Vischer, N. and Gadella, T. W., Jr. (2006). Contribution ofmicrotubule growth polarity and flux to spindle assembly and functioning in plantcells. J. Cell. Sci. 119, 3193-3205.

Dumont, J. and Desai, A. (2012). Acentrosomal spindle assembly andchromosome segregation during oocyte meiosis. Trends Cell Biol. 22, 241-249.

Dumont, S. and Mitchison, T. J. (2009). Force and length in the mitotic spindle.Curr. Biol. 19, R749-R761.

Dumont, J., Petri, S., Pellegrin, F., Terret, M.-E., Bohnsack, M. T., Rassinier, P.,Georget, V., Kalab, P., Gruss, O. J. and Verlhac, M.-H. (2007). A centriole- andRanGTP-independent spindle assembly pathway in meiosis I of vertebrateoocytes. J. Cell Biol. 176, 295-305.

Dumont, J., Oegema, K. and Desai, A. (2010). A kinetochore-independentmechanism drives anaphase chromosome separation during acentrosomalmeiosis. Nat. Cell Biol. 12, 894-901.

Duncan, R. E. and Persidsky, M. D. (1958). The achromatic figure during mitosis inmaize endosperm. Am. J. Bot. 45, 719-729.

Gilbert, S. F. (2006). The Nematode Caenorhabditis elegans. In DevelopmentalBiology, pp. 234-251. Sunderland, MA: Sinauer Associates.

Gilliland, W. D., Hughes, S. E., Cotitta, J. L., Takeo, S., Xiang, Y. and Hawley,R. S. (2007). The multiple roles of Mps1 in drosophila female meiosis. PLoSGenet. 3, e113.

Gordon, D. J., Resio, B. and Pellman, D. (2012). Causes and consequences ofaneuploidy in cancer. Nat. Rev. Genet. 13, 189-203.

Gould, G. W. (2016). Animal cell cytokinesis: the role of dynamic changes in theplasma membrane proteome and lipidome. Semin. Cell Dev. Biol. 53, 64-73.

Hassold, T. and Hunt, P. (2001). To err (meiotically) is human: the genesis ofhuman aneuploidy. Nat. Rev. Genet. 2, 280-291.

Hayashi, T., Sano, T., Kutsuna, N., Kumagai-Sano, F. and Hasezawa, S. (2007).Contribution of anaphase B to chromosome separation in higher plant cellsestimated by image processing. Plant Cell Physiol. 48, 1509-1513.

Higgins, D. M., Nannas, N. J. and Dawe, R. K. (2016). The maize divergentspindle-1 (dv1) gene encodes a kinesin-14A motor protein required for meioticspindle pole organization. Front. Plant Sci. 7, 1277.

Holubcova, Z., Blayney, M., Elder, K. and Schuh, M. (2015). Error-pronechromosome-mediated spindle assembly favors chromosome segregationdefects in human oocytes. Science 348, 1143-1147.

Hyde, K. J. and Schust, D. J. (2015). Genetic considerations in recurrentpregnancy loss. Cold Spring Harb. Perspect. Med. 5, a023119.

Kalt, M. R. (1973). Ultrastructural observations on the germ line of xenopus laevis.Z. Zellforsch. Mikrosk. Anat. 138, 41-62.

Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. and Brand, A. H. (2000).Rotation and asymmetry of themitotic spindle direct asymmetric cell division in thedeveloping central nervous system. Nat. Cell Biol. 2, 7-12.

Kiyomitsu, T. (2015). Mechanisms of daughter cell-size control during cell division.Trends Cell Biol. 25, 286-295.

Knoblich, J. A. (2008). Mechanisms of asymmetric stem cell division. Cell 132,583-597.

Kotak, S., Busso, C. and Gonczy, P. (2012). Cortical dynein is critical for properspindle positioning in human cells. J. Cell Biol. 199, 97-110.

