© 2016. Published by The Company of Biologists Ltd.
Anaphase asymmetry and dynamic repositioning of the division plane during maize meiosis
Natalie J. Nannas1, David M. Higgins1, and R. Kelly Dawe1,2
1
Department of Plant Biology, University of Georgia, Athens, GA 30605
2
Department of Genetics, University of Georgia, Athens, GA 30605
Corresponding author: R. Kelly Dawe, [email protected]
Key words: chromosome, spindle, phragmoplast, meiosis
SUMMARY STATEMENT
Meiotic chromosomes are segregated asymmetrically in anaphase and the phragmoplast
dynamically adjusts its location to correct for metaphase spindle misplacement within the cell
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volume.
JCS Advance Online Article. Posted on 8 September 2016
ABSTRACT
The success of an organism is contingent upon its ability to transmit genetic material through
meiotic cell division. In plant meiosis I, the process begins in a large spherical cell without physical
cues to guide the process. Yet two microtubule-based structures, the spindle and phragmoplast,
divide the chromosomes and the cell with extraordinary accuracy. Using a live-cell system and
fluorescently labeled spindles and chromosomes, we found that the process self corrects as meiosis
proceeds. Metaphase spindles frequently initiate division off-center, and in these cases anaphase
progression is asymmetric with the two masses of chromosomes traveling unequal distances on
the spindle. The asymmetry is compensatory, such that the chromosomes on the side of the spindle
that is farthest from the cell cortex travel a longer distance at a faster rate. The phragmoplast forms
equidistant between the telophase nuclei rather than at the original spindle mid-zone. This
asymmetry in chromosome movement implies a structural difference between the two halves of a
bipolar spindle, and may allow meiotic cells to dynamically adapt to metaphase errors and
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accurately divide the cell volume.
INTRODUCTION
Organisms package their genetic material into chromosomes that must be faithfully
separated into two new cells during division. While this process is critical in mitosis where
mistakes can lead to 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 (Nagaoka et al., 2012), less is
known about the meiotic regulation of spindle assembly and chromosome segregation. Female
meiosis in mammals (Schatten and Sun, 2009), C.elegans (Sumiyoshi et al., 2002), Drosophila
(Casal et al., 1990) and Xenopus (Kalt, 1973), are different from their mitotic and male meiotic
divisions. They must assemble spindles, position them within the cell volume and segregate
chromosomes, all without the organizational support of centrosomes (Dumont and Desai, 2012).
The lack of centrosomes may account for the higher rates of mis-segregation seen in female
meiosis compared to male meiosis (Hassold and Hunt, 2001). A study on human oocytes found
that less than 20% of meiosis I spindles are stable. The majority of spindles could not focus their
poles, formed multi-polar spindles, or had a high frequency of lagging chromosomes (Holubcová
et al., 2015).
Female meiosis in animals is also known for unequal cellular division (Brunet and Verlhac,
2011). In mammals, the meiotic spindle is positioned near the cell periphery to produce one large
egg 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
and Verlhac, 2011). Meiotic spindles are positioned at the cell cortex by actin microfilaments
(Longo and Chen, 1985), their nucleator FORMIN2 (Dumont et al., 2007;Leader et al., 2002), and
both chromosomes (Longo and Chen, 1985;Maro et al., 1986) and microtubules (Azoury et al.,
2008) to position the spindle at the cell cortex for asymmetric division. In other asymmetric cell
divisions such as the first zygotic division of C.elegans (Cowan and Hyman, 2004) or Drosophila
neuroblast development (Yu et al., 2006), spindles are positioned within the cell volume by cortical
pulling forces (Kiyomitsu, 2015). Astral microtubules interact with the minus-end directed motor
dynein, which is asymmetrically anchored on one side of the cell cortex by G-coupled protein
receptors (Kotak et al., 2012), and the spindle is pulled into position (Siller and Doe, 2009).
Cortical pulling also used to position spindles symmetrically, but in these divisions dynein
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myosins (Simerly et al., 1998;Weber et al., 2004). A dense actin network interacts directly with
localization is non-polarized. Accurate spindle positioning within the cell volume is critical
because the spindle mid-zone has been shown to set the location of the cleavage furrow and thus
the size and shape of resulting daughter cells (Burgess and Chang, 2005).
Much less is known about spindle positioning and chromosomes segregation in plants,
where all cell divisions occur in the absence of centrosomes (Schmit, 2002;Wasteneys,
2002;Zhang and Dawe, 2011). Meiosis in plants such as maize have been heavily studied at the
level of fixed specimens (Dawe, 1998;Zamariola et al., 2014) but far less studied at the level of
live cells (Yu et al., 1997;Yu et al., 1999), leaving gaps in our understanding of spindle and
chromosomes movements.
In this study, we imaged live cell division in male maize meiosis I and II. Maize meiotic cells are
easily collected from immature tassels and provide an excellent system to study acentrosomal
spindles. Early attempts to live image maize meiosis were limited to observing only chromosome
movements with a cell-permeant DNA stain (Yu et al., 1997), but a newly developed fluorescent
tubulin fusion (Mohanty et al., 2009) allowed this first study of live meiotic spindle dynamics.
Imaging meiosis I and II spindles revealed an unexpected and previously undocumented
phenomenon: the separation of chromosomes is not consistently symmetric. The observed
asymmetry is limited to anaphase A, and correlates with the position of the spindle relative to the
cell cortex. In cases where the spindle is positioned significantly off-center, the mass of
chromosomes furthest from the edge of the cell moves faster and farther, helping to adjust the
position of the chromosomes and correct the initial asymmetry. Additional data show that the
phragmoplast, the plant equivalent of the cleavage furrow which determines the plane of division
zone, helping to assure that cell division accurately divides the cell into equal parts.
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(Otegui et al., 2005), forms midway between the chromosome masses and not at the spindle mid-
RESULTS
Chromosome segregation dynamics in male maize meiosis I and II
Chromosome segregation in male maize meiosis was studied via live imaging.
