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Actin-driven chromosomal motility leads to symmetry
breaking in mammalian meiotic oocytes
Hongbin Li1, Fengli Guo1, Boris Rubinstein1 and Rong Li1,2
Movement of meiosis I (MI) chromosomes from the oocyte centre
to a subcortical location is the first step in the establishment of
cortical polarity. This is required for two consecutive rounds of
asymmetric meiotic cell divisions, which generate a mature egg
and two polar bodies1. Here we use live-cell imaging and genetic
and pharmacological manipulations to determine the forcegenerating mechanism underlying this chromosome movement.
Chromosomes were observed to move toward the cortex in a
pulsatile manner along a meandering path. This movement is
not propelled by myosin-II-driven cortical flow but is associated
with a cloud of dynamic actin filaments trailing behind the
chromosomes/spindle. Formation of these filaments depends on
the actin nucleation activity of Fmn2, a formin-family protein
that concentrates around chromosomes through its aminoterminal region. Symmetry breaking of the actin cloud relative
to chromosomes, and net chromosome translocation toward the
cortex require actin turnover.
Oocyte meiotic divisions are highly asymmetric, and symmetry breaking
initiates with chromosome movement from the oocyte centre towards
the cortex in many vertebrate and invertebrate organisms1,2. The subcortically located chromosomes then elicit a signal to induce assembly of
an actomyosin-rich cortical domain for polar-body extrusion, as shown
in mouse oocytes3. Chromosomal translocation to the cortex is microtubule-dependent in Caenorhabditis elegans, Drosophila melanogaster
and sea cucumber oocytes2,4,5, but in mouse oocytes, is known to rely
on F-actin6,7. The actin cytoskeleton drives cellular and intracellular
motility through three distinct mechanisms. First, actin filaments or
bundles serve as oriented tracks for transport motors, such as myosin
V8. This form of actin-based motility is used to traffic cellular organelles
and membrane vesicles. Second, contractile networks consisting of actin
and type II myosin can drive cortical flow and transport of signalling
complexes9 and structures such as centrosomes10. A contractile actin
network is also thought to drive the first phase of chromosome congression in starfish oocytes11. Third, actin can generate propulsive force
directly through polymerization at filament barbed ends. This type of
mechanism is crucial for leading-edge movement in motile cells, as well
1
2
as intracellular movement of certain vesicles and pathogens, such as
Listeria monocytogenes and vaccinia virus12.
We first used four-dimensional (4D) tracking to obtain quantitative parameters characterizing this process. DNA was stained with
Hoechst bisbenzamide in live MI oocytes at a low concentration that
had no toxic effects (see Methods). 4D imaging was performed using a
two-photon microscope, and the movement of chromosomes towards
the cortex after formation of the metaphase plate was tracked at 5-min
intervals using the Imaris software (Fig. 1a; Supplementary Information,
Movies 1, 2). Along the initial position-destination (ID) axis (Fig. 1b),
the displacement of meiotic chromosomes increased with time (Fig. 1c),
with an average total displacement of 17.0 ± 1.8 µm (n = 16) and an average velocity of 0.05 ± 0.01 µm min–1 (velocity = displacement/time).
However, the average length of the path covered by the movement was
much larger, 46.4 ± 4.7 µm, with an average speed of 0.13 ± 0.01 µm
min–1 (speed = path length/time). The straightness of the path, defined
as displacement/path length, for chromosomes in each oocyte is shown
in Fig. 1d and averaged 0.37 ± 0.05 (Table 1). The efficiency of movement
during each time-interval was further analysed using a correlation analysis to generate a Pearson’s coefficient (see Methods) between speed and
displacement (Fig. 1d). This coefficient should be close to 1 for movement
with maximum straightness; however, the average Pearson’s coefficient
was 0.50 ± 0.13, suggesting that the movement was inefficient. We plotted
the angle of direction change between two consecutive time-points (θ)
over time (Fig. 1b). There were large changes in θ along the movement
path for all oocytes (Fig. 1e), suggesting that the movement was unlikely
to be guided by a straight pre-established actin track.
We next examined the pattern of speed during chromosome movement
to the cortex. The average speed was highly consistent (Fig. 1f; Table 1);
however, chromosome speed in individual oocytes oscillated between about
0.06 µm min–1 and 0.25 µm min–1 in the course of the movement (Fig. 1g).
