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Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters


Abstract and Figures

The cleavage furrow in Xenopus zygotes is positioned by two large microtubule asters that grow out from the poles of the 1st mitotic spindle. Where these asters meet at the midplane they assemble a disc-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin which instructs the cleavage furrow. Here, we investigate the mechanism that keeps the two asters separate, and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg ortholog of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in Xenopus egg extracts. Prc1E was required for Kif4A recruitment, but not vice versa. Microtubule plus end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote "anti-parallel pruning" which enforces radial organization within asters, and generates boundaries to microtubule growth at between asters.
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Prc1E and Kif4A Control Microtubule
Organization Within and Between Large
Xenopus Egg Asters
Nguyen P.A.1,2, Field C.M.1,2, Mitchison T.J.1,2,*
1Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
2Marine Biological Laboratory, Woods Hole, MA 02543, USA.
* Correspondence to:
Running Title: Prc1/Kif4A in large embryo asters
Keywords: Kif4A, Prc1, Xenopus laevis, zygote, egg extract, microtubule aster,
cytokinesis, cleavage
Supplemental Material can be found at:
The cleavage furrow in Xenopus zygotes is positioned by two large microtubule asters that grow
out from the poles of the 1st mitotic spindle. Where these asters meet at the midplane they
assemble a disc-shaped interaction zone consisting of anti-parallel microtubule bundles coated
with chromosome passenger complex (CPC) and centralspindlin which instructs the cleavage
furrow. Here, we investigate the mechanism that keeps the two asters separate, and forms a
distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1
and Kif4A. Prc1E, the egg ortholog of Prc1, and Kif4A were recruited to anti-parallel bundles at
interaction zones between asters in Xenopus egg extracts. Prc1E was required for Kif4A
recruitment, but not vice versa. Microtubule plus end growth slowed and terminated
preferentially within interaction zones, resulting in a block to interpenetration that depended on
both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of
large asters growing in isolation, apparently to compensate for the direction-randomizing
influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with
catastrophe factors, promote “anti-parallel pruning” which enforces radial organization within
asters, and generates boundaries to microtubule growth at between asters.
Symmetric cell division requires precise positioning of the cleavage furrow at the mid-
plane of a cell. During anaphase, animal somatic cells assemble a bipolar microtubule array in
the cell centre called the cytokinesis midzone or central spindle (Glotzer, 2009). Midzone
microtubules are orientated with plus ends toward the midplane, and a small antiparallel
overlap at the equator (McIntosh and Euteneuer, 1984)(Mastronarde et al., 1993). The plus end
directed kinesin-family motor Kif23 (a.k.a. MKLP1) transports the furrow-stimulating factor
RacGap1 (together they constitute the centralspindlin complex) to the center of the midzone
(Mishima et al., 2002), while the plus end directed motor Kif20A (a.k.a. MKLP2) transports the
chromosome passenger complex (CPC) (Gruneberg et al., 2004). Centralspindlin and the CPC
then signal locally to the equatorial cortex to initiate the furrow (Jantsch-Plunger et al.,
2000)(Yüce et al., 2005)(Canman et al., 2008)(Lewellyn et al., 2011)(Argiros et al.,
2012)(Basant et al., 2015)(Henson et al 2016). In addition, astral microtubules and/or
kinetochores may relax the cortex and inhibit furrow formation at the poles in some systems
(White and Borisy, 1983)(Rodrigues et al., 2015). In cells forced to undergo monopolar
cytokinesis, monopolar midzones assemble with microtubule plus ends oriented towards the
side of the cell proximal to chromatin. Centralspindlin and CPC accumulate on that side, where
they trigger highly asymmetric and incomplete cleavage (Canman et al., 2003)(Hu et al.,
2008)(Shrestha et al., 2012).
An important question in cytokinesis mechanism is how midzone microtubules, or their
equivalents in large embryo cells, are spatially organized. The conserved cytokinesis proteins
Prc1 and Kif4A play important roles in promoting anti-parallel organization at the midplane and
determining midzone microtubule length (Kurasawa et al., 2004)(Hu et al., 2011)(Subramanian
et al., 2013). Their functions have been extensively studied in somatic cells, and using pure
proteins, but not in very large egg cells. Prc1 (a member of the Ase1/MAP65 family of
microtubule cross-linkers) promotes anti-parallel bundling in the midzone (Jiang et al.,
1998)(Mollinari et al., 2002)(Subramanian et al., 2010). Prc1 can also organize parallel
microtubule bundles in dividing cells. In cell undergoing monopolar cytokinesis, Prc1 is required
to polarize the midzone and trigger furrowing (Shrestha et al., 2012), and during normal division
it transiently “tags” parallel bundles at plus ends (Subramanian et al 2013). Kif4A (a member of
the Kinesin-4 family of plus end directed motors with plus end polymerization-inhibiting activity)
antagonizes growth of midzone plus ends (Bringmann et al., 2004)(Hu et al., 2011). Prc1 and
Kif4A interact dynamically during cytokinesis but do not form a stable complex (Zhu and Jiang,
2005). Together, they can generate anti-parallel overlaps of defined length in a pure protein
system (Bieling et al., 2010), and also “tag”, and halt growth, of parallel bundles at plus ends,
(Subramanian et al 2013).
The large size of frog eggs make them an interesting system to study aster organization
and cytokinesis signaling. The 1st mitotic spindle is small compared to the egg. At anaphase
onset, large microtubule asters grow out from centrosomes at the spindle poles and expand
towards the cortex, taking ~15min to reach it in frog zygotes (Mitchison et al., 2012). As the
asters grow, the centrosomes and nuclei at their centres are pulled away from the midplane of
the cell by dynein (Wühr et al., 2010). In a Xenopus zygote fixed between 1st anaphase and 1st
cytokinesis, microtubules from the two asters overlap and interact, forming a disc of antiparallel
bundles at the midplane. This disc, which we will call the aster interaction zone, recruits CPC
and Centralspindlin. It expands outwards towards the cortex as the asters enlarge, and initiates
a furrow where, and when, it touches the cortex (Nguyen et al., 2014)(Field et al., 2015). The
interaction zone between asters is functionally equivalent to the midzone in a dividing somatic
cell, but it extends out much further from the position previously occupied by the metaphase
The interaction zone between asters in frog eggs appears to limit microtubule growth
and prevent interpenetration of the two asters, as evidenced by lower microtubule density in the
zone compared to nearby regions (Mitchison et al., 2012). Using EB1 tracking in an extract
system, we directly observed that microtubules from one aster tend to stop at the interaction
zone, and do not cross between asters (Nguyen et al., 2014). This block to microtubule
interpenetration between asters is presumably important for correct CPC and centralspindlin
localization. It may also serve to generate microtubule length asymmetries that promote dynein-
mediated movement of centrosomes away from the midplane (Wühr et al., 2010). Based on
prior work in somatic cells and with pure proteins (cited above), Prc1 and Kif4A are candidates
for mediating the block to microtubule growth between asters. Prc1 has been little characterized
in Xenopus eggs, where it is present as the egg-specific homolog Prc1E (Nguyen et al 2014).
Xenopus Kif4A (previously called Xklp1) was shown to be expressed in eggs and essential for
normal cleavage (Vernos et al., 1995). That paper focused on Kif4A localization to mitotic
chromosomes, and lead to the concept of “chromokinesins”.
Here, we use the Xenopus egg extract system to probe the role of Prc1 and Kif4A in
controlling microtubule dynamics within, and between, large interphase asters. In (Nguyen et al
2014) we reported that Prc1E and Kif4A are recruited to anti-parallel bundles between asters,
and that Kif4A is required for normal microtubule organization and CPC transport. We did not
investigate the effect of Prc1E depletion on Kif4A localization or vice versa, and we did not
investigate the effect of either depletion on EB1 comet dynamics. These experiments are
reported below. In the course of this analysis we made an unexpected finding, that Prc1E and
Kif4A work together to enforce radial order in expanding single asters, presumably by
recognition and pruning of accidental anti-parallel overlaps. Our findings extend understanding
of the cell division biology of the conserved Prc1/Kif4A module, and reveal interesting
adaptations to promote microtubule organization and cleavage in very large egg cells.
Prc1E and Kif4A expression in Xenopus zygotes
We first identified the egg forms of Prc1 and Kif4A and generated antibodies to localize
them in zygotes and deplete them in extracts. NCBI identifiers for each protein are listed in
Supplementary Table S1. By proteomic analysis, Xenopus eggs express do not express
detectable levels of somatic Prc1 (inferred from proteomics in (Wühr et al., 2014)). We
confirmed this by western blot with an anti-peptide antibody (not shown). Instead, eggs express
a Prc1 isoform, called “Protein Regulator of Cytokinesis-Like (PRC1L)” in NCBI databases, that
we will call Prc1E to reflect its egg specific expression. Prc1E is ~45% identical to Prc1 and is
present in eggs at ~60nM (inferred from proteomics in (Wühr et al., 2014)). Prc1E is gradually
replaced by somatic Prc1 during development (inferred from mRNA and proteome analysis in
(Peshkin et al., 2015)). Somatic Kif4A is present at ~100nM in eggs, and there is no evidence
for egg-specific isoforms by proteomics (Wühr et al., 2014). We raised affinity-purified rabbit
antibodies to a C-terminal peptide from Prc1E and to the entire non-motor region of Kif4A.