Lane, S. I. R., Yun, Y. and Jones, K. T. (2012). Timing of anaphase-promotingcomplex activation in mouse oocytes is predicted by microtubule-kinetochoreattachment but not by bivalent alignment or tension. Development 139,1947-1955.

Lawrence, C. J., Morris, N. R., Meagher, R. B. and Dawe, R. K. (2001). Dyneinshave run their course in plant lineage. Traffic 2, 362-363.

Leader, B., Lim, H., Carabatsos, M. J., Harrington, A., Ecsedy, J., Pellman, D.,Maas, R. and Leder, P. (2002). Formin-2, polyploidy, hypofertility and positioningof the meiotic spindle in mouse oocytes. Nat. Cell Biol. 4, 921-928.

Li, R. (2013). The art of choreographing asymmetric cell division. Dev. Cell 25,439-450.

Li, X. and Dawe, R. K. (2009). Fused sister kinetochores initiate the reductionaldivision in meiosis I. Nat.Cell Biol. 11, 1103-1108.

Lloyd, C. and Hussey, P. (2001). Microtubule-associated proteins in plants—whywe need a MAP. Nat. Rev. Mol. Cell Biol. 2, 40-47.

Longo, F. J. and Chen, D.-Y. (1985). Development of cortical polarity in mouseeggs: involvement of the meiotic apparatus. Dev. Biol. 107, 382-394.

Maiato, H. and Lince-Faria, M. (2010). The perpetual movements of anaphase.Cell. Mol. Life Sci. 67, 2251-2269.

Maro, B., Howlett, S. K. and Houliston, E. (1986). Cytoskeletal dynamics in themouse egg. J. Cell Sci. 1986, 343-359.

Matos, I., Pereira, A. J., Lince-Faria, M., Cameron, L. A., Salmon, E. D. andMaiato, H. (2009). Synchronizing chromosome segregation by flux-dependentforce equalization at kinetochores. J. Cell Biol. 186, 11-26.

Mineyuki, Y., Murata, T. and Wada, M. (1991). Experimental obliteration ofthe preprophase band alters the site of cell division, cell plate orientation andphragmoplast expansion in adiantum protonemata. J. Cell Sci. 100, 551-557.

Mohanty, A., Yang, Y., Luo, A., Sylvester, A.W. and Jackson, D. (2009). Methodsfor generation and analysis of fluorescent protein-tagged maize lines. InTransgenic Maize, pp. 71-89. New York, NY: Springer.

Muller, S. and Jurgens, G. (2016). Plant cytokinesis—No ring, no constriction butcentrifugal construction of the partitioning membrane. Semin. Cell Dev. Biol. 53,10-18.

Nagaoka, S. I., Hassold, T. J. and Hunt, P. A. (2012). Human aneuploidy:mechanisms and new insights into an age-old problem. Nat. Rev. Genet. 13,493-504.

Otegui, M. and Staehelin, L. A. (2000). Cytokinesis in flowering plants: more thanone way to divide a cell. Curr. Opin. Plant Biol. 3, 493-502.

Otegui, M. S., Verbrugghe, K. J. and Skop, A. R. (2005). Midbodies andphragmoplasts: analogous structures involved in cytokinesis. Trends Cell Biol. 15,404-413.

Ou, G., Stuurman, N., D’Ambrosio, M. and Vale, R. D. (2010). Polarized myosinproduces unequal-size daughters during asymmetric cell division. Science 330,677-680.

Schatten, H. and Sun, Q.-Y. (2009). The functional significance of centrosomes inmammalian meiosis, fertilization, development, nuclear transfer, and stem celldifferentiation. Environ. Mol. Mutagen. 50, 620-636.

Schmit, A.-C. (2002). Acentrosomal microtubule nucleation in higher plants. Int.Rev. Cytol. 220, 257-289.

4023

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4014-4024 doi:10.1242/jcs.194860

Journal

ofCe

llScience

Segbert, C., Barkus, R., Powers, J., Strome, S., Saxton,W.M. andBossinger, O.(2003). KLP-18, a Klp2 kinesin, is required for assembly of acentrosomal meioticspindles in caenorhabditis elegans. Mol. Biol. Cell 14, 4458-4469.