The pulsatile motion can be more easily seen in the example in Fig. 1h,
which shows an average periodicity (time between two adjacent peaks)
of 14.6 min. The average periodicity in speed for all oocytes observed was
15.0 ± 0.7 min (n = 16). This periodic pattern of movement was previously
observed for the actin polymerization-driven motility of certain Listeria
mutants and large beads coated with an Arp2/3 complex activator13,14.
Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA.
Correspondence should be addressed to R.L. (e-mail: rli@stowers-institute.org)
Received 23 May 2008; accepted 21 August 2008; published online 5 October 2008; DOI: 10.1038/ncb1788
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a
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Destination
Displacement
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Figure 1 Quantitative characterization of chromosome movement by 4D
tracking. (a) An example of tracking chromosome movement in 3D space over
time using the Imaris software. The green ball represents the centre of mass
of the chromosomes at the initial position, and the trajectory of the movement
is shown. The scale bar represents 2 µm. (b) A schematic diagram explaining
parameters characterizing chromosome movement. Orange balls represent
consecutive positions of chromosomes. I, initial point; D, destination point;
displacement is the distance moved along the ID axis; θ1, θ2 are angle
changes in movement direction at each position. (c) Displacement along the
ID axis from five representative oocytes were plotted over time. (d) Pearson’s
coefficient and straightness distribution in eight representative oocytes.
(e) Direction changes as defined in b from five representative oocytes were
plotted over time. (f) Average speeds of chromosome movement were similar
for the oocytes observed. Different coloured boxes represent different oocytes.
Box range, 25–75%; small box, mean; whisker range, 5–95% (standard
data representation by OriginPro). (g) Speed of chromosome movement for
individual oocytes was plotted over time. (h) Speed over time in one of the
oocytes from g (blue) is shown at a higher time resolution after subtracting
the basal speed. Peaks (fast movement) are separated by pauses. The average
time between two pulses was 14.6 min in the example shown.
Pharmacological inhibitors were next used to examine how actin is
involved in meiotic chromosome movement. We first confirmed that
treatment of oocytes with the actin polymerization inhibitor latrunculin
A (Lat A) (10 µM) completely abolished chromosome migration, producing an average displacement of 2.0 ± 1.2 µm (Fig. 2a; Table 1) and an
average speed of 0.04 ± 0.03 µm min–1 (n = 15), compared with 0.13 ± 0.01
µm min–1 for untreated oocytes (Fig. 2b; Table 1). Treatment of oocytes
with nocodazole to depolymerize microtubules resulted in even faster
chromosome movement, compared with untreated oocytes (Table 1;
Supplementary Information, Fig. S1a, b).
As myosin II-dependent cortical flow was previously shown to
be required for positioning centrosomes in mammalian cells10, and
for establishing and maintaining anterior–posterior polarity in
C. elegans zygotes9, we tested whether myosin II was required for
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Table 1 Dynamic parameters of meiotic chromosome movement
Untreated
Moved to cortex
Displacement (µm)
Velocity (µm min–1)
Speed (µm min–1)
Straightness
70%
17.0 ± 1.8
0.05 ± 0.01
0.13 ± 0.01
0.37 ± 0.05
Lat A
0
2.0 ± 1.2
0.004 ± 0.001
0.04 ± 0.03
0.06 ± 0.01
Jas
0
3.0 ± 0.4
0.005 ± 0.002
0.09 ± 0.03
0.05 ± 0.02
Fmn2–/–
0
4.0 ± 1.3
0.004 ± 0.003
0.06 ± 0.001
0.16 ± 0.038
WGA
69%
19.5 ± 3.2
0.04 ± 0.01
0.14 ± 0.02
0.43 ± 0.12
Nocodazole
53.5%
22.0 ± 3.6
0.05 ± 0.004
0.23 ± 0.03
0.35 ± 0.14
F-Lifeact-injected
69%
17.2 ± 1.1
0.049 ± 0.05
0.12 ± 0.03
0.40 ± 0.02
For each treatment or genetic background, 80–120 (for second column) and 12–120 (for third to sixth columns) oocytes were examined (data shown are mean ± s.e.m.). Displacement is the
distance between initial and destination points; velocity is displacement/time; speed is path length/time; straightness is displacement/path length.
meiotic chromosome movement by treating oocytes with ML-7, a
myosin II light chain kinase inhibitor, or blebbistatin, an inhibitor
of myosin-II ATPase. As these inhibitors can be absorbed by mineral
oil, the treatments were performed in medium without oil, which
did not permit time-lapse imaging of the movement. Neither ML-7
(20 µM) nor blebbistatin (50–75 µM) blocked chromosome movement to the cortex (Fig. 2c). At this concentration range, blebbistatin
completely inhibited polar-body extrusion, suggesting that myosin
II motor activity was dispensable for chromosome movement to the
cortex. Wheat germ agglutinin (WGA), a lectin that binds to cell surface glycoproteins and thus blocks cortical flow10, also had no effect,
as shown with 4D tracking, even though WGA completely blocked
polar body extrusion (Fig. 2c; Table 1). This further suggests a lack
of involvement of cortical flow in chromosome movement.