These antibodies were described and characterized previously using standard methods
(Nguyen et al., 2014), (Mitchison et al., 2013). For this study, we performed
immunoprecipitation-mass spectrometry analysis, from which we concluded that our antibodies
recognize native protein, are specific for their primary target over all cell division proteins, and
that Prc1E and Kif4A do not co-immunoprecipitate, suggesting they do not form a stable
complex in solution.
Having identified Prc1E and Kif4A as the form of this conserved cytokinesis module that
is expressed in eggs, we probed their localization in fixed zygotes. We focused on the period
between 1st mitosis and 1st cleavage, when a pair of asters grows out from the poles of the
mitotic spindle to position the furrow. The boundary region between asters excluded our Prc1E
antibody (not shown), even though this protein localizes to anti-parallel microtubule bundles in
extract (see below). We suspect this negative result on localization in fixed zygotes was an
artefact caused by lack of antibody penetration, which is known for somatic midzone staining
(Saxton and McIntosh, 1987), or by failure of our antibody to recognize a phosphorylated form
of Prc1E. Kif4A was enriched on anti-parallel bundles between asters, where it mostly co-
localized with the AurkB subunit of the CPC (Fig. 1). The CPC sometimes appeared to be more
focused at the center of antiparallel bundles than Kif4A (Fig 1A,C). Weak Kif4A staining was
observed throughout asters, suggesting Kif4A does not require anti-parallel bundles for
recruitment to microtubules. Kif4A was also present on mitotic chromosomes shortly after
anaphase onset, consistent with its function as a chromokinesin (Fig 1A).
Prc1E and Kif4A localization in interphase egg extracts
To more reliably localize Prc1E and Kif4A, and probe their dynamics and function, we turned to
an actin-intact egg extract system that reconstitutes spatially organized cytokinesis signalling
(Field et al., 2014)(Field et al., 2017). We used magnetic beads coated with anti-Aurora A
kinase (AurkA) IgG as model centrosomes (Tsai and Zheng, 2005). Prc1E and Kif4A were
visualized by addition of green and red fluorescent recombinant fusion proteins whose
biochemical characterization is shown in supplementary Fig. 1a. Later depletion-add back
experiments (Fig 5) showed they were functionally competent. For visualization experiments we
added these to extract at <20% of the concentration of endogenous proteins to minimize
perturbation. We reported previously that Prc1E and Kif4A are enriched in anti-parallel bundles
between asters in the extract system (Nguyen et al 2014). Here we report dynamics and more
careful analysis using kymographs.
Both Prc1E and Kif4A were recruited to microtubules and localized throughout asters.
When the asters grew into each other, Prc1E became weakly enriched, and Kif4A strongly
enriched, on anti-parallel bundles between the asters (Fig. 2a 19, 23min, Supplementary Video
1). The two proteins co-localized on some bundles, but their overall distribution differed,
consistent their interacting transiently on microtubules but not forming a stable complex in
solution (Zhu and Jiang, 2005)(Bieling et al., 2010). To quantify microtubule morphology and
Prc1E/Kif4A recruitment over time we generated kymograph plots along the line connecting the
two aster centres, shown in the blue rectangle (Fig. 2a‟). These plots confirmed Prc1E and
Kif4A recruitment to anti-parallel bundles between the asters. Both were recruited at the same
time, and Kif4A was always more enriched in the interaction zone. We also observed bright
streaks of Prc1E and Kif4A at the free edges of growing asters (Fig. 2a, zoom-ups within yellow
square). These probably correspond to Prc1/Kif4A positive “plus end tags” observed in dividing
somatic cells (Subramanian et al., 2013) which result from Kif4A transport to plus ends, followed
by stabilization of plus ends by the two proteins. We also co-imaged the CPC with Prc1E and
tubulin, adding a GFP tagged DasraA subunit of the CPC for visualization (Fig. 2b and
Supplementary Video 2). Unlike Prc1E and Kif4A, the CPC localized exclusively to a narrow
region at the interaction zone, and was not present in streaks elsewhere in asters. The tight
localization at interaction zones was quantified using kymograph plots within the blue rectangle
(Fig. 2b‟).
Plus end dynamics at interaction zones between asters
To provide a quantitative assay for the block to microtubule growth between asters we
imaged growing plus ends between two asters with EB1-GFP and tracked them using
PlusTipTracker software (Applegate et al., 2011). Figure 3a and Supplementary Video 3 show
EB1 trajectories colour-coded by the orientation of microtubule growth. Quantification of EB1
growth direction (Fig 3e) showed that more than 90% of the growing microtubules within each
aster were oriented with their plus ends pointing radially away from the nucleating site, and
orientation switched abruptly over ~40m at the boundary between asters. Similar directionality
data was reported previously using TIRF imaging (Nguyen et al., 2014). We next mapped EB1
comet velocities (Fig 3b, f), initiations (Fig 3b, h) terminations (Fig 3c, h) and density (Fig 3g).
EB1 comets tended to slow down as they entered the interaction zone, as evidenced by
enrichment of blue colours at the midline in Fig 3b and the central dip in Fig 3f. Mean growth
rates in one experiment were 25.8 ± 9.9 μm/min (± SD, n = 13632 comets) outside the
interaction zone and 22.9 ±1 1.0 μm/min (± SD, n = 3172 comets) inside it, representing an 11%
reduction (the two rate distributions were statistically significantly different, P < 10-45 from KS
test). Full biological repeat experiments showed qualitatively similar slowdowns (11.3 ± 2.3%
reduction, mean ± SD, n = 5 interaction zones, P < 0.001 from one-sample t-test; also see Fig.
3h). EB1 track initiations, which correspond to nucleation and/or rescue events, were uniformly
distributed (Fig 3c,h). Termination events, which correspond to catastrophes or pauses, were
enriched at the centre of the interaction zone (Fig 3d, h). Comet density was lower near the
aster centres, but not specifically increased in the interaction zone (Fig 3g). From analysis of
multiple representative zones we observed no strong spatial regulation of either EB1 comet
density (10 ± 39% reduction, mean ± SD from n = 5 interaction zones, P = 0.59 from one-
sample t-test) or of the spatial frequency of initiation events (15 ± 33% increase, P = 0.37).
However, we saw a consistent increase in the frequency of termination events in or near the
interaction zone midline (86 ± 33% increase, P = 0.004). Thus, plus ends tended to slow down
and terminate more within interaction zones than elsewhere in asters, but the density of growing
plus ends, and the frequency of nucleation/rescue (which are hard to distinguish by EB1
tracking), are not spatially regulated. Spatially uniform nucleation of plus ends throughout large
asters was quantified previously, and shown to drive aster growth by mathematical modelling
(Ishihara et al., 2014)(Ishihara et al., 2016).
A limitation of EB1 tracking is that it does not report on catastrophes or
depolymerisation. To gain longer time scale information on dynamics at boundaries between
asters we imaged labelled tubulin for a prolonged time interval, using a lower magnification to
minimize photobleaching and focal plane drift. We found that once asters grew into contact, the
total microtubule density at boundaries remained approximately constant for 40 min (Fig 3i). We
were not able to quantify microtubule density from tubulin images, but these long time scale
data rule out a continuous build-up of anti-parallel bundles microtubules over time. This
approximate steady state in total microtubule density implies that all plus end polymerization
reported by the EB1 probe must be balanced by an equal amount of depolymerization, most
likely by catastrophe at plus ends. We previously estimated that individual microtubules inside
interphase asters in the extract system undergo catastrophes at a rate of ~3min-1 (Ishihara et al
2016). Since the rates of EB1 comet initiation and termination is not very different in the
boundary region between asters compared to the bulk aster (Fig 3h), and both regions are
approximately at steady state in microtubule density (Fig 3i), we estimate that the catastrophe
rate in anti-parallel bundles must be of similar order, i.e. >1 min-1. Thus, we infer that inhibition
of plus end growth in anti-parallel bundles must be followed by catastrophe. Alternatively, it is
possible that anti-parallel bundles form early and then do not turn over or recruit new
microtubules, but this seems unlikely given that EB1 comets appear to track into anti-parallel
bundles continuously.
Kif4A requires Prc1E for localization to anti-parallel bundles
To test the function of Prc1E and Kif4A in aster organization we depleted each protein to
less than 5% of its initial abundance. We depleted Kif4A previously in (Nguyen et al 2014), and
noted strong disorganization of anti-parallel bundles between asters, and also defects in
focusing the CPC at the center of the aster interaction zone. We did not deplete Prc1E or test
the effect of Kif4A depletion on Prc1E localization, or on EB1 comets trajectories. Fig 4 shows
an overview of depletion effects and Fig 5 a detailed analysis of microtubule dynamics. Western
blots quantification of depletions and add-backs are shown in supplementary Fig 1b-e which are
directly associated with Fig 5. Mass spectrometry analysis of these immunopreciptates showed
that Prc1E and Kif4A did not significantly co-immunoprecipitate (data not shown), so we could
measure the effect of depletions independently.
Fig 4a shows an overview of the effects of depleting Prc1E or Kif4, or inhibiting the
AurkB subunit of the CPC with the small molecule ZM447439. We observed no obvious effect
on nucleation from AurkA beads or on microtubule polymerization rates (measured by EB1
tracking) for any of these perturbations. Formation of regions of low microtubule density
between asters were inhibited by all three perturbations, suggesting the Prc1E, Kif4 and CPC
activity are all required for formation of sharp boundaries between asters.