Microtubules were labeled with cyan fluorescent protein (CFP) fusion with β-tubulin (Mohanty et
al., 2009), and chromosomes were labeled with SYTO12, a green nucleic acid stain that penetrates
live cells (Yu et al., 1997). Meiotic cells (meiocytes) were extruded from anthers into a culture
media previously demonstrated to support cell growth (De La Peña, 1986;Yu et al., 1997), and
imaged by fluorescence microscopy. Spindle morphology and chromosome movements were
tracked over time, and movies were captured of both meiosis I (Fig. 1A, Supplemental Movie 1)
and meiosis II (Fig. 1B, Supplemental Movie 2). We confirmed that the CFP-tubulin tag did not
affect spindle dynamics by comparing to unlabeled fixed cells (Fig. S1A,B). All characterization
and data analysis presented below is based on 41 live cells.
In both meiosis I and II, chromosomes align on the spindle metaphase plate, separate into
two masses in anaphase, and decondense into two new nuclei separated by a growing phragmoplast
(n=41). Anaphase I lasted an average of 12.7 ± 3.2 minutes, and anaphase II lasted an average of
11.0 ± 3.7 minutes; this timing is not statistically different (Student’s t-test, p=0.36). Anaphase
was defined as the period of time from chromosomes separation until spindle breakdown. Only
one lagging chromosome was observed out of 41 imaged cells (2%) (Fig. 1C); it remained in the
center of the spindle and was not pulled to one pole. Maize meiotic chromosome segregation is
thus more stable than human female meiosis where these “persistent” lagging chromosomes have
been observed in 40% of cells (Holubcová et al., 2015). In 17% of cells, at least one chromosome
was observed trailing the main mass after initial separation but always caught up to the mass within
reported in studies on fixed cells (Yu and Dawe, 2000; Li and Dawe, 2009), we cannot rule out
the possibility that culturing and imaging the meiocytes may impact chromosome segregation. We
also observed astral-like microtubules in some spindles that appeared to contact and curl around
the cell cortex (Fig. S2A).
Spindle dynamics are similar in both meiosis I and II. Metaphase spindle length in meiosis
I is 34.2 ± 3.6μm and 35.3 ± 5.2μm in meiosis II (Fig. 2A). Eukaryotic chromosomes are separated
in anaphase by two mechanisms, movement of chromosomes to the spindle poles (anaphase A)
and elongation of the spindle with the poles pushing apart (anaphase B) (Fig. 2B). The usage of
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5-10 minutes (Fig. 1C). Because these slow moving chromosomes have not been previously
these two mechanisms and their degree of contribution to chromosome separation varies from
species to species (Maiato and Lince-Faria, 2010). We found that spindles do not elongate from
their metaphase length during anaphase (Fig. 2A). In fact, spindles are significantly shorter 20
minutes after anaphase onset in both meiosis I (24.9 ± 7.0μm, p<10-5) and meiosis II (23.7 ± 5.2μm,
p=0.01) (Fig. 2C). The segregation of maize chromosomes appears to be exclusively achieved
through anaphase A movement, with no observed anaphase B contribution.
Chromosome movement to the poles (anaphase A) exhibits differences between meiosis I
and II. While spindle length is the same, chromosomes in meiosis II are pulled significantly further
apart than in meiosis I. Homologous chromosomes are pulled an average of 5.6 ± 2.1μm from the
metaphase plate to the poles in meiosis I and sister chromatids are pulled 6.7± 0.7μm (Fig. 2D,
p=0.001). If we normalize by spindle length, where being pulled 50% of spindle length means
being pulled from the metaphase plate all the way to the poles, meiosis I chromosomes are pulled
an average of 32 ± 12% and meiosis II chromosomes are pulled 40 ± 8% (Fig. S3A, p=0.009).
Meiosis II chromosomes are pulled closer to the poles of the spindle, both in terms of raw distance
and as a percentage of spindle length.
Asymmetric chromosome segregation in anaphase A
We found that the standard deviation of chromosome segregation distance (anaphase A
distance) is larger in meiosis I than meiosis II (Fig. 2D), suggesting more variation between the
distances traveled by each mass of chromosomes. We tracked the movement of the two masses
through time, and found that chromosomes moved asymmetrically on the spindle in anaphase (Fig.
3A). Fig. 3B shows an example of an asymmetric division where the chromosome masses are
(T=5-10 min). The asymmetry was quantified by designating one chromosome mass as “A” and
determining that it traveled distance DA and designating the other mass as “B” and that it traveled
distance DB. The difference in segregation distance is then ΔDA-B= DA-DB (Fig. 3C). A small ΔDAB
value means that the two masses of chromosomes traveled approximately equal distances toward
their respective poles, while a large value means the chromosomes segregated asymmetrically.
The asymmetry in chromosome movement is statistically significant. The average total
distance traveled by mass A (DA) is statistically greater than that of mass B (DB) (Fig. 3D, 6.6 ±
1.9μm vs. 4.9 ± 1.7μm, p<10-4). Mass A also travels further than mass B over multiple time points
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outline in white and tracked in three dimensions from metaphase (T=0 min) through anaphase
throughout anaphase, beginning 10 minutes after the metaphase-anaphase transition (p<0.004)
(Fig. 3E). The average difference in chromosome segregation distance (ΔDA-B) was twice as large
in meiosis I than in meiosis II (Fig. 3F, 1.8 ± 1.5μm vs. 0.9 ± 0.6μm, p=0.02), meaning
chromosomes segregate more asymmetrically in meiosis I (Fig. 3F). Approximately half of
meiosis I cells segregate like meiosis II with less than 2μm difference, while the other half travel
quite asymmetrically with a range of 2 to 7μm difference in chromosome paths (Fig. 3G).
To better compare chromosome movements between cells, DA and DB was normalized by
total movement (DA+ DB). In symmetrically segregating cells, DA and DB are nearly equal, thus
contributing 50%-50% to chromosome movement. The percentage of total chromosome
movement attributed to each chromosome mass was plotted and ordered by symmetry (Fig. 3H).