To test whether dynamic actin is required for chromosome migration, oocytes were treated with the actin depolymerization inhibitor
jasplakinolide (Jas). The effect of Jas treatment was similar to that
of Lat A on chromosome migration to the cortex, with an average
displacement of 3.0 ± 0.4 µm (Table 1). No cell division or polar-body
extrusion was observed (data not shown). However, in Jas-treated
oocytes, meiotic chromosomes moved at a higher average speed
(0.09 ± 0.03 µm min–1) than in Lat A-treated cells (Fig. 2b; Table 1).
Comparison of the trajectories of chromosome movement in control
and Lat A-treated oocytes showed significant chromosome movement
in Jas-treated oocytes, but the net displacement was minimal due to
constant changes in direction (Fig. 2d; Supplementary Information,
Movie 3). This result suggests that actin turnover may be important for
a decisive symmetry-breaking event, which is required for persistent
movement towards the cortex.
It is well documented that actin filaments are present on the cortex of
mouse oocytes after germinal vesicle breakdown. After chromosomes
have moved to the cortex, actin forms a cortical cap6,7; however, previous studies with phalloidin-labelling were unable to show F-actin
in the vicinity of chromosomes in the cytosol7,15,16 (Supplementary
Information, Fig. S1a). To immunostain mouse oocytes for actin, we
used anti-β-actin as the primary antibody and FluoNanogold-antimouse Fab´-Alexa Fluor 488 as the secondary antibody. In addition to
cortical actin, cytosolic patches of actin were observed 6 h after release
from the interphase arrest, some apparently in the vicinity of the chromosomes (Fig. 3a; Supplementary Information, Movie 4). This staining was further confirmed using pre-embed labelling immuno-electron
microscopy after gold enhancement (Fig. 3b). However, the paucity of
actin structures observed suggests that cytosolic actin in oocytes may be
too dynamic to preserve under formaldehyde fixation.
To observe F-actin during chromosome movement in live oocytes, we
used the recently developed Lifeact peptide, which binds specifically to
F-actin but does not alter actin dynamics17. Oocytes injected with FITClabelled Lifeact (F-Lifeact) showed chromosome movement with normal
speed and displacement (Table 1). In the FITC channel, a cloud of F-actin
occupying a circular area, with a radius of about 12–18 µm, was observed
around the meiotic chromosomes after germinal vesicle breakdown
(Fig. 3c, d). Injection of control FITC–dextran 4 did not show this pattern of localization (Supplementary Information, Fig. S1c). Furthermore,
this actin cloud was not present in Lat A-treated oocytes (Fig. 3f). Twocolour 4D movies showed that the actin cloud initially distributed symmetrically around the chromosome/spindle region, but as chromosomes
initiated movement towards the cell periphery, the actin distribution
became asymmetric, with actin concentrated towards the back of the
chromosomes/spindle (Fig. 3c, d; Supplementary Information, Movie 5).
When viewed along a plane parallel to the trajectory of the movement,
the space between the chromosome mass and actin cloud was expected
to be occupied by the spindle structure (Supplementary Information,
Fig. S1a). The symmetry-breaking event described above can be represented quantitatively in a plot of the distance between the centre of the
chromosomal mass and the centre of the actin cloud mass over time
(Fig. 3h, red line). Initially the two centres of mass were close to each
other, but a sudden increase in the distance between them correlated
with a large chromosome displacement towards the cortex (Fig. 3).
Actin continued to concentrate behind the chromosomes/spindle as
they moved towards the cortex (Fig. 3c, d; Supplementary Information,
Movie 5). In Jas-treated cells, a much denser actin cloud maintained
a roughly symmetric distribution around the chromosomes (Fig. 3e;
Supplementary Information, Movie 6).
We used thin-section electron microscopy to observe cytoskeletal structures in the vicinity of the chromosomes in better detail.