We next probed dependency relationships for localization to bundles using fluorescently
labelled proteins. Kif4A no longer localized to bundles when Prc1E was depleted (Fig 4c). Prc1E
still localized when Kif4A was depleted, though the bundles were longer and more disorganized
(Fig 4d). Inhibition of AurkB caused profound randomization of bundle position relative to the
midline between the asters, but Prc1E and Kif4A still localized to bundles, and to some extent
still co-localized (Fig 4e). These data suggest a model where Prc1E recognizes anti-parallel
bundles and then recruits Kif4A, consistent with the model previously developed using pure
proteins (Bieling et al., 2010). AurkB activity was not required for recruitment of either protein to
bundles. However, AurkB activity was required for focusing Prc1E/Kif4A-positive bundles into a
sharp line that bisects the distance between the asters.
Prc1E and Kif4A are required to block plus end growth between asters
To quantify the effect of immunodepletion on microtubule growth between asters we
used EB1 comet tracking. The top panels in 5a show tracking data, the bottom panels
quantification of those data. The effects of depletions and add-backs are quantified as a bar
graph in Fig. 5g where we report the distance over which EB1 directionality switches, using the
arbitrary distance metric D60, the distance over which EB1 comet directionality switches from
60%:40% to 40%:60%. For comparison, fig 5g includes the effects of small molecule inhibition
(ZM-447439) of the AurkB subunit of the CPC, which we previously showed increases D60
(Nguyen et al., 2014).
Depletion of either Prc1E or Kif4A caused microtubules from each aster to grow into the
neighbouring aster much more than depletion using control IgG or anti-Kif23 (the kinesin subunit
of Centralspindlin) (Fig. 5a-d). There was still a switch in microtubule orientation between the
asters when Prc1E or Kif4A was depleted, but the distance over which this occurred was much
larger. Thus, both proteins are required for a tightly organized block to microtubule
interpenetration at the boundary between asters. EB1 comet visualization was superior using
TIRF imaging of plus ends tracking along the surface of partially passivated coverslips. Videos
4-6 show movies of EB1 and tubulin between asters in control, Prc1E depleted and Kif4A
depleted extracts. The block to plus end growth between asters in control extracts, and loss of
this block after depleting either protein, are clearly visualized.
The Prc1E depletion phenotypes was rescued by adding back the appropriate
recombinant expressed proteins, either as a GFP fusion or unlabelled protein (Fig. 5e). Kif4A
add back also rescued (data not shown). This confirms both the specificity of the
immunodepletion, and the functional attributes of the recombinant proteins. The effect of Prc1E
depletion was also rescued by adding back the somatic isoform of Xenopus Prc1 protein (Fig.
5f). In general, we were unable to determine a functional difference between somatic and egg
isoforms of Prc1 using the extract system.
The Prc1/Kif4A module enforces radial order within large asters
In the course of measuring the effects of Prc1E and Kif4A depletion on interaction zones
between asters we noticed that the morphology of isolated asters was also strongly perturbed.
In control IgG depleted extracts containing fewer nucleating beads isolated asters grew with
high radial order to the edge of the camera field (Fig 6a). In extracts depleted of either Prc1E or
Kif4A, microtubules grew radially from the AurkA bead out to a radius of ~40 μm. Microtubules
continued to assemble at the periphery, but instead of forming a well-organised aster, they
appeared disorganised at larger radii. This effect is evident by the disorganized EB1 tracks at
the periphery in Fig 6 b,c.
To quantify radial order in asters, we measured the directions of EB1 comet trajectories
within a quadrant of an isolated aster with its centre at the top left corner (Fig. 6a-e). We then
generated radial order heat maps by plotting radial order parameters (R) calculated from the
distribution of angular deviations of EB1 trajectories from the radial direction (Fig. 6a‟-e‟; see
Methods). We also plotted average radial order parameters against the distance from the aster
centre at the AurkA bead (Fig. 6f; see Methods). Radial order may be inaccurately measured at
small radii (<10 m) due to growth of microtubules in the Z plane close to the bead, so the initial
rise in these plots probably does not represent low order near the bead.
Mock depleted asters maintained high radial order out to the largest radius measured with our
camera (~150-200μm), with a slight decrease at large radii. Depletion of Kif4A or Prc1E strongly
disrupted radial order. Limited order was maintained within ~40 μm radius, presumably due to
frequent re-nucleation from the AurkA bead. Radial order was progressively lost as radius
increased, though growth direction never completely randomized. The effect of depleting both
Prc1E and Kif4A on radial order was rescued by adding back appropriate recombinant proteins
(Fig. 6d, e; data not shown for Kif4A add-back). Both the embryonic and somatic isoforms of
Prc1 were able to rescue radial order. We conclude from these data that Kif4A and Prc1E are
necessary to maintain radial order within large asters as they expand, presumably to counter
directional randomizing effects of nucleation away from the centrosome. Loss of radial order
probably caused the gross disorganization of microtubules between asters we observed in
(Nguyen et al 2014) when Kif4A was depleted, and could not account for in that paper.
Pruning of anti-parallel microtubule overlaps by Prc1E and Kif4A
We finally turned to single microtubule imaging to better understand the function of
Prec1E and Kif4A in asters. We sought regions in 2- or 3-colour confocal movies where the
microtubule density was low enough to image individual microtubules, then analysed them by
visual inspection and kymographs. Finding regions that were interpretable at the single
microtubule level was difficult because microtubule density was high, and most microtubules
occurred in bundles of more than two. Figure 7 shows two examples near the edge of growing
asters where a microtubule growing in the wrong direction enters an anti-parallel overlap, and
then the overlap recruits Kif4A. In Fig 7a (Supplementary Video 7) a single microtubule growing
in the wrong direction is evident in the first frame. It then enters an anti-parallel bundle that first
recruits Kif4A, then shrinks. In Fig 7b (Supplementary Video 8) the incorrect microtubule may
nucleate within in a bundle, which we suspect is more common. The bundle recruits Prc1E and
Kif4A, and then proceeds to shrink, with loss of microtubule density. These movies support an
anti-parallel pruning model for Prc1E/Kif4A function in asters, though we were not able to find
enough clear examples to make any statistically justified statements concerning single
microtubule mechanism.
Our data confirm much of what is already known about Prc1 and Kif4A function from
published work from our laboratory and many others, e.g. the localization of both factors to anti-
parallel bundles prior to cytokinesis (Figs 1,2), a Prc1E requirement for recruitment of Kif4A to
anti-parallel bundles (Fig 4), a role for both protein in organizing anti-parallel bundles (Fig 4,5)
and in restricting plus end growth in anti-parallel bundles (Fig 5). In some cases, these
confirmatory findings come with greatly improved quantification compared to previous work,
or/and extend work from somatic cells to the Xenopus egg system with its huge spatial scale,
e.g. precise quantification of plus ends dynamics between asters (Fig 3) and showing that
Prc1E and Kif4A keep asters distinct in frog eggs by inhibiting plus end growth at shared
boundaries (Fig 5). Data in this paper also reveal new mechanisms and allow us to build new
hypothesis, e.g. evidence for depolymerisation as well as stabilization in anti-parallel bundles
(Fig 3i) and an unexpected role of the two proteins in enforcing radial order in isolated asters
(Fig 6).
In Figure 8 we propose an “anti-parallel pruning” model to account for the role of Prc1E
and Kif4 in generating the boundary between asters, and enforcing radial order within them.
This model combines Bieling and Surrey‟s molecular model from seminal biochemical
reconstitution (Bieling et al., 2010) with new information from the egg extract system. We
propose that after Prc1E and Kif4 recognize anti-parallel bundles and slow plus end growth the
plus ends eventually undergo catastrophe, so at least one of the anti-parallel microtubules is
removed. We were not able to image catastrophes and depolymerisation directly, except in
occasional movies (Fig 7). It must nevertheless occur on the majority of microtubules that join
anti-parallel bundles in the interaction zone because new plus ends continually enter anti-
parallel bundles, yet total microtubule density remains approximately constant for many minutes
(Fig 3i, video 4). Catastrophes were not observed on plus ends coated with Prc1+Kif4A in pure
protein systems (Bieling et al., 2010, Subramanian et al 2013). We suspect this was due to lack
of catastrophe factors, though we note that pure protein work used pH 6.8 PIPES buffer, which
is known to artificially stabilize microtubules, and might also supress catastrophes. Higher pH is
required to observe the catastrophe-promoting activity of stathmin, a different factor that
negatively regulates plus end dynamics (Howell et al 1999).
Our model assumes that Prc1E and Kif4A transiently function together in anti-parallel
overlaps, even though they do not strongly co-localize on average (Fig 2). Consistent with this
assumption, we were able to observe examples of transient co-localization preceding anti-
parallel pruning (Fig 7). Our model neglects other functions of both proteins. For example,
Prc1E may contribute to the bundling of microtubules we observe throughout asters, and may
also help recruit CPC and centralspindlin to a subset of microtubules, as seen in somatic cells
(Shrestha et al., 2012).
Our model for the block to interpenetration between asters (Fig 8 red box). is essentially
an extension of current ideas about somatic midzone organization applied to the much larger
spatial scale of Xenopus zygotes, with the addition of a catastrophe step following recognition
by Prc1E + Kif4A as discussed above. Our model for the role of Prc1E and Kif4A in enforcing
radial order within asters (Fig 8 blue box) is a new idea, and may be specific to very large egg
cells. We propose radial order is enforced by the same anti-parallel pruning mechanism as the
block to interpenetration between asters. Interphase asters must grow to radii of >500m to
center the sperm pro-nucleus and orient the cleavage furrow in frog zygotes (Wühr et al.,
2010),(Mitchison et al., 2012). Radial order is presumably important to promote orderly aster
growth and recruit cytokinesis signalling complexes selectively at the boundary between asters.