Shamina, N., Gordeeva, E., Kovaleva, N., Seriukova, E. and Dorogova, N.(2007). Formation and function of phragmoplast during successive cytokinesisstages in higher plant meiosis. Cell Biol. Int. 31, 626-635.

Siller, K. H. and Doe, C. Q. (2009). Spindle orientation during asymmetric celldivision. Nat. Cell Biol. 11, 365-374.

Simerly, C., Nowak, G., de Lanerolle, P. and Schatten, G. (1998). Differentialexpression and functions of cortical myosin IIA and IIB isotypes during meioticmaturation, fertilization, andmitosis inmouse oocytes and embryos.Mol. Biol. Cell9, 2509-2525.

Smirnova, E. A. and Bajer, A. S. (1992). Spindle poles in higher plant mitosis. CellMotil. Cytoskeleton 23, 1-7.

Smith, L. G., Hake, S. and Sylvester, A. W. (1996). The tangled-1 mutation alterscell division orientations throughout maize leaf development without altering leafshape. Development 122, 481-489.

Staehelin, L. A. and Hepler, P. K. (1996). Cytokinesis in higher plants. Cell 84,821-824.

Staiger, C. J. and Cande, W. Z. (1991). Microfilament distribution in maize meioticmutants correlates with microtubule organization. Plant Cell 3, 637-644.

Stumpff, J., Ghule, P. N., Shimamura, A., Stein, J. L. and Greenblatt, M. (2014).Spindle microtubule dysfunction and cancer predisposition. J. Cell. Physiol. 229,1881-1883.

Sumiyoshi, E., Sugimoto, A. and Yamamoto, M. (2002). Protein phosphatase 4 isrequired for centrosome maturation in mitosis and sperm meiosis in C. elegans.J. Cell Sci. 115, 1403-1410.

Traas, J. A., Doonan, J. H., Rawlins, D. J., Shaw, P. J., Watts, J. and Lloyd,C. W. (1987). An actin network is present in the cytoplasm throughout the cellcycle of carrot cells and associates with the dividing nucleus. J. Cell Biol. 105,387-395.

Wasteneys, G. O. (2002). Microtubule organization in the green kingdom: Chaos orself-order? J. Cell Sci. 115, 1345-1354.

Weber, K. L., Sokac, A. M., Berg, J. S., Cheney, R. E. and Bement, W. M. (2004).A microtubule-binding myosin required for nuclear anchoring and spindleassembly. Nature 431, 325-329.

Yu, H.-G. and Dawe, R. K. (2000). Functional redundancy in the maize meiotickinetochore. J. Cell Biol. 151, 131-142.

Yu, H.-G., Hiatt, E. N., Chan, A., Sweeney, M. and Dawe, R. K. (1997).Neocentromere-mediated chromosome movement in maize. J. Cell Biol. 139,831-840.

Yu, H.-G., Muszynski, M. G. and Kelly Dawe, R. (1999). The maize homologueof the cell cycle checkpoint protein MAD2 reveals kinetochore substructureand contrasting mitotic and meiotic localization patterns. J. Cell Biol. 145,425-435.

Yu, F., Kuo, C. T. and Jan, Y. N. (2006). Drosophila neuroblast asymmetric celldivision: recent advances and implications for stem cell biology.Neuron 51, 13-20.

Yun, Y., Lane, S. I. and Jones, K. T. (2014). Premature dyad separation inmeiosis IIis the major segregation error with maternal age in mouse oocytes. Development141, 199-208.

Zamariola, L., Tiang, C. L., De Storme, N., Pawlowski, W. and Geelen, D. (2014).Chromosome segregation in plant meiosis. Front. Plant Sci. 5, 279.

Zhang, H. and Dawe, R. K. (2011). Mechanisms of plant spindle formation.Chromosome Res. 19, 335-344.

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