Plotting this data shows that there are not two discrete populations of symmetric and asymmetric
cells; instead, we observed a continuum of asymmetry from 50%-50% to 80%-20% as the most
extreme. A plot of all cells and their non-normalized chromosome segregation distances, as well
as their spindle lengths can be found in Fig. S3B. The asymmetry in chromosome segregation
distance (ΔDA-B) is not correlated with spindle length (Fig. S3C), total anaphase A distance (Fig.
S3D), or the presence of slow-moving chromosomes (Fig.S1C).
The timing of anaphase was not correlated with asymmetry (Fig. S3E), and chromosomes
that were traveling further did not spend a longer time moving (p=0.4), suggesting that the two
masses of chromosomes move at different rates. The data show that in 95% of the observed cells
(39/41), the chromosome mass that traveled further (A) did so at a faster rate than the chromosome
mass that moved the shorter distances (B) (p<0.0001, Fisher’s exact test). Both the average rate of
segregation and the maximum speed was greater for chromosome mass A than mass B (Fig. 3I,
continuum of symmetry phenotypes rather than two discrete populations (Fig. 3H), we compared
the top 25% most asymmetric divisions with the 25% least asymmetric divisions (symmetric
divisions). By comparing top and bottom quartiles for the phenotype, we found that in symmetric
divisions there is no difference in mass A and B speeds at any point in anaphase (Fig. S3F, p>0.7)
but in asymmetric divisions mass A is faster than mass B at both 5 and 10 minutes 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 chromosome mass A from 5 minutes (0.58 μm min-1) to 10 minutes (0.90
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p=0.003 and p=0.05, respectively, see Fig. S3G,H for plot of all cells). Because cells show a
μm min-1, p=0.01). The difference in the initial rate of movement between mass A and B suggests
that the chromosome masses maybe under different force conditions at metaphase.
Asymmetry is a corrective mechanism resulting from offset spindles.
As they segregate at different rates, the two masses of chromosomes may be experiencing
different forces at the metaphase-anaphase transition. Forces are generated on chromosomes in
metaphase as microtubules depolymerize and pull the paired chromosomes toward the poles but
are resisted by chiasma in meiosis I and cohesin in meiosis II. Microtubule polymerization,
chromosome elasticity, and viscous drag also generate force which all balance to align
chromosomes at metaphase (Dumont and Mitchison, 2009). It is possible that the chromosomes in
asymmetrically dividing cells were not aligned in the center of the spindle at the start of anaphase,
and thus experienced unequal forces driving them apart (Fig. 4A). The three-dimensional location
of the spindle center was measured and subtracted from the three-dimensional location of the
metaphase chromosomes to determine the chromosome-spindle offset (ΔChrom-Spindle). If the
asymmetric movement is caused by misalignment of the chromosomes, we would expect the offset
distance to be greater in the top quartile of asymmetric divisions, as one mass must travel further
to approach its pole (DA> DB, Fig. 4A). Chromosomes were offset from spindle center by 0.7±
0.4μm in asymmetric divisions and by 0.7 ± 0.3μm in symmetric divisions (Fig. 4B), with no
significant difference in offset. Thus, the asymmetric movement is not due to a failure to align
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 by the spindle mid-zone in both animals and plants
meiosis, it is possible that the asymmetric segregation is a post-metaphase correction mechanism
to pull chromosomes into equal volumes when the spindle is not centered in the cell (Fig. 4C). If
this were true, we would expect spindles in asymmetric divisions to be more offset from the cell
center (ΔSpindle-Cell) than in symmetric divisions. Asymmetric spindles are indeed more offset
(3.3 ± 2.2μm) than symmetric divisions (1.5 ± 0.9μm, p=0.04) (Fig. 4D).
We also expect that the further moving chromosome mass (A) should travel in the direction
of larger cell volume (Fig. 5A). We measured the distance from spindle pole to cell cortex
(distances X and Y), and found this to be true. In the top quartile of asymmetric divisions, all cells
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(Kiyomitsu, 2015). Given that four equally sized products is the optimal outcome in maize male
had greater chromosome movement on the side of the spindle further from the cell cortex (distance
X), which is significant compared to randomly oriented movement (Fisher’s exact test, p=0.01)
(Fig. 5B). The difference in pole-cell wall distance (X-Y) is greater in asymmetric divisions both
in terms of absolute distance (X-Y, p=0.003) and normalized for cell size (X-Y/X+Y, p=0.002)
(Fig. 5C). In cells that have spindles offset by more than a standard deviation, asymmetry of
chromosome movement is significantly greater than the average of all observed cells (p=0.01, Fig.
5D).
Most convincingly, the extent to which spindles are offset correlates with asymmetric
movement such that the greater the spindle is shifted towards one wall, the greater the difference
in chromosome movement. The position of the spindle in the cell can explain ~59% of the observed
anaphase asymmetry (an R2 of 0.588, Fig. 5E). Two cells were excluded from this analysis due to
confounding features that may increase asymmetry in ways unrelated to spindle position (red on
Fig. 5E). One has a persistent lagging chromosome with possible merotelic attachments that
impede chromosome movement towards one pole and the other has a major spindle morphology
defect. Images of these divisions can be found in Fig. S2B and C. No other cells showed significant
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 in C.elegans, the whole spindle is shifted during anaphase to
achieve the desired location within the cell volume (Li, 2013). To determine if offset spindles shift
toward cell center, we measured the pole to wall distance (X) throughout anaphase (T=0-20 min)
in the top quartile of asymmetric divisions. Distance at each time point (XT) was normalized by
the initial pole-wall distance (X0), such that a value less than 1 indicates a decrease in distance and
distance is not significantly different at the end of anaphase than at the beginning. Because our
spindles do not shift (Fig. 5F) or elongate (Fig. 2A,C), the observed asymmetric movement is due
solely to unequal movement of chromosomes toward their poles. This represents a novel
mechanism for positioning chromosomes within a cell volume.