Individual microtubules in the aster are short and unstable, with an estimated average length of
~16μm (Ishihara et al., 2016). We do not know the nucleation mechanism(s) of these
microtubules. From imaging EB1 track initiations we suspect nucleation occurs mostly within
bundles (Ishihara et al., 2014) , and there is probably a preference for parallel nucleation, as
observed in CSF-arrested egg extracts (Petry et al., 2013). However, the nucleation
mechanisms likely differ, because TPX2 and Augmin, which play central roles in nucleating
microtubules in meiosis-II spindles in CSF extract, did not seem to play a major role in
interphase asters (Ishihara et al., 2014). We observed ~3-10% of plus ends growing with the
incorrect orientation in control asters (Fig 3a,e), so we suspect nucleation away from
centrosomes can occur with incorrect orientation, at least in extracts, and that the anti-parallel
pruning function of Prc1E and Kif4A removes these incorrectly orientated microtubules.
Activity of the AurkB subunit of the CPC was required for assembly of spatially focused
interaction zones between asters (Fig 4e, 5g). However, AurkB activity was not required for
recruitment of Prc1E and Kif4A to microtubule bundles inside asters (Fig 4e), or for growth of
well-organized asters with correctly oriented plus ends (Nguyen et al., 2014). We propose that
the anti-parallel pruning function of Prc1E and Kif4A does not depend on AurkB activity, though
the CPC helps focus it in CPC-positive interaction zones. When asters from different mitotic
spindles, which we term “non-sister” asters, meet in polyspermic Xenopus zygotes, they fail to
recruit CPC and centralspindlin to their shared boundary, and they also fail to trigger cleavage
furrows (Field et al., 2015). However, non-sister asters appeared not to cross into each other as
they grew, and they formed distinct interaction zones at the shared boundary in microtubule
images, albeit less focused than those between sister asters. We propose that Prc1E and Kif4A
generate the boundary between non-sister asters in the same way they do between sisters, by
anti-parallel pruning, though the boundary is less sharp in the absence of the spatial focusing
activity of the CPC.
Many questions remain concerning the organization and dynamics of large interphase
asters and the interaction zones that form between them. For example, we proposed that
interaction zones constitute a bistable self-organizing system, which propagates outwards with a
memory of initial conditions, to explain how information on proximity to chromatin is propagated
from the mitotic spindle to the cortex (Field et al., 2015). The egg extract system will be useful to
further address spatial communication from spindle to cortex, which was first characterized in
detail by Rappaport in echinoderm eggs (Rappaport, 1996), and lies at the heart of the cleavage
pattern of early embryos.
Protein expression plasmid constructs
The X. laevis Prc1E coding sequence (codon optimized for E. coli and insect cell
expression; GenScript, NJ) was subcloned into pET-28b bacterial expression vector with an N-
terminal Strep-tag (WSHPQFEK), creating Strep-Prc1E. To create the Strep-Prc1 construct, the
Prc1E sequence was replaced with the X. laevis Prc1 somatic coding sequence from a
published construct (Bieling et al., 2010) kindly provided by Thomas Surrey (London Research
Institute, UK). To create the Strep-mCherry-Prc1E construct, a mCherry coding sequence with a
C-terminal AAA linker was inserted N-terminal to the Prc1E coding sequence. All subcloning
was performed with Gibson cloning.
Proteins and antibodies
X. laevis Kif4A-GFP-His was purified from Sf21 insect cells as described (Bieling et al.,
2010) (Nguyen et al., 2014). X. laevis His-GFP-DasraA, human EB1-GFP-His and human EB1-
mCherry-His were purified from E. coli as described (Nguyen et al., 2015). X. laevis Strep-
mCherry-Prc1E, Strep-Prc1E and Strep-Prc1 were purified as previously described for a similar
Strep-GFP-Prc1E construct (Nguyen et al., 2014). Tubulin was purified from bovine brain and
labelled with Alexa488, Alexa568, or Alexa647 dyes (Life Technologies, NY) as described
(Hyman et al., 1991). Protein concentrations were determined by Bradford assay with BSA
standards, and refer to dimers for tubulin and monomers for all the other proteins. See Table S1
for sequence information on the Xenopus proteins studied in this paper.
Affinity purified C-terminal peptide antibodies (rabbit) were produced against X. laevis
Prc1E (C-FKEEMTKKSSHSEAVFNSTVNENL) (NeoBioLab, MA). Antibodies were raised in
rabbits to GST-fused X. laevis Prc1 (C-terminal 160 amino acids), and affinity purified using the
Prc1 C-terminal tail fused to MBP. Affinity purified antibodies (rabbit) against C-terminal tails of
X. laevis Kif4A, Kif23, and Aurora kinase B were from a previous study (Nguyen et al., 2014).
Tubulin antibodies (Sigma #T6074) were purchased. Antibodies were labelled on column with
Alexa488, Alexa568, or Alexa647 dyes (Life Technologies, NY) as described (Groen et al.,
Passivation of glass coverslips
Fully passivated PEG-coated coverslips were prepared either by following previous
relatively labour-intensive protocols for covalently coating coverslips with silane-PEG (Bieling et
al., 2010) or by a much simpler coating with poly-(L-lysine)-PEG (PLL-PEG). For the latter
method, coverslips were stored in 95% ethanol. On the day of use they were withdrawn from the
ethanol with forceps, flame dried in a Bunsen burner for a few secs, then cooled to RT. They
were incubated on one side with 100 μg/mL PLL-PEG {PLL(20)-g[3.5]-PEG(2), SuSoS,
Switzerland} dissolved in 10 mM Hepes, pH 7.7, for 10-30 min at room temperature. Coverslips
were rinsed several times in deionized water by transferring to drops on parafilm, then blow
dried with nitrogen gas, and used within 24 hrs. For TIRF imaging where we needed
microtubules to track along the glass we used either coated with kappa casein or partial PEG
passivation according to a published protocol (Portran, 2014). For these experiments, we made
squashed between a fully passivated 22mm2 coverslip and a kappa casein coated, or partially
passivated, 18mm2 coverslip, and imaged with less passivated coverslip.
Aster assembly assay
Metaphase arrested Xenopus egg extracts with intact actin were prepared as described
(Field et al., 2014). Interphase asters were assembled between PEG-passivated coverslips as
previously described (Nguyen et al., 2014) with modifications in the coverslip passivation
procedure described above. Briefly, M-phase extract was supplemented with fluorescent
probes, treated with 0.4mM CaCl2 to mimic fertilization and induce metaphase-to-interphase
transition. Activated extract was supplemented with Protein A Dynabeads (Life Technologies,
NY) coupled to Aurora kinase A antibody, which served as artificial centrosomes (Tsai and
Zheng, 2005). Extract was then spread between two PEG-passivated glass coverslips. Aster
assembly reactions were monitored with a widefield, spinning disc confocal, or TIRF (Total
Internal Reflection Fluorescence) microscope at 20°C. For widefield and confocal imaging,
asters were assembled between two fully passivated PEG-coated coverslips. For TIRF imaging,
asters were assembled between a fully passivated PEG coverslip (top, 18 × 18 mm) and a
partially passivated coverslip (bottom, 22 × 22 mm). Partial passivation allowed microtubules to
track along the coated surface, within the TIRF illumination field. For visualization, fluorescent
probes were used at the following concentrations: 250 nM Alexa488-, 568- or 647-tubulin, 40
nM EB1-GFP or EB1-mCherry, 10 nM Kif4-GFP, 20 nM mCherry-Prc1E, 20 nM GFP-DasraA.
Immunodepletions, protein addbacks, and drug inhibition
Prc1E, Kif4A and Kif23 were depleted in 2 rounds using 2x20 μg of antibodies
conjugated to 50μL Protein A Dynabeads (Life Technologies, NY) per 50 μL of extracts.
Depletions using beads coated with random rabbit IgG served as controls. Depletions were
confirmed by immunoblots. Kif4A depletion was rescued by adding back ~100 nM Kif4A-GFP,
and Prc1E depletion was rescued by adding back ~65 nM purified Prc1E or Prc1. For inhibition
of Aurora B kinase, 100 μM ZM447439 (Tocris Bioscience, MN; 10 mM stock solution in 10 mM
glycine, pH 7) was added to calcium activated extracts. Aster assembly assays were performed
with treated extracts as described above.
All immunodepletion and drug inhibition experiments were performed with n ≥ 3
biological repeats, using extracts prepared from eggs produced by different female frogs.
Ideally, all the treatments ought to be performed on the same extract to reduce extract-to-extract
(and frog-to-frog) variability, and the samples from each treatment ought to be imaged at the
same time to reduce the variability introduced by the “aging” of extracts while stored on ice. In
practice, however, this is impossible due to limitations posed by the amount of extract produced
from a single female frog and our ability to handle samples. Therefore, we routinely performed
only 3-4 treatments (including control buffer addition or IgG depletion) per experiment. Although
we aimed to acquire data for all treatments simultaneously, 1-2 samples out of the 3-4 would
often be destroyed via mishandling, thus the data for those treatments had to be acquired in the
next round of aster assembly reaction.