Filaments of 7–10 nm in width were frequently observed in striated
bundles of 0.2–1.5 µm in length and 0.08–0.15 µm in width in the vicinity of meiotic chromosomes (Supplementary Information, Fig. S2a, b).
On-section labelling immuno-electron microscopy using an anti-actin
antibody showed that these structures contained actin (Supplementary
Information, Fig. S2h). Consistent with these being actin-associated
structures, the filament bundles were sensitive to Lat A treatment
(Supplementary Information, Fig. S2e, g). Similar structures were also
reported in a previous study, which showed the presence of both actin
and keratin in the observed bundles18.
Fmn2 (a formin-family protein) was previously shown to be required
for the movement of meiotic chromosomes to the cortex and polarbody extrusion in mouse oocytes15,19. As many members of the formin
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P = 0.057
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Speed (µm min–1)
Displacement (µm)
b
Untreated
Fmn2–/–
Lat A
Jas
0.15
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n = 13
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No drug
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d
Untreated
Lat A
Jas
Fmn2–/–
Figure 2 Testing the requirement for actin polymerization, depolymerization
and myosin-II in meiotic chromosome movement. (a) Displacements along
the ID axis were plotted over time in oocytes treated with Lat A (n = 15),
Jas (n = 13), or in Fmn2–/– (n = 14) oocytes. Shown is the displacement of
all the oocytes observed at each time point (mean ± s.e.m). (b) Comparison
of overall average speeds between different conditions described in a.
(c) Myosin II and cortical flow are dispensable for chromosome movement
to the cortex. The histograms shown are percentages of the observed
oocytes that performed polar body extrusion or chromosome movement to
the cortex as visualized by Hoechst staining. The total numbers of oocytes
observed were 78, 89, 158, 83 for untreated, ML-7-treated, blebbistatintreated and WGA-treated, respectively. A representative Hoechst-stained
oocyte image is shown above each histogram, except for WGA treatment
where a movement trajectory is shown (data are mean ± s.d.) (d) A
representative trajectory of chromosome movement is shown for each
condition as indicated. The scale bars represent 2 µm.
family can nucleate actin assembly through their conserved FH1 and
FH2 domains20, Fmn2 was an obvious candidate for the nucleating
protein for assembly of the actin around the chromosomes. Indeed,
in oocytes from Fmn2–/– mice, neither the actin cloud nor the filament bundles surrounding the chromosomes was observed (Fig. 3g;
Supplementary Information, Fig. S2f, g). 4D-tracking experiments
further confirmed that Fmn2–/– oocytes were deficient in chromosome
movement (Fig. 2a, b, d; Table 1). To test whether Fmn2 can indeed
nucleate actin filaments, a recombinant Fmn2 fragment containing the
FH1 and FH2 domains (FH1FH2) was produced and tested using the
pyrene–actin assembly assay. Actin polymerization was stimulated in a
concentration-dependent manner in the presence of FH1FH2 (Fig. 4a);
mutation of residues Ile 1215, Arg 1295 and Lys 1371 in the FH2 domain
(conserved residues directly contacting actin)21,22 to Ala (FH1FH2IRK)
abolished this activity (Fig. 4a). To test whether the actin nucleation
activity of Fmn2 is required for chromosome movement towards the
cortex, mRNA encoding full-length Fmn2, the FH1FH2 fragment or
the full-length Fmn2IRK mutant was injected into Fmn2–/– oocytes. The
full-length wild-type mRNA rescued chromosome movement to the
cortex in 55% of the Fmn2–/– oocytes (n = 67), and even the FH1FH2
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300
Figure 3 Visualization of actin in the vicinity of meiotic chromosomes.
(a) Immunofluorescent staining of actin (green) and DNA with DAPI. (b)
Visualization of actin by pre-embed labellling immuno-electron microscopy.