The phragmoplast is established equidistant from chromosomes, not at the spindle mid-zone
Following chromosomes segregation in anaphase, cytokinesis structures form at the
spindle mid-zone to split the cell in two (Otegui et al., 2005). These include the cleavage furrow
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shifting of the spindle towards cell center (Fig. 5F). The data show that the average pole-wall
in animals (Gould, 2016) and the phragmoplast in plants (Müller and Jürgens, 2016). Our
observations indicate that chromosomes are asymmetrically pulled away from the spindle midzone, suggesting the distance from the phragmoplast to the chromosomes might also be
asymmetric (Fig. 6A, Option 1). However, anaphase asymmetry (ΔDA-B) clearly does not correlate
to the cytokinesis asymmetry (ΔPA-B) (Fig. 6B, R2=0.0007) and instead the phragmoplast forms
equidistant from the chromosome masses (Fig. 6A Option 2, 6C). The distance from phragmoplast
to chromosome mass strongly correlates with the midpoint distance between chromosomes,
calculated by averaging DA and DB (Fig. 6C, R2=0.8424). By establishing itself relative to the
position of the chromosomes, the phragmoplast may be able to provide a back-up mechanism to
segregate chromosomes, and ensure that each daughter cell receive at least some genetic material.
We observed two instances where the spindle failed to segregate chromosomes in which the
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phragmoplast was able to push chromosomes apart into two masses (Fig. 6D).
DISCUSSION
We investigated the dynamics of chromosome segregation in male maize meiosis, a system
that segregates chromosomes with an acentrosomal spindle (Zhang and Dawe, 2011). We found
that the two masses of chromosomes do not segregate consistently equal distances (Fig. 3D).
Instead, we see a large variation in anaphase A distance, with masses of chromosomes travelling
asymmetric distances on the spindle. In the most extreme case, one mass of chromosomes traveled
80% of the total anaphase A distance with the other mass traveled only 20% (Fig. 3H). The
asymmetry observed here is different from the high incidence of lagging chromosomes seen in
human (Holubcová et al., 2015) and mouse (Yun et al., 2014) oocytes, as well as in the perturbed
meiosis in other species (Dumont et al., 2010). In these prior studies, the two masses of
chromosomes moved apart equally with individual chromosomes lagging behind; here we see that
all ten of chromosomes on one side of the spindle frequently move at different speeds than the
chromosomes other side (Fig. 3I).
To our knowledge, asymmetrical segregation during anaphase A has not been previously
described. Prior live-imaging studies of meiosis in human (Holubcová et al., 2015), mouse (Lane
et al., 2012), C.elegans (Dumont et al., 2010;Segbert et al., 2003), and Drosophila (Gilliland et al.,
2007) showed no evidence of asymmetry, and the prior data on mitosis also suggests that anaphase
A is predictably symmetric. There are, however, a few documented examples of unequal
chromosome movement through the action of anaphase B. In Drosophila (Kaltschmidt et al., 2000)
and C.elegans (Ou et al., 2010) neurogenesis, chromosomes are segregated by a centrally
positioned spindle, and in anaphase B one side of the spindle elongates further than the other,
creating a large neuroblast and a small ganglion mother cell (Kaltschmidt et al., 2000;Li, 2013;Ou
centrosome that nucleates more microtubules to pull the chromosomes further in one direction (Cai
et al., 2003), and in C. elegans, polarization of myosin II squeezes one side of the spindle, allowing
the other side to preferentially elongate (Ou et al., 2010). Additionally, in a study on mitotic
tobacco culture cells, unequal anaphase B elongation pulled chromosomes further on one side
(Hayashi et al., 2007). While there is evidence for anaphase B in mitotic maize cells (Duncan and
Persidsky, 1958), we have found no evidence for anaphase B in meiosis as the spindle does not
elongate (Fig. 2A,C). Our observation indicate that all of the asymmetry occurs in anaphase A
movement (Fig. 2E-I, Fig. 5F). Maize does not have centrosomes to create an imbalance in
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et al., 2010). In Drosophila, the asymmetry in spindle morphology is created by a larger
microtubule nucleation, and there is no evidence of polarized actin in maize meiosis (Staiger and
Cande, 1991), so neither of these established mechanisms can explain the asymmetry seen here.
The function of asymmetric chromosome segregation is typically to move chromosomes
into unequal cell volumes to produce daughter cells of different sizes and/or cell fates (Li, 2013).
Asymmetry generally arises from the orientation and positioning of the spindle (Siller and Doe,
2009). In mouse meiosis, the spindle is positioned near the cell periphery to create one large egg
and three small polar bodies (Brunet and Verlhac, 2011). In the first zygotic division of C. elegans,
the spindle is offset from cell center, producing a small posterior cell that gives rise to the germline
and muscle, and a larger anterior cell that becomes all other somatic tissue (Gilbert, 2000;Li, 2013).
Asymmetry and orientation of the spindle is also used in stem cells to produce one self-renewing
and one differentiating daughter (Knoblich, 2008). Maize male meiosis presents the opposite
problem, where the spindle is frequently offset from the cell center but the final products of meiosis
are highly uniform in size. If the purpose of anaphase asymmetry in maize is to place the new
nuclei in equal volumes, it should be correlated with how offset the spindle is within the cell (Fig.
5A). We find that there is indeed such a correlation, as the greater the spindle is shifted towards
the edge of the cell, the greater the asymmetry (Fig. 5E). When the spindle is highly offset (the top
quartile of asymmetric divisions), chromosomes always move further on the side of the spindle
with greater pole-wall spacing (Fig. 5B). The spindle itself does not move (Fig. 5F), but the rate
and distance of chromosome movement within the spindle serves to correct for irregularities in
spindle position and help to place chromosomes into opposite hemispheres of the cell.
The phragmoplast forms the new cell wall after cell division in plants. In mitotic cells, the
position of both the spindle and phragmoplast is dictated by the preprophase band, a microtubule
(Azimzadeh et al., 2008;Camilleri et al., 2002;Traas et al., 1987) and TANGLED (Cleary and
Smith, 1998;Smith et al., 1996). Meiotic plant cells lack a preprophase band (Chan and Cande,
1998), and analysis of fixed specimens suggests that phragmoplasts are formed from the central
fibers of the spindle remaining after chromosomes segregation (Otegui and Staehelin,
2000;Shamina et al., 2007;Staehelin and Hepler, 1996). While in most cell types, the position of
the metaphase plate marks the location of the spindle mid-zone, due to anaphase asymmetry, these
positions often differ in our cells. We find that the phragmoplast appears halfway between the two
chromosome masses after anaphase rather than at the original spindle mid-zone (Fig. 6 A-C).