Time-lapse fluorescence microscopy
Widefield images were obtained using a 10x Plan Apo 0.45 NA objective lens (Nikon) on
an upright Nikon Eclipse 90i microscope equipped with a Prior Lumen 200 metal arc lamp, a
Prior ProScan III motorized XY stage, a Hamamatsu ORCA-ER cooled CCD camera, and driven
by Metamorph image acquisition software (Molecular Devices, CA). Spinning disc confocal
images were obtained using a 40x oil Plan Apo 1.30 NA objective lens (Nikon) on an upright
Nikon Eclipse E800 microscope equipped with a Melles Griot Krypton/Argon ion laser (488nm,
568nm, 647nm), a Yokogawa CSU-10 spinning disc (Perkin Elmer, MA), a Hamamatsu ORCA-
ER cooled CCD camera, and driven by Metamorph. TIRF images were obtained using a 60x
Apo TIRF 1.49 NA objective lens (Nikon) on a Nikon Ti-E motorized inverted microscope
equipped with a Nikon motorized TIRF illuminator, Perfect focus, a Prior Proscan II motorized
stage, Agilent MLC400B laser launch (488nm, 561nm, 647nm), an Andor DU-897 EM-CCD
camera driven by NIS-Elements image acquisition software. Two separate TIRF setups were
available at the Nikon Imaging Center at Harvard Medical School and the Marine Biological
Fixed Immunofluorescence of fixed Xenopus zygotes (Fig 1)
Zygotes were fixed and stained approximately 100 min after fertilization as previously
described (Field et al., 2015). Briefly, zygotes were fixed in a methanol/EGTA solution for 24 hrs
at room temperature, rehydrated in a series of methanol/TBS mixtures, hemisected, bleached
and then incubated with directly labeled antibodies for at least 24 hrs at 4°C. Antibodies were
used at approximately the following concentrations: Alexa488-anti-tubulin (1-2μg/mL),
Alexa568-anti-Prc1E (1-2μg/mL), Alexa647-anti-Kif4A (1-2μg/mL). Fixed zygotes were mounted
in a mixture of benzyl alcohol and benzyl benzoate (Aldrich) and imaged with a laser scanning
confocal microscope at the Nikon Imaging Center at Harvard Medical School. Imaging used a
Nikon Ti-E inverted microscope with a Nikon A1R point scanning confocal head, driven by NIS-
Elements image acquisition software.
Image analysis: microtubule plus end dynamics from EB1 comet tracking (Figs. 3, 5, 6)
Time-lapse image sequences of EB1-GFP were acquired, processed, and analyzed as
described (Nguyen et al., 2014). Briefly, extract was supplemented with 40 nM EB1-GFP and
250 nM Alexa647-tubulin. Asters were assembled between fully passivated PEG coverslips as
described above. Lower AurkA bead density was used to study isolated asters, and higher bead
density to study interaction zones. Multiple isolated asters or interaction zones were imaged
between 20 and 50 min of the assembly reaction at 20°C, alternating between control and
treatment conditions. A spinning disc confocal microscope with a 40x oil objective (NA = 1.30)
was used to acquire images with 2x2 binning. Time-lapse image sequences were acquired of
EB1-GFP with 1.5 sec intervals for a total duration of 2 min (81 frames total). Tubulin images
were acquired at the beginning and end of each sequence.
EB1 image sequences were registered using the StackReg ImageJ plugin with rigid
body transformation (Thévenaz et al., 1998). The plusTipTracker MATLAB software (Applegate
et al., 2011) was used to perform automated detection and frame-to-frame linking of EB1
comets. Tracks were filtered out and excluded from analysis based on the criteria and
parameters listed in (Nguyen et al., 2014). Remaining tracks were then plotted and colored
according to their mean direction (Fig. 3a) or instantaneous velocity averaged over 3
consecutive frames (Fig. 3b).
The degree of interpenetration was quantified by dividing all EB1 comets detected in
frames 2-81 into two groups based on the direction of their displacement relative to the previous
frame (Fig. 3a‟, inset showing „blue‟ and „red‟ directions). Rectangular regions of interests (ROIs;
90 μm x 10 μm) parallel to the bead-bead axis and traversing the interaction zone were overlaid.
Each ROI was divided into 9 cells (10 μm x 10 μm each), and the fraction of EB1 comets
moving in the „blue‟ direction was determined and plotted as grey dots against the distance from
the interaction zone midline (see fig. S3 in ref. (Nguyen et al., 2014). The procedure was
repeated for multiple non-overlapping, neighbouring ROIs within an interaction zone, giving rise
to all the grey dot data. The data points were fitted to a sigmoidal distribution (Fig. 3a‟, blue
 
 ,
where is distance and  is the fraction of EB1 comets moving in the „blue‟ direction. A
complementary sigmoidal distribution described the fraction of EB1 comets moving in the other,
„red‟, direction (Fig. 3a‟, red curve). The exact position of the interaction zone midline was
defined where the blue and red curves intersected. A „D60‟ parameter was defined as the
difference between the interpolated distances where the red and blue curves crossed 60%. This
parameter served as a measure for the depth of interpenetration.
The instantaneous velocities associated with each EB1 comet detected in frames 2-81
were averaged within each square cell. The total number of EB1 comets was counted, and the
comet density determined for each cell. The number of EB1 comets comprising the initiation or
the termination of a growth track was counted and expressed as the fraction of total EB1 comets
for each cell. EB1 comet speeds, densities, track terminations and initiations were averaged for
cells in neighboring ROIs at a given distance from the interaction zone midline. These mean
values were plotted in Fig. 3b‟ and 3c‟, where the error bars indicate SEMs for EB1 speeds and
SDs for densities, track terminations and initiations.
To obtain the % difference in plus end dynamics measurements inside an interaction
zone compared to outside, the mean value of each measurement obtained at the exact
interaction zone midline (0 μm) was divided by the mean of the mean values obtained at -40, -
30, -20, 20, 30, and 40 μm from the interaction zone midline. The mean values obtained at -10
and 10 μm were considered transition values, and thus were omitted from the calculations. The
% difference measurements were averaged for multiple (n ≥ 3) interaction zones per condition;
the mean values were plotted with standard deviations as error bars in Fig. 4). All analyses were
performed in MATLAB.
Image analysis: quantification of radial order within isolated asters
The first approach to quantifying radial order in asters involved tracking EB1 comets
over 2 min (81 frames at 1.5 sec intervals) and measuring their directions (angles of frame-to-
frame displacement) within a quadrant of an isolated aster. Radial order heat maps were
created by dividing the field of view into square cells measuring 5x5 μm2 (Fig. 6a‟-e‟). A radial
order parameter (R) was calculated from the distribution of angular deviations of EB1 comet
directions from the radial direction within each square cell:
 
  
 ,
where   . The variable is the radial direction of each EB1 comet (i.e. angular
coordinate within a polar coordinate system with the aster centre as the pole) and is the
angle of displacement of each comet with respect to the previous frame. The variable ranges
from  to  in radians. For the heat maps, all EB1 comets detected from frame 2 to 81 were
considered within each square cell. In directional statistics, circular variance is defined as (1 -
R), which is a measure of the spread of a population of angles . R is the length of the mean
resultant vector of the population of angles and ranges from 0 to 1. A MATLAB toolbox called
„CircStat‟ was used to compute the resultant vector length (R) (Berens, 2009). To quantify the
dependence of radial order on the distance from the aster centre, the field of view was divided
into concentric rings with a width of 5 μm, centring on the AurkA bead. Radial order parameters
(R) were obtained for each ring area, considering EB1 comets detected within 10 consecutive
frames (e.g. frames 2 to 11, 12 to 21, …, 72 to 81). The mean R values were obtained from all 8
time ranges and plotted against distance from the bead, along with standard deviations as error
bars (Figure 6f).
We thank Aaron Groen for providing GFP-DasraA, antibodies against AurkA and Kif23 kinase,
and help with the manuscript. We also thank Edwin Tan for making Kif23 antibodies and David
Miyamoto for making Kif4A antibodies. Keisuke Ishihara and Martin Loose helped with PLL-
PEG coverslip passivation and TIRF imaging. We thank the Nikon Imaging Center at HMS and
Nikon at MBL for microscopy support, and the NXR at MBL for Xenopus care. This work was
supported by NIH GM39565 (TJM) and MBL fellowships from the Evans Foundation, MBL
Associates and the Colwin Fund (TJM, CMF).
Applegate, K.T., Besson, S., Matov, A., Bagonis, M.H., Jaqaman, K., and Danuser, G. (2011).
plusTipTracker: Quantitative image analysis software for the measurement of microtubule
dynamics. J. Struct. Biol. 176, 168184.
Argiros, H., Henson, L., Holguin, C., Foe, V., and Shuster, C.B. (2012). Centralspindlin and
chromosomal passenger complex behavior during normal and Rappaport furrow specification in
echinoderm embryos. Cytoskeleton (Hoboken) 69, 840853.
Basant, A., Lekomtsev, S., Tse, Y.C., Zhang, D., Longhini, K.M., Petronczki, M., and Glotzer, M.
(2015). Aurora B kinase promotes cytokinesis by inducing centralspindlin oligomers that
associate with the plasma membrane. Dev. Cell 33, 204215.
Berens, P. (2009). Circstat: A MATLAB toolbox for Circular Statistics. Journal of Statistical
Software 31, 121.
Bieling, P., Telley, I.A., and Surrey, T. (2010). A minimal midzone protein module controls
formation and length of antiparallel microtubule overlaps. Cell 142, 420432.
Bringmann, H., Skiniotis, G., Spilker, A., Kandels-Lewis, S., Vernos, I., and Surrey, T. (2004). A
kinesin-like motor inhibits microtubule dynamic instability. Science 303, 15191522.