The right panel shows cortical actin (arrow) and the left panel shows actin
(arrow) in the chromosomal region. The cortex and a chromosome are outlined
with white lines. (c) Time-lapse imaging of F-actin by F-Lifeact peptide (green)
and chromosomes (blue). The range line is a stationary position near the
equator of the oocyte. (d) 3D reconstruction of panels in c. (e–g) Visualization
of F-actin with F-Lifeact peptide (green) and chromosomes (blue) in Jastreated (e), Lat A-treated (f) and Fmn2–/– (g) oocytes. (h) Symmetry breaking
of the actin cloud temporally correlates with chromosome displacement
towards the cortex. Displacement of the centre of mass of the chromosomes
(blue) along the ID axis, and the distance between the centres of mass of
chromosomes and actin cloud (red) were plotted over time. The red arrow
points to a major step in symmetry breaking and chromosome movement. The
scale bars represent 5 µm (a), 1 µm (b) and 20 µm (c–g).
fragment rescued 30% (n = 82) of the injected oocytes; however, the
full-length Fmn2IRK mutant did not show any rescuing activity (n = 108;
Fig. 4b). These results suggest that the actin nucleation activity of Fmn2
is required for chromosome movement to the cortex.
As injection of mRNA encoding GFP–Fmn2 rescued chromosome
movement in Fmn2–/– oocytes (Fig. 4b), we used this construct to observe
Fmn2 localization. Before germinal vesicle breakdown, GFP–Fmn2 was
diffuse throughout the oocyte (Fig. 4c). After germinal vesicle breakdown, GFP–Fmn2 localized, similarly to F-actin, around the meiotic
chromosomes in a circular area with a radius of 14–17 µm (Fig. 4d).
On-section labelling immuno-electron microscopy using an anti-GFP
antibody showed the presence of gold particles on or near the chromosomes (Supplementary Information, Fig. S3). In oocytes where chromosomes were on their way to the cortex, GFP–Fmn2 concentrated behind
the chromosomes/spindle (Fig. 4e); however, time-lapse imaging was
unsuccessful as the fluorescence signal was low. To identify the region of
Fmn2 required for the observed localization, mRNA encoding the GFPtagged N-terminal 1–734 amino acids was injected. This N-terminal
fragment was sufficient for localization to the chromosome vicinity and
the location was insensitive to Lat A treatment (Fig. 4f, g). In contrast,
the remaining C-terminal half (amino acids 735–1578, containing FH1
and FH2) showed only a diffuse distribution in the cytosol (Fig. 4h).
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a
150
70
100
50
0
0
2
4
P < 10-4
60
Percent moved
Fluorescence intensity (a.u)
b
BSA, 1.6 µM
FH1FH2, 1.6 µM
FH1FH2, 0.4 µM
FH1FH2IRK, 1.6 µM
FH1FH2IRK, 0.4 µM
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Time (min)
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P < 2 x 10-4
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FH1FH2
Fmn2
Fmn2IRK Fmn2–GFP
e
h
Figure 4 Chromosome movement requires the actin nucleation activity of
Fmn2 and localization of Fmn2 to the chromosome vicinity. (a) Pyrene actin
assembly assay showing FH1FH2 fragment but not mutant FH1FH2IRS was
able to stimulate actin polymerization. (b) The ability of injected mRNA
encoding various forms of Fmn2, as indicated along the horizontal bar, to
rescue chromosome movement in Fmn2–/– oocytes. The histograms shown
are percentages of Fmn2–/– oocytes, in which the chromosomes moved to
the cortex, over the total oocytes injected with each of the mRNAs. The
numbers of injected oocyte were 58 (control); 82 (FH1FH2), 67 (full-length
Fmn2), 108 (Fmn2IRK) and 77 GFP–Fmn2. (c–e) Fmn2–GFP showed diffuse
distribution in oocytes before GVBD (c) and localized around chromosomes
in MI (d, e). (f, g) GFP-tagged N-terminal 734 amino acids of Fmn2 were
sufficient for localization to the chromosome vicinity in the absence (f)
or presence of Lat A (g). (h) GFP-tagged C-terminal half of Fmn2, which
contains FH1 and FH2 domains, showed a diffuse distribution in the cytosol.
The scale bars represent 10 µm (c–h).
Taken together, the results described above do not favour the model
that chromosomes are moved as cargo along a pre-existing actin track
or through an actin/myosin-II-based contractile network. Instead,
our observations are consistent with a model in which chromosome
movement to the cortex is driven by actin polymerization. An interesting property of chromosome movement is the periodicity in speed.
A similar pattern of movement was previously observed for the actin
polymerization-driven movement of Listeria or VCA-coated beads13,14.
An elastic analysis of these motility systems suggests that such a movement pattern may be explained by intrinsic physical parameters, such
as the diameter of the beads14. Another important property of actin
polymerization-driven motility observed with beads is the capacity for
spontaneous symmetry breaking from a cloud of actin that initially
forms symmetrically around the beads14,23,24. A stochastic model based
on Brownian ratchet25 predicted that a moderate filament depolymerization rate would be critical for the conversion of random motions to
directional motions by a polymerizing actin network. This prediction is
consistent with our observation that Jas prevented symmetry breaking
of the actin cloud and movement of meiotic chromosomes away from
the oocyte centre.