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array around the cell periphery (Mineyuki et al., 1991), and its associated proteins, TONNEAU
Dynamic establishment of the phragmoplast relative to chromosomes allows cells to correct the
division plane if the spindle is improperly positioned (Fig. 5B, E) and provides a back-up
mechanism for segregation when the spindle fails to fully separate chromosomes (Fig. 6D).
We find that chromosomes move farther and faster on the side of the spindle most distant
the cell cortex (Fig. 3I and J, Fig. 5A and B). Maize meiotic cells must have a sensing mechanism
to integrate spindle position with chromosome segregation distances. One option is astral
microtubules that reach from the spindle 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 and Hussey, 2001;Smirnova and Bajer, 1992), but recent microscopy
has revealed astral-like microtubule that reach from the acentrosomal pole towards the cell cortex
in Arabidopsis (Chan et al., 2005) and tobacco (Dhonukshe et al., 2005). We observed similar
astral connections to the cortex in our time-lapse data (Fig. S1A). In animals, astral microtubules
work in concert with cortical 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 modulating
microtubule dynamics. Astral-like microtubules could bind regulatory molecules such as KLP10
or CLASP, which regulate microtubule flux rates in Drosophila and produce a similar asymmetry
phenotype with unequal movement of chromosomes towards poles when depleted (Matos et al.,
2009). While very little is known about microtubule flux regulation in plants (Dhonukshe et al.,
2006), our data suggest that such regulators may exist and are sensitive to spindle position with
the cell. Overall, our findings demonstrate that positional signals are transduced from the cortex
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to modulate chromosome dynamics as late as anaphase to correct errors in spindle placement.
METHODS and MATERIALS
Maize Lines and Genotyping
A maize line (Zea mays ssp. mays) containing CFP fused to the N-terminus of beta-tubulin (βTUB1) was generated by the Anne Sylvester lab (University of Wyoming). Plants were genotyped
for the CFP-tubulin transgene using a CTAB DNA extraction protocol (Clarke, 2009) on leaf tissue
and primers that annealed in CFP (5’-GGAGTACAACTACATCAGCCACAACGTC) and tubulin
(5’-CCGGACTGACCGAAGACGAAGTTGT). The maize line containing dv1 was obtained from
the Maize Genetics Cooperation Stock Center (University of Illinois), and genotyped as previously
described (Higgins et al., 2016). All chemicals and reagents, unless otherwise stated, were
purchased from Sigma Aldrich (St. Louis, MO, USA).
Live Imaging
Male meiotic cells were harvested from immature tassels as previously described (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) that contained a final concentration of 2μM SYTO12 Green DNA dye
(Invitrogen Molecular Probes, Grand Island, NY, USA). Cells were staged for meiosis I and
meiosis II, loaded onto poly-L-lysine (Sigma Aldrich, St. Louis, MO, USA) coated coverslips
(Corning, Corning, NY) and sealed onto microscope slides (Fisher Scientific, Waltham, MA,
USA). Cells were imaged on a Zeiss Axio Imager.M1 fluorescence microscope with a 63x PlanAPO Chromat oil objective. Images were collected every 3-7 minutes in 3-dimensions using a
20μm Z range and 1μm step size with 50ms exposure for CFP and 30ms exposure for SYTO12
meiosis II cell (Supplemental Movie 4) demonstrate that cells were not compressed between
coverslip and slide during imaging. Sample size was 41 cells, and the asymmetric and symmetric
categories were defined as the top and bottom quartile of the asymmetric phenotype. Sample size
was sufficient for statistical power in both Student’s t-test and Fisher’s exact test used as described
in text.
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and 2x2 binning. 3D volume renderings of an example meiosis I cell (Supplemental Movie 3) and
Image Analysis
Images were analyzed using Slidebook software (Intelligent Imaging Innovations, Denver, CO,
USA). Cells, spindles, chromosomes, and phragmoplasts were identified as objects by
thresholding. Cells, labelled by diffuse cytoplasmic CFP-tubulin monomers, were thresholded at
approximately 35% above CFP background, spindle and phragmoplast signals at approximately
50% above background, and chromosomes at approximately 5% above FITC background. Object
statistics were extracted including center of volume (x, y, z coordinates of the center of the object)
and longest chord (distance between the two furthest pixels within the object). Spindle length was
measured as the longest chord within the spindle object after constrained iterative deconvolution
using a calculated point spread function. Chromosome movements were calculated as the threedimensional distance between the center of volume at different time points, and chromosome speed
was calculated as this distance divided by time. The total distance travelled in anaphase (DA or DB)
was calculated as the distance between the initial position on the metaphase plate and the final
position near the spindle pole. The asymmetry value (ΔDA-B) is the difference in these distances:
ΔDA-B = DA- DB. Congression of chromosomes on the metaphase plate (ΔChromosome-Spindle)
was calculated as the difference between the chromosome center of volume and the spindle center
of volume at metaphase. The offset of the spindle from the cell center (ΔSpindle-Cell) was
calculated as the difference between the spindle center of volume and the cell center of volume.
Cytokinesis ΔPA-B is the difference in distance from each chromosome mass to the phragmoplast
center of volume. The xyz position of the spindle pole was determined by the edge of the
thresholded spindle outline (50% above deconvolved background CFP signal) as the furthest point
from the center of the spindle, measured using Slidebook ruler function. The distance from pole to
outline of the cell (35% above non-deconvolved background CFP signal) within the same z plane
as the pole using Slidebook ruler function.