Canman, J.C., Cameron, L.A., Maddox, P.S., Straight, A., Tirnauer, J.S., Mitchison, T.J., Fang,
G., Kapoor, T.M., and Salmon, E.D. (2003). Determining the position of the cell division plane.
Nature 424, 10741078.
Canman, J.C., Lewellyn, L., Laband, K., Smerdon, S.J., Desai, A., Bowerman, B., and Oegema,
K. (2008). Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.
Science 322, 15431546.
Field, C.M., Nguyen, P.A., Ishihara, K., Groen, A.C., and Mitchison, T.J. (2014). Xenopus egg
cytoplasm with intact actin. Meth. Enzymol. 540, 399415.
Field, C.M., Groen, A.C., Nguyen, P.A., and Mitchison, T.J. (2015). Spindle-to-cortex
communication in cleaving, polyspermic Xenopus eggs. Mol. Biol. Cell 26, 36283640.
Field, C.M., Pelletier, J.F., and Mitchison, T.J. (2017). Xenopus extract approaches to studying
microtubule organization and signaling in cytokinesis. Methods Cell Biol. 137, 395435.
Glotzer, M. (2009). The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat.
Rev. Mol. Cell Biol. 10, 920.
Groen, A.C., Ngyuen, P.A., Field, C.M., Ishihara, K., and Mitchison, T.J. (2014). Glycogen-
supplemented mitotic cytosol for analyzing Xenopus egg microtubule organization. Meth.
Enzymol. 540, 417433.
Gruneberg, U., Neef, R., Honda, R., Nigg, E.A., and Barr, F.A. (2004). Relocation of Aurora B
from centromeres to the central spindle at the metaphase to anaphase transition requires
MKlp2. J. Cell Biol. 166, 167172.
Henson, J.H., Buckley, M.W., Yeterian, M., Weeks, R.M., Simerly, C.R., and Shuster, C.B.
(2016). Central Spindle Self-Organization and Cytokinesis in Artificially Activated Sea Urchin
Eggs. Biol. Bull. 230, 85-95.
Howell B, Larsson N, Gullberg M, Cassimeris L (1999). Dissociation of the tubulin-sequestering
and microtubule catastrophe-promoting activities of oncoprotein 18/stathmin. Mol Biol Cell. 10,
Hu, C.-K., Coughlin, M., Field, C.M., and Mitchison, T.J. (2008). Cell polarization during
monopolar cytokinesis. J. Cell Biol. 181, 195202.
Hu, C.-K., Coughlin, M., Field, C.M., and Mitchison, T.J. (2011). KIF4 regulates midzone length
during cytokinesis. Curr. Biol. 21, 815824.
Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., and
Mitchison, T. (1991). Preparation of modified tubulins. Meth. Enzymol. 196, 478485.
Ishihara, K., Nguyen, P.A., Groen, A.C., Field, C.M., and Mitchison, T.J. (2014). Microtubule
nucleation remote from centrosomes may explain how asters span large cells. Proc. Natl. Acad.
Sci. U.S.A. 111, 1771517722.
Ishihara, K., Korolev, K.S., and Mitchison, T.J. (2016). Physical basis of large microtubule aster
growth. Elife 5.
Jantsch-Plunger, V., Gönczy, P., Romano, A., Schnabel, H., Hamill, D., Schnabel, R., Hyman,
A.A., and Glotzer, M. (2000). CYK-4: A Rho family gtpase activating protein (GAP) required for
central spindle formation and cytokinesis. J. Cell Biol. 149, 13911404.
Jiang, W., Jimenez, G., Wells, N.J., Hope, T.J., Wahl, G.M., Hunter, T., and Fukunaga, R.
(1998). PRC1: a human mitotic spindle-associated CDK substrate protein required for
cytokinesis. Mol. Cell 2, 877885.
Kurasawa, Y., Earnshaw, W.C., Mochizuki, Y., Dohmae, N., and Todokoro, K. (2004). Essential
roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation.
EMBO J. 23, 32373248.
Lewellyn, L., Carvalho, A., Desai, A., Maddox, A.S., and Oegema, K. (2011). The chromosomal
passenger complex and centralspindlin independently contribute to contractile ring assembly. J.
Cell Biol. 193, 155169.
Mastronarde, D.N., McDonald, K.L., Ding, R., and McIntosh, J.R. (1993). Interpolar spindle
microtubules in PTK cells. J. Cell Biol. 123, 14751489.
McAlister, G.C., Nusinow, D.P., Jedrychowski, M.P., Wühr, M., Huttlin, E.L., Erickson, B.K.,
Rad, R., Haas, W., and Gygi, S.P. (2014). MultiNotch MS3 enables accurate, sensitive, and
multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem.
86, 71507158.
McIntosh, J.R., and Euteneuer, U. (1984). Tubulin hooks as probes for microtubule polarity: an
analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle. J.
Cell Biol. 98, 525533.
Mishima, M., Kaitna, S., and Glotzer, M. (2002). Central spindle assembly and cytokinesis
require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev. Cell 2,
Mitchison, T., Wühr, M., Nguyen, P., Ishihara, K., Groen, A., and Field, C.M. (2012). Growth,
interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells.
Cytoskeleton (Hoboken) 69, 738750.
Mitchison, T.J., Nguyen, P., Coughlin, M., and Groen, A.C. (2013). Self-organization of
stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol. Mol.
Biol. Cell 24, 15591573.
Mollinari, C., Kleman, J.-P., Jiang, W., Schoehn, G., Hunter, T., and Margolis, R.L. (2002).
PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle
midzone. J. Cell Biol. 157, 11751186.
Nguyen, P.A., Groen, A.C., Loose, M., Ishihara, K., Wühr, M., Field, C.M., and Mitchison, T.J.
(2014). Spatial organization of cytokinesis signaling reconstituted in a cell-free system. Science
346, 244247.
Nguyen, P.A., Field, C.M., Groen, A.C., Mitchison, T.J., and Loose, M. (2015). Using supported
bilayers to study the spatiotemporal organization of membrane-bound proteins. Methods Cell
Biol. 128, 223241.
Peshkin, L., Wühr, M., Pearl, E., Haas, W., Freeman, R.M., Gerhart, J.C., Klein, A.M., Horb, M.,
Gygi, S.P., and Kirschner, M.W. (2015). On the Relationship of Protein and mRNA Dynamics in
Vertebrate Embryonic Development. Dev. Cell 35, 383394.
Petry, S., Groen, A.C., Ishihara, K., Mitchison, T.J., and Vale, R.D. (2013). Branching
microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. Cell 152, 768
Portran, D. (2014). Micropatterning microtubules. Methods Cell Biol. 120, 3951.
Rappaport, R. (1996). Cytokinesis in animal cells (Cambridge University Press).
Rodrigues, N.T.L., Lekomtsev, S., Jananji, S., Kriston-Vizi, J., Hickson, G.R.X., and Baum, B.
(2015). Kinetochore-localized PP1-Sds22 couples chromosome segregation to polar relaxation.
Nature 524, 489492.
Saxton, W.M., and McIntosh, J.R. (1987). Interzone microtubule behavior in late anaphase and
telophase spindles. J. Cell Biol. 105, 875886.
Shrestha, S., Wilmeth, L.J., Eyer, J., and Shuster, C.B. (2012). PRC1 controls spindle
polarization and recruitment of cytokinetic factors during monopolar cytokinesis. Mol. Biol. Cell
23, 11961207.
Subramanian, R., Wilson-Kubalek, E.M., Arthur, C.P., Bick, M.J., Campbell, E.A., Darst, S.A.,
Milligan, R.A., and Kapoor, T.M. (2010). Insights into antiparallel microtubule crosslinking by
PRC1, a conserved nonmotor microtubule binding protein. Cell 142, 433443.
Subramanian, R., Ti, S.-C., Tan, L., Darst, S.A., and Kapoor, T.M. (2013). Marking and
measuring single microtubules by PRC1 and kinesin-4. Cell 154, 377390.
Thévenaz, P., Ruttimann, U.E., and Unser, M. (1998). A pyramid approach to subpixel
registration based on intensity. IEEE Trans Image Process 7, 2741.
Ting, L., Rad, R., Gygi, S.P., and Haas, W. (2011). MS3 eliminates ratio distortion in isobaric
multiplexed quantitative proteomics. Nat. Methods 8, 937940.
Tsai, M.-Y., and Zheng, Y. (2005a). Aurora A kinase-coated beads function as microtubule-
organizing centers and enhance RanGTP-induced spindle assembly. Curr. Biol. 15, 21562163.
Tsai, M.-Y., and Zheng, Y. (2005b). Aurora A kinase-coated beads function as microtubule-
organizing centers and enhance RanGTP-induced spindle assembly. Curr. Biol. 15, 21562163.
Vernos, I., Raats, J., Hirano, T., Heasman, J., Karsenti, E., and Wylie, C. (1995). Xklp1, a
chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome
positioning. Cell 81, 117127.
White, J.G., and Borisy, G.G. (1983). On the mechanisms of cytokinesis in animal cells. J.
Theor. Biol. 101, 289316.
Wühr, M., Tan, E.S., Parker, S.K., Detrich, H.W., 3rd, and Mitchison, T.J. (2010). A model for
cleavage plane determination in early amphibian and fish embryos. Curr. Biol. 20, 20402045.
Wühr, M., Freeman, R.M., Presler, M., Horb, M.E., Peshkin, L., Gygi, S.P., and Kirschner, M.W.
(2014). Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference
database. Curr. Biol. 24, 14671475.