Immunolocalization
Immunolocalization was performed as previously described (Higgins et al., 2016). Anthers from
immature tassels were fixed for 60 minutes in 4% paraformaldehyde/PHEMS buffer, washed three
times in 1xPBS, and dissected for meiotic cells. Staged meiocytes were adhered to poly-L-lysine
coverslips by centrifugation at 100g for 1 minute, then permeabilized for one hour in a 1% Triton
Journal of Cell Science • Advance article
cell cortex was measured by determining the shortest xy distance from the pole to the thresholded
X-100, 1mM EDTA, 1xPBS solution. Coverslips with affixed cells were blocked in 10% goat
serum for 90 minutes, incubated with a monoclonal antibody against sea urchin α-tubulin (Asai et
al., 1982) at 37°C overnight, blocked again with 10% goat serum, then incubated with a
Rhodamine-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson
ImmunoResearch Inc., West Grove, PA, USA) for 150 minutes. Between each step, coverslips
were washed three times with 1xPBS solution. Coverslips were mounted with ProLong Gold with
DAPI (Thermo Fisher Scientific, Waltham, MA, USA) and imaged as described above except
there was no binning and exposure times were optimized for each channel and ranged from 0.1-1
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seconds.
ACKNOWLEDGEMENTS
We thank Caroline Jackson for genotyping the CFP-tubulin plants and Amy Hodges for
genotyping the dv1 plants, as well as Jonathan Gent and other members of the Plant Biology
Department for discussion of the data.
COMPETING INTERESTS
Authors declare no competing interests.
AUTHOR CONTRIBUTIONS
Conceptualization 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- review and editing:
N.J.N, D.M.H., R.K.D; Visualization: N.J.N; Funding acquisition and Resources: N.J.N, R.K.D.;
Supervision: R.K.D;
FUNDING
This study was supported by a National Science Foundation: fellowship IOS-1400616 to Nannas
Journal of Cell Science • Advance article
and grants MCB-1412063 and IOS-0922703 to Dawe.
Journal of Cell Science • Advance article
Figures
Figure 1. Live imaging of maize male meiosis. Meiosis was imaged in live male maize cells via
CFP-tubulin (β-TUB1) to label spindles (green) and SYTO12 DNA stain to label chromosomes
(magenta). The cell volume is visible by diffuse, unincorporated CFP-tubulin monomers in the
cytoplasm. Timing of anaphase in meiosis I (A) and meiosis II (B) is not statistically different
(Student’s t-test, p=0.36), lasting approximately 12 minutes from chromosome separation to
spindle breakdown. In both meiosis I and II, chromosomes are pulled towards the poles (anaphase
A) but there is no visible elongation of the spindle (anaphase B). The phragmoplast, a microtubulebased structure that directs cell division, appears between the new nuclei after spindle breakdown
in both meiosis I and II. Images are frames from live movies that can be found in Supplemental
Movies 1 and 2. (C) Of the 41 live meiosis imaged, only one cell showed a lagging chromosome
that remained behind on the spindle and was not pulled to a pole (white arrow). In approximately
20% of cells, a slow moving chromosome initially trailed behind the main chromosome mass, but
rejoined the main mass during anaphase (white arrows). The scale bar in all images represents
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10μm, sample size n=41 cells.
Figure 2. Characterization of spindle and chromosome dynamics in meiosis I vs. meiosis II.
(A) Maize meiotic spindles do not elongate in anaphase. The mean length of meiosis II spindles
does not statistically change from metaphase to anaphase (p=0.37), while meiosis I spindles
shorten slightly (p=0.02). (B) Chromosomes aligned on the metaphase spindle are segregated by
two types of movements, movement of chromosomes to the poles (anaphase A) and elongation of
mechanisms with varying levels of contribution to separate their chromosomes. (C) Spindle length
tracked through time in meiosis I (black circles) and meiosis II (gray boxes) shows a shortening
rather than elongating spindles (meiosis I, p<10-5 and meiosis II, p=0.01) demonstrating there is
no anaphase B in male maize meiosis. The blue box marks anaphase. (D) Chromosomes are
separated entirely by anaphase A movements, which is on average a longer distance in meiosis II
than meiosis I (p=0.001). All error bars represent standard deviation from the mean, statistical
significance is marked with a star, and p-values are calculated by Student’s t-test, sample size
n=35 for meiosis I and n=6 for meiosis II.
Journal of Cell Science • Advance article
the spindle that drives the poles apart (anaphase B). Organisms utilize one or both of these
travel unequal distances on the spindle (green) from their starting position on the metaphase plate.
An example movie is shown in (B) where one mass of chromosomes, whose path is denoted by
the orange line, travels a shorter distance than the mass of chromosomes marked with the blue
path. (C) A schematic of asymmetry; the further moving chromosome mass (A) is in blue and
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Figure 3. Asymmetric anaphase A chromosome segregation. (A) Chromosomes (magenta)
moves distance DA, the shorter moving chromosome mass (B) is in orange and moves distance DB.
The asymmetry is calculated as DA-DB and represented as ΔDA-B. (D) The mean distance traveled
by chromosome mass A is statistically greater than mass B (p<10-4, n=41). (E) Chromosome
asymmetry becomes statistically significant within 10 minutes of the metaphase-anaphase
transition (p<0.004, n= 28). The grey box represents anaphase, which starts at the metaphaseanaphase transition (time = 0 minutes). (G) A histogram of asymmetry values shows that meiosis
I has a wide range of variation in this asymmetry phenotype, n=41. (H) The range of asymmetry
is displayed by normalizing anaphase A distance to 100%. In symmetric divisions, each
chromosome mass (mass A=blue and mass B=orange bars) travel 50% of the total anaphase
distance; in asymmetric division, a greater percentage of anaphase A distance is attributed to
chromosome mass A. The data is sorted by increasing asymmetry, n=41. The most asymmetric
division is represented by the left-most bar where mass A travels 80% of anaphase A distance
while mass B travels only 20%. (I) On average, chromosome mass A moves statistically faster
than mass B, both in average speed (p=0.003) and maximum speed (p=0.05), n= 41. See
Supplemental Figure 3G and H for individual cell data. (J) In the top quartile of asymmetric
divisions (n=10), chromosome mass A moves faster than mass B at both 5 and 10 minutes post
metaphase-anaphase transition (T=5 min, p=0.037)(T=10 min, p=0.019), and chromosome mass
A speeds up over time (5 vs. 10 min, p=0.038). All error bars represent standard deviation from
the mean, statistical significance is marked with a star, and p-values are calculated by Student’s t-
Journal of Cell Science • Advance article
test. See Supplemental Figure 3I for individual cell data.