Yüce, O., Piekny, A., and Glotzer, M. (2005). An ECT2-centralspindlin complex regulates the
localization and function of RhoA. J. Cell Biol. 170, 571582.
Zhu, C., and Jiang, W. (2005). Cell cycle-dependent translocation of PRC1 on the spindle by
Kif4 is essential for midzone formation and cytokinesis. Proc. Natl. Acad. Sci. U.S.A. 102, 343
Figure 1. Kif4A localization at aster interaction zones in zygotes
Xenopus eggs were fixed between 1st mitosis and 1st cleavage (70-100 min post fertilization),
triple stained for Kif4A, AurkB (a subunit of the CPC) and tubulin, and imaged by laser scanning
confocal microscopy in a clearing solvent.
A: Anaphase-telophase. Kif4A is enriched at anti-parallel bundles between asters. At this stage
it is still present on mitotic chromosomes (Chr).
B: Shortly before the asters reach the cortex. Kif4A is enriched at the interaction zone between
asters, where it co-localizes with the CPC.
C: Similar stage to B, different embryo. Higher magnification of the interaction zone between
asters illustrating anti-parallel microtubule bundles with Kif4A and CPC enriched at the midline.
Figure 2. Localization of Prc1E and Kif4A in interphase egg extracts.
(a) Spinning disc confocal time-lapse sequence of asters nucleated from AurkA beads in
interphase extract. Probes: microtubules (Alexa647-tubulin), mCherry-Prc1E, and Kif4A-GFP
(Supplementary Video 1). (b) Widefield sequence using GFP-DasraA subunit to visualize the
CPC (Supplementary Video 2). (a’ and b’) Kymograph analysis along a 30 μm wide line (cyan
box in a and b). Note that the CPC is more focused in interaction zones than Prc1E or Kif4A.
Figure 3. Visualization of microtubule dynamics between asters.
(a)-(h) show analysis of EB1-GFP trajectories imaged by spinning disc confocal microscopy at
interaction zones between two asters. (a) EB1 comet trajectories coloured by mean direction.
(b) EB1 comet trajectories coloured by instantaneous velocity. (c) EB1 comet initiations
coloured by velocity for 5 sec after initiation. (d) EB1 comet termination coloured by velocity 5
sec before termination. (e) Spatial distributions EB1 comet trajectories classified by direction
(grey dots: fraction of EB1 comets moving from left to right, blue curve: sigmoidal fit to grey
dots, red curve: sigmoidal fit to fraction of EB1 comets moving in opposite direction). (f) mean
instantaneous growth rates (mean ± SEM). (g) EB1 comet density. (h) fractions of EB1 comets
that are growth initiation/terminations (mean ± SD), n = 12 neighbouring ROIs (see Methods for
data analysis).
(i) Tubulin-A647 image sequence of a field of asters growing and interacting. 20x widefield.
Interaction zones between asters were established by 24min in this example. Note that the
microtubule density between asters remained approximately constant for a further 40min,
showing that there is no increase in the density of anti-parallel bundles over time. Images were
linearly rescaled to 8-bits to correct for mild photobleaching over the long image sequence.
Figure 4. Immunodepletion of Prc1E and Kif4A
(a) Low-mag widefield tubulin images of asters depleted as shown, or treated with 100 μM ZM-
447439 (AurkB inhibitor). Prc1E and Kif4A depletion had no discernalbe effect on nucleation of
microtubule density. AurkB inhibition caused descrease of MT density over time.
(b-d) High-mag TIRF images showing microtubules (Alexa647-tubulin), mCherry-Prc1E, Kif4A-
GFP under the depletion/inhibition above. Zoom-ups of boxed areas are shown on the right.
Note Kif4A localization to bundles depended on Prc1E. Prc1E localization did not depend on
Kif4A, but bundles were more spread out in its absence. Both proteins were recruited to bundles
when AurkB was inhibited, but the bundles are much more spread out.
Figure 5. Prc1E and Kif4A are required for the block to interpenetration between asters
(a-f) Analyses of microtubule growth directions by EB1 tracking of spinning disc confocal
images sequences as per Fig. 2a. Depletions and add-backs as noted.
(g) The D60 parameter, a metric for the degree of interpenetration, was defined as the
difference between the interpolated distances where the red and blue curves crossed 60% (see
panel a). Plot of mean D60 values (± SD) measured for each treatment (n ≥ 3 interaction zones
each). The last two bars (control and AurkB inhibition with 100 μM ZM-447439) were re-plotted
from (Nguyen et al., 2014) to provide a comparison. Asterisks indicate significant of different
from control IgG depletion based on unpaired t-test analyses, *p<0.01, **p<0.001.
Figure 6. Prc1E and Kif4A enforce radial order in isolated asters
(a-e) Plus end growth trajectories within isolated asters coloured by mean direction. Imaging
and EB1 tracking methods similar to Fig. 2a. Asterisks indicates position of the nucleating
center, Immunodepletion/add back conditions as labelled.
(a’-e’) Radial order heat maps showing the local radial order quantified by the R parameter;
each square cell measures 5x5 μm2 (see Methods). Redder colours represent higher radial
order. White pixels contained too few comet tracks to measure radial order.
(f) Radial order parameter R as a function of distance from the aster centre for the five asters
shown above. Error bars are standard deviations (see Methods for analysis). Data are truncated
at <20m where microtubule growth components in the z axis complicate analysis.
Figure 7. Examples of anti-parallel overlaps in growing asters being eliminated.
Examples of anti-parallel microtubule overlap formation and elimination at the edge of growing
asters imaged by spinning disc confocal microscopy (Supplementary Videos 7 and 8). Proteins
visualized: (a) Alexa647-tubulin and Kif4A-GFP, Alexa647-tubulin, (b) Kif4A-GFP, and mCherry-
Prc1E Arrowheads indicate: likely plus ends (red), likely minus ends (cyan), microtubule growing
out radially from aster (empty), microtubule growing in opposite direction (full).
(a’,b’) Kymographs of examples above along the radially growing microtubule. Events indicated:
likely catastrophe (c), likely stabilization (st), likely rescue (r).
Figure 8. Model for function of Prc1E and Kif4A within and between asters
(Left box) General function of Prc1E and Kif4A in anti-parallel bundles.
(Middle box) Block to interpenetration at the interaction zone between asters
(Right box) Enforcement of radial order within single asters by pruning of anti-parallel overlaps
... During meiosis and mitosis, spindles in tmi mutants adopt an abnormal morphology, namely a reduced length during meiosis and a lack of the characteristic spindle symmetry during early embryonic mitosis (depicted for mitosis in Fig. 9, center). These results are consistent with recent studies using Xenopus extracts that show the maternal Prc1l orthologue (named Prc1E in this study), together with the kinesin Kif4A, required to regulate the organization of spindle asters and the extent of interdigitation in the early Xenopus embryo (Nguyen et al. 2018). ...
... Moreover, these sites of coordinated microtubule bundling appear enriched in Prc1l protein. Previous studies in Xenopus have also identified localization of the Xenopus Prc1l interacting partner Kif4A at the interdigitating tips of the FMA in this organism, although this study was not able to detect Prc1l labeling at the furrow in embryos, presumably due to an antibody exclusion artefact (Nguyen et al. 2018). Together with previous work (Nguyen et al. 2018), our studies (which was not certified by peer review) is the author/funder. ...
... Previous studies in Xenopus have also identified localization of the Xenopus Prc1l interacting partner Kif4A at the interdigitating tips of the FMA in this organism, although this study was not able to detect Prc1l labeling at the furrow in embryos, presumably due to an antibody exclusion artefact (Nguyen et al. 2018). Together with previous work (Nguyen et al. 2018), our studies (which was not certified by peer review) is the author/funder. All rights reserved. ...
We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1family of microtubule-associated proteins. Embryos from tmi/prc1l homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that tmi/prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal tmi/prc1l function is also essential for the reorganization of vegetal pole microtubules required for embryonic axis induction. While Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1 in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.
... Here, we investigated whether these three proteins are required for nuclear separation, lending support to an aster-aster interaction model (Baker et al., 1993) which has been reconstituted in Xenopus laevis egg extract (Nguyen et al., 2014(Nguyen et al., , 2018. We performed a combination of gene knockdown, micromanipulation, and perturbation by exogenous protein addition in embryo explants to enable previously unachieved time-lapse visualization of nuclear and cytoskeletal dynamics. ...
... (K) The truncated Feo protein significantly reduced nuclear separation, as measured by the area of quadrilaterals shown in J, as compared with the full-length protein construct (black). and dynamics of microtubules and prevent invasion of neighboring asters by antiparallel microtubule cross-linking (Nguyen et al., 2018). The same protein module is responsible for recruiting cytokinesis signaling complexes and formation of the cleavage furrow (Nguyen et al., 2014). ...
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The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin-driven cytoplasmic movement transporting nuclei or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule cross-linking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less-dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo-inhibited embryos, following micromanipulation-assisted repositioning. A dimerization-deficient Feo abolishes nuclear separation in embryo explants, while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A cross-linking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.
... PRC1 crosslinks MTs but leaves them free to slide, while KIF4A inhibits the growth of plus ends and compacts PRC1 toward plus ends. In frog egg extract, the PRC1-KIF4A module is required to prevent the MTs of one aster from invading their neighbors at the boundaries (Nguyen et al. 2018). This module also acts within the main body of the aster to prune out antiparallel MT interactions and thus enforce the radial polarity of the aster. ...