Figure 4. Asymmetry is related to spindle position, not chromosome congression. (A) A model
showing chromosome offset within the spindle (ΔChrom-Spindle) may explain asymmetry.
Asymmetry may 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 is no difference in chromosome offset between asymmetric and
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 have significantly larger spindle offsets than
symmetric divisions (p=0.04). Error bars represent standard deviation from the mean, statistical
significance is marked with a star, and p-values are calculated by Student’s t-test, sample size n=10
for both asymmetric and symmetric divisions.
Journal of Cell Science • Advance article
symmetric divisions. (C) A model showing how spindle offset within the cell (ΔSpindle-Cell) may
Figure 5. Asymmetric chromosome movement determined by distance from cell cortex. (A)
may move further at anaphase (chromosome mass A, blue). Chromosomes on the side closest to
the cortex (Y) would then move the shorter distance (chromosome mass B, orange). The pole-wall
offset was normalized by 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 A was most distant from the cell cortex are
colored black. The trend is most apparent for highly asymmetric divisions. (C) The normalized
pole-wall offset is statistically greater in asymmetric divisions than symmetric divisions (p=0.002,
n=10 for each). (D) In cells that have spindles offset from the cortex by more than a standard
Journal of Cell Science • Advance article
A model showing how chromosomes on the side of the spindle furthest from the cell cortex (X)
deviation (“Offset”, n=5), asymmetry is statistically larger than compared to all observed cells
(“All”, n=41) (p=0.01). (E) Asymmetry is correlated with normalized pole-wall offset. Two data
points (red) were excluded from the analysis due to spindle errors (see text for discussion, n=39).
(F) The distance from pole to wall does not significantly change from the beginning of anaphase
(T=0) to the end of anaphase (T=20). The pole-wall distance (X) was normalized by the initial
distance (ΔX0-T) such that a value less than 1 means the distance has decreased (n=10). Error bars
represent standard deviation from the mean, statistical significance is marked with a star, and p-
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values are calculated by Student’s t-test.
chromosomes in anaphase is often asymmetric. In these instances, phragmoplast could either form:
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Figure 6. Phragmoplast is established equidistant from chromosomes. (A) The separation of
1) at the position of the original metaphase plate (spindle mid-zone) or 2) midway between the
chromosomes. If the phragmoplast is established at the mid-zone (Option 1), the asymmetry seen
in anaphase (ΔDA-B) should be roughly equal to the asymmetry 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) is not correlated with anaphase asymmetry (ΔDA-B), thus it is not located at the spindle
midzone. Red dots indicate cells excluded from correlation analysis, see text and Fig. S2B and C
for explanation of excluded cells (n=39). (C) Plot to test Option 2: the scatter plot shows that
phragmoplast position (distance from chromosome mass A to the phragmoplast, PA) is strongly
correlated with the midpoint between chromosomes ([DA+DB]/2). Red dots indicate cells excluded
from correlation analysis (n=39). (D) The phragmoplast is capable of pushing chromosomes apart
Journal of Cell Science • Advance article
when the spindle fails to separate chromosomes.
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J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
wild-type
dv1
B
ΔSpindle-Cell (μm)
A
8.0
4.0
2.0
Asymmetry value, ΔD
A-B
Asymmetry, ΔDA-B
0.0
C
8.0
8.0
6.0
6.0
Top 25% of
asymmetric
divisions
*
6.0
Wild-type
dv1
CFP-tubulin
Cells with slow-moving chromosome
Cell with lagging chromosome
Botttom 25% of
asymmetric
divisions
4.0
4.0
2.0
2.0
0.0
0.0
Individual cell movies
Supplemental Figure 1. CFP-tubulin tag and slow moving chromosomes do not interfere
with spindle dynamics. (A) Data from CFP-labeled spindles were compared to immuno-labeled
spindles from wild type and dv1 mutants. The dv1 mutant is known to affect spindle formation
and positioning (Clark, 1940;Higgins et al., 2016). Scale bar represents 10µm. (B) Spindle
cell (∆Spindle-Cell); see Fig. 4C for diagram. There was no statistical difference between wildtype (n=33) and CFP-tubulin cells (p=0.3, n=41), while dv1 showed a greater offset and thus a
defect in spindle positioning (p˂10-4, n=33). All error bars represent standard deviation from the
mean, statistical significance is marked with a star, and p-values are calculated by Student’s ttest. (C) In approximately 20% of cells, at least one slow-moving chromosome was identified
that trailed behind the main chromosome mass at initial separation, but later caught up (see
Figure 1C). The presence of these slow-moving chromosomes was not correlated with the
asymmetry observed in segregating chromosomes. A plot of all 41 imaged cells ordered by
asymmetry (ΔDA-B) shows that cells with slow-moving chromosomes (black bars) are not
disproportionally represented in the top quartile of asymmetric divisions. With the cell
containing a lagging chromosome excluded (red bar), there are 2 cells with slow-moving
chromosomes in the top quartile and two in the bottom quartile of asymmetric divisions.
Journal of Cell Science • Supplementary information
positioning within the cell was measured by the offset between the center of the spindle and the
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
A
0 min
10 min
B
0 min
15 min
C
0 min
10 min
Supplemental Figure 2. Cell with astral-like microtubules and cells excluded from
correlation analyses. (A) Astral-like microtubules (white arrows) can be seen projecting from
Spindles (green) but not chromosomes are shown in this cell. (B) This cell was excluded from
correlation analyses due to spindle morphology defects. A large bundle of microtubules was not
organized into the spindle (T=0) and persisted into anaphase (T=10). Chromosomes moved
slower and for a shorter distance on the side with the defect. (C) This cell had a persistent
lagging chromosome that never reached the pole along with the other chromosomes (white
arrows, also shown in Fig. 1C), and it was also excluded from analyses. Only chromosomes
(magenta) are shown to highlight the lagging chromosome. Merotelic attachments (attachments
to both poles) likely impeded segregation of this chromosome, and potentially affected the
segregation of the whole chromosome mass. The chromosome mass in contact with the lagging
chromosome traveled slower and for a shorter distance than the other mass, thus this cell was
excluded due to confounding effects on asymmetric segregation. Scale bars represent 10µm.
Journal of Cell Science • Supplementary information
acentrosomal poles and curling around the cell cortex in metaphase (T=0) and anaphase (T=10).
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
C
Anaphase A distance
(% spindle length)
40%
Meiosis I
30%
Meiosis II
20%
10%
D
0%
B
7.0
Metaphase Plate/Spindle Mid-zone
6.0
R² = 0.0019
5.0
4.0
3.0
2.0
1.0
0.0
25
30
35
40
45
Spindle Length (μm)
8.0
Asymmetry value, ΔDA-B
*
50%
8.0
Asymmetry value , ΔDA-B
A
7.0
6.0
R² = 0.0361
5.0
4.0
3.0
2.0
1.0
0.0
0
5
10
15
20
F
-20
20
-15
15
-10
10
DA
-5
5
1.01.000
0.50.500
0.00.000
-0.500
-0.5
-1.000
-1.0
-1.500
-1.5
Asymmetry
Chromosome mass B
15
15
20
20
25
25
Series1
Spindle Length
Series2
Chromosome mass B
Symmetric divisions
0
10
1.5
1.5
Chromosome mass A
*
*
30
*
Chromosome mass A
Chromosome mass B
1.0
1
H
0
0.0
T= 5 1 10
5 2 10
5 310
5 410
Series1
Series2
Asymmetric Divisions
Symmetric Divisions
2.000
2.0
1.500
1.5
1.000
1.0
0.500
0.5
0.000
0.0
-0.500
-0.5
-1.000
-1.0
-1.500
-1.5
Asymmetry
Max anaphase speed
Series1
Chromosome mass A
Series2
Chromosome mass B
Asymmetric divisions
1.51.5 Cell1
1.0 1
0.50.5
0.0 0
-0.5-0.5
-1.0 -1
-1.5-1.5
Time = 5 10 min
20
Anaphase Time (min)
0.5
0.5
DB
Average anaphase speed
Chromosome mass A
2
Rate (μm/min)
10
10
Rate (μm/min)
Rate (μm/min)
I
1.51.500
55
Distance/Length (μm)
Chromosome mass A
G
00
R² = 0.0368
Chromosome mass B
Journal of Cell Science • Supplementary information
-25
25
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Anaphase speed (μm/min)
Rate (μm/min)
E
Asymmetry value, ΔDA-B(μm)
Total Anaphase A Distance (μm)
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
Supplemental Figure 3. Asymmetry in chromosome segregation. (A) The distance
chromosomes are segregated in anaphase A is statistically greater in meiosis II (n=6) than
meiosis I (n=35) when normalized by spindle length (p=0.009). (B) Data from all movies
collected, n=41. Metaphase spindle length is displayed as the black bar with the spindle midzone/metaphase plate centered at 0μm. The path of the chromosome masses is overlaid on
spindle length with the blue bar representing the distance traveled by mass A (longer traveling)
and orange bar representing mass B (shorter traveling). Asymmetry (∆DA-B) is not correlated
with (C) spindle length, (D) total anaphase A distance, or (E) total time of anaphase (n=41). (F)
In the top quartile of asymmetric divisions, chromosome mass A moves faster than mass B at
both 5 and 10 minutes post metaphase-anaphase transition (T=5 min, p=0.037, T=10 min,
p=0.019, n=10), and chromosome mass A speeds up over time (5 vs. 10 min, p=0.038). In the
top quartile of symmetric divisions (n=10), there is no statistical difference between mass A and
B at any time point. All error bars represent standard deviation from the mean, statistical
significance is marked with a star, and p-values are calculated by Student’s t-test. Rate data from
individual cells used to calculate the displayed averages can be found in (I). (G) Data from all
cells (n=41) used to calculate average anaphase speed displayed in Figure 3I is plotted and
ordered by asymmetry. The rate for chromosome mass A movement is plotted as a positive value
used to calculate maximum anaphase speed in Figure 3I (n=41 cells) is plotted in (H).
Journal of Cell Science • Supplementary information
(blue bars) and chromosome mass B is plotted as a negative value (orange bars). Similarly, data
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
Supplemental Movie 1. Live meiosis I. Meiosis I was lived imaged in male maize cells via
green. Chromosomes are labelled with SYTO12 DNA stain and pseudo-colored magenta. The
scale bar is 20 μm.
Journal of Cell Science • Supplementary information
fluorescence microscopy. Spindles are labelled via CFP-tubulin (β-TUB1) and pseudo-colored
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
Supplemental Movie 2. Live meiosis II. Meiosis II was lived imaged in male maize cells via
green. Chromosomes are labelled with SYTO12 DNA stain and pseudo-colored magenta. The
scale bar is 20 μm.
Journal of Cell Science • Supplementary information
fluorescence microscopy. Spindles are labelled via CFP-tubulin (β-TUB1) and pseudo-colored
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
Supplemental Movie 3. 3D volume view of meiosis I cell. A 3D volume view of a metaphase I
cell rotates around the y-axis to show the depth of the z-dimension and demonstrate that cells
SYTO12 (green) and the metaphase I chromosomes (bright green) can be seen aligned in the
middle of the cell volume. All movies captured a 20μm z-dimensional cross-section of meiotic
cells. The grid scale is 10 μm.
Journal of Cell Science • Supplementary information
were not compressed between the coverslip and slide. The cell volume is illuminated with
J. Cell Sci. 129: doi:10.1242/jcs.194860: Supplementary information
Supplemental Movie 4. 3D volume view of meiosis II cell. A 3D volume view of a metaphase
II cell rotates around the y-axis to show the depth of the z-dimension and demonstrate that cells
SYTO12 (green) and the metaphase II chromosomes (bright green) can be seen aligned in the
middle of the cell volume. All movies captured a 20μm z-dimensional cross-section of meiotic
cells. The grid scale is 10 μm.
Journal of Cell Science • Supplementary information
were not compressed between the coverslip and slide. The cell volume is illuminated with