The purpose of this review is to explore self-organizing mechanisms that pattern microtubules (MTs) and spatially organize animal cell cytoplasm, inspired by recent experiments in frog egg extract. We start by reviewing conceptual distinctions between self-organizing and templating mechanisms for subcellular organization. We then discuss self-organizing mechanisms that generate radial MT arrays and cell centers in the absence of centrosomes. These include autocatalytic MT nucleation, transport of minus ends, and nucleation from organelles such as melanosomes and Golgi vesicles that are also dynein cargoes. We then discuss mechanisms that partition the cytoplasm in syncytia, in which multiple nuclei share a common cytoplasm, starting with cytokinesis, when all metazoan cells are transiently syncytial. The cytoplasm of frog eggs is partitioned prior to cytokinesis by two self-organizing modules, protein regulator of cytokinesis 1 (PRC1)-kinesin family member 4A (KIF4A) and chromosome passenger complex (CPC)-KIF20A. Similar modules may partition longer-lasting syncytia, such as early Drosophila embryos. We end by discussing shared mechanisms and principles for the MT-based self-organization of cellular units. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see for revised estimates.
... Regarding KIF4, it plays important roles in DNA repair and DNA replication maintaining genetic stability and is also essential for regulation of mitosis and meiosis [55][56][57]. Abnormalities in KIF4 are associated with a variety of diseases, including cancer, HIV infection, Alzheimer's disease [58]. Interestingly, KIF4 is abnormally expressed in various cancers, where KIF4 is often up-regulated but can also be down-regulated in certain cancers, suggesting distinctive regulatory mechanisms for different cancers. ...
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Background Though considerable efforts have been made to improve the treatment of epithelial ovarian cancer (EOC), the prognosis of patients has remained poor. Identifying differentially expressed genes (DEGs) involved in EOC progression and exploiting them as novel biomarkers or therapeutic targets is of great value. Methods Overlapping DEGs were screened out from three independent gene expression omnibus (GEO) datasets and were subjected to Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses. The protein-protein interactions (PPI) network of DEGs was constructed based on the STRING database. The expression of hub genes was validated in GEPIA and GEO. The relationship of hub genes expression with tumor stage and overall survival and progression-free survival of EOC patients was investigated using the cancer genome atlas data. Results A total of 306 DEGs were identified, including 265 up-regulated and 41 down-regulated. Through PPI network analysis, the top 20 genes were screened out, among which 4 hub genes, which were not researched in depth so far, were selected after literature retrieval, including CDC45, CDCA5, KIF4A, ESPL1. The four genes were up-regulated in EOC tissues compared with normal tissues, but their expression decreased gradually with the continuous progression of EOC. Survival curves illustrated that patients with a lower level of CDCA5 and ESPL1 had better overall survival and progression-free survival statistically. Conclusion Two hub genes, CDCA5 and ESPL1, identified as probably playing tumor-promotive roles, have great potential to be utilized as novel therapeutic targets for EOC treatment.
... Compartments are also separated from each other by boundary zones that are largely devoid of microtubules, ER, or mitochondria. These boundary zones are established by the overlap between neighboring astral microtubule structures, inhibiting plus-end microtubule growth and promoting microtubule plus-end capping (Nguyen et al., 2014(Nguyen et al., , 2018. Together, these findings reveal an important role of the microtubule cytoskeleton for cytoplasmic self-organization in Xenopus egg extracts by building cell-like compartments that are clearly separated from each other. ...
Cytoplasm is a gel-like crowded environment composed of various macromolecules, organelles, cytoskeletal networks, and cytosol. The structure of the cytoplasm is highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules are restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the crowded nature of the cytoplasm at the microscopic scale, large-scale reorganization of the cytoplasm is essential for important cellular functions, such as cell division and polarization. How such mesoscale reorganization of the cytoplasm is achieved, especially for large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, is only beginning to be understood. In this review, we will discuss recent advances in elucidating the molecular, cellular, and biophysical mechanisms by which the cytoskeleton drives cytoplasmic reorganization across different scales, structures, and species.
The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.
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Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.
Protein regulator of cytokinesis 1 (PRC1) is a microtubule bundling protein that is involved in the regulation of the central spindle bundle and spindle orientation during mitosis. However, the functions of PRC1 during meiosis have rarely been studied. In this study, we explored the roles of PRC1 during meiosis using an oocyte model. Our results found that PRC1 was expressed at all stages of mouse oocyte meiosis, and PRC1 accumulated in the midzone/midbody during anaphase/telophase I. Moreover, depleting PRC1 caused defects in polar body extrusion during mouse oocyte maturation. Further analysis found that PRC1 knockdown did not affect meiotic spindle formation or chromosome segregation; however, deleting PRC1 prevented formation of the midzone and midbody at the anaphase/telophase stage of meiosis I, which caused cytokinesis defects and further induced the formation of two spindles in the oocytes. PRC1 knockdown increased the level of tubulin acetylation, indicating that microtubule stability was affected. Furthermore, KIF4A and PRC1 showed similar localization in the midzone/midbody of oocytes at anaphase/telophase I, while the depletion of KIF4A affected the expression and localization of PRC1. The PRC1 mRNA injection rescued the defects caused by PRC1 knockdown in oocytes. In summary, our results suggest that PRC1 is critical for midzone/midbody formation and cytokinesis under regulation of KIF4A in mouse oocytes.
Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss‐of‐function screenings with analysis of MT‐dynamics in human cells to investigate the molecular mechanisms underlying MT‐flux. We report that kinesin‐7/CENP‐E at kinetochores (KTs) is the predominant driver of MT‐flux in early prometaphase, while kinesin‐4/KIF4A on chromosome arms facilitates MT‐flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin‐5/EG5 and kinesin‐12/KIF15, and our data suggest that the MT‐flux driving force is transmitted from non‐KT‐MTs to KT‐MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT‐flux rate correlates with spindle length, and this correlation depends on the establishment of stable end‐on KT‐MT attachments. Strikingly, we find that MT‐flux is required to regulate spindle length by counteracting kinesin 13/MCAK‐dependent MT‐depolymerization. Thus, our study unveils the long‐sought mechanism of MT‐flux in human cells as relying on the coordinated action of four kinesins to compensate for MT‐depolymerization and regulate spindle length.
In contrast to the homeokinesis of mitosis, asymmetric division of cytoplasm is the conspicuous feature of meiosis in mammalian oocytes. Protein regulator of cytokinesis 1 (PRC1) is an important regulator during mitotic spindle assembly and cytoplasmic division, but its functions in oocyte meiosis and early embryo development have not been fully elucidated. In this study, we detected PRC1 expression and localization and revealed a nuclear, spindle midzone‐related dynamic pattern throughout meiotic and mitotic progressions. Treatment of oocytes with the reagents taxol or nocodazole disturbed the distribution of PRC1 in metaphase II oocytes. Further, PRC1 depletion led to failure of first polar body (PB1) extrusion and spindle migration, aneuploidy, and defective kinetochore‐microtubules (MT) attachment and spindle assembly. Overexpression of PRC1 resulted in PB1 extrusion failure, aneuploidy, and serious defects of spindle assembly. To investigate PRC1 function in early embryos, we injected Prc1 morpholino into zygotes and 2‐cell stage embryos. Depletion of PRC1 in zygotes impaired 4‐cell, morula, and blastocyst formation. Loss of PRC1 in single or double blastomeres in 2‐cell stage embryos significantly impaired cell division, indicating its indispensable role in early embryo development. Co‐immunoprecipitation showed that PRC1 interacts with polo‐like kinase 1 (PLK1), and functional knockdown and rescue experiments demonstrated that PRC1 recruits PLK1 to the spindle midzone to regulate cytoplasmic division during meiosis. Finally, Kif4 knockdown down‐regulates PRC1 expression and leads to PRC1 localization failure. Taken together, our data suggest PRC1 plays an important role during oocyte maturation and early embryonic development by regulating chromosome dynamics and cytoplasmic division.
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Microtubule asters-radial arrays of microtubules organized by centrosomes-play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks.
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A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ~10,000 proteins and ~28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.
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Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment where aster pairs from the same spindle (sister asters) have chromatin between them, while asters pairs from different spindles (non-sisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, Chromosome Passenger Complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubules bundles between asters from the same spindle. Next, a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane CPC-positive microtubule bundles out to the cortex to position the furrow. © 2015 by The American Society for Cell Biology.
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Cell division requires the precise coordination of chromosome segregation and cytokinesis. This coordination is achieved by the recruitment of an actomyosin regulator, Ect2, to overlapping microtubules at the centre of the elongating anaphase spindle. Ect2 then signals to the overlying cortex to promote the assembly and constriction of an actomyosin ring between segregating chromosomes. Here, by studying division in proliferating Drosophila and human cells, we demonstrate the existence of a second, parallel signalling pathway, which triggers the relaxation of the polar cell cortex at mid anaphase. This is independent of furrow formation, centrosomes and microtubules and, instead, depends on PP1 phosphatase and its regulatory subunit Sds22 (refs 2, 3). As separating chromosomes move towards the polar cortex at mid anaphase, kinetochore-localized PP1-Sds22 helps to break cortical symmetry by inducing the dephosphorylation and inactivation of ezrin/radixin/moesin proteins at cell poles. This promotes local softening of the cortex, facilitating anaphase elongation and orderly cell division. In summary, this identifies a conserved kinetochore-based phosphatase signal and substrate, which function together to link anaphase chromosome movements to cortical polarization, thereby coupling chromosome segregation to cell division.
We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ∼11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues.
The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main--and not necessarily mutually exclusive--candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper-like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus.
Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.