Examples of actin polymerization-driven motility that have been well
studied involve actin nucleation by the Arp2/3 complex, which leads to
assembly of a dendritic network composed of branched actin filaments20.
However, in recent studies, filament bundles containing actin and keratin were observed in the vicinity of meiotic chromosomes. The formation
of these bundles around chromosomes depends on both Fmn2 and actin
polymerization, as indicated by latA sensitivity. Thus, one interesting
question for future research is how Fmn2 might mediate co-assembly of
actin and intermediate filaments to exert force on meiotic chromosomes.
In addition, it remains to be determined whether actin directly exerts
force on meiotic chromosomes. The observed cloud of F-actin or Fmn2
did not appear to be always in tight association with the chromosomes,
and it is therefore possible that an as-yet unidentified structure serves as
a scaffold for the actin-based force. In conclusion, meiotic chromosomes
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are another natural cargo for an actin polymerization-driven motility
system. More remarkably, symmetry breaking in this system to achieve
an overall displacement is also the origin of symmetry breaking at the
cellular level, which enables oocytes to establish cortical polarity and
undergo asymmetric meiotic cell divisions.
METHODS
Oocyte dissection, preparation and culture. All experiments involving higher
vertebrates were approved by the Institutional Animal Care and Use Committee
of the Stowers Institute for Medical Research. Ovaries were collected from 4–7week-old wild-type FVB, Fmn2+/+ and Fmn2–/– female mice 44–48 h after injecting
with pregnant mare serum gonadotropin (5 IU; Sigma). Mice were euthanized
by CO2 asphyxiation followed by cervical dislocation. Oocytes were released by
puncturing the ovaries with needles in M2 medium, which contained 0.2 mM
3-isobutyl-1-methylxanthine (IBMX, Sigma) to maintain prophase arrest.
Time-lapse imaging. Oocytes from FVB mice were cultured in a chamber with
appropriate humidity and 5% CO2 at 37 °C. To visualize DNA, oocytes were
cultured in M16 medium (Specialty Medium) containing Hoechst bisbenzimide
33258 (0.5 µg ml–1; Sigma). We examined the toxicity of Hoechst (1–200 µg ml–1)
on mouse oocyte maturation. Oocytes stained with 200 µg ml–1 Hoechst showed
a normal rate of polar-body extrusion (76%, n = 60), which is comparable to
unstained oocytes (70%, n = 70). The time required for maturation (9.1 ± 0.2 h)
was also similar to that in unstained oocytes (8.5 ± 0.3 h). In time-lapse imaging, Hoechst was added after GVBD but before chromosome movement to
further reduce possible toxicity. In drug-treatment experiments, the medium
was supplemented with 200 µg ml–1 wheat germ agglutinin (WGA, Sigma),
20 µM ML-7 (Sigma), 10 µM Lat A (Molecular probes), or 1 µM or 10 µM Jas
(Molecular probes), 10 µM nocodazole (Sigma) and 75–100 µM blebbistatin (+/–)
(Biomol). Imaging was performed with a LSM 510 META (Carl Zeiss) with a
Plan Apochromat ×20/0.75 objective. Hoechst was excited at 775 nm using a
2-photon coherent chameleon laser at low power (4%) and emission was collected
at 435–485 nm. To visualize F actin, 10 pl of 1 mg ml–1 FITC–Lifeact peptide17
was injected into oocytes and was excited at 488 nm with 10% argon laser power.
Emission was collected at 500–550 nm with Plan 40 × 1.2 NA C-Apochromat. All
4D images were tracked and anaysed using Imaris (Bitplane).
Statistical analysis. Statistical analysis, including determination of Pearson’s coefficient, was performed using Excel and OriginPro 7.5 software. To determine P
values, a heteroscedatic two-tailed test was used.
Immunofluorescent staining and electron microscopy. After removal of the
zona pellucida using Tyrode’s acidic solution (Sigma), oocytes were fixed for
30 min in 3.8% formaldehyde (Alfa Aesar) in PBS and blocked with 3% BSA, 1%
normal goat serum, 0.1 M glycine and 0.01% Triton X-100 for 1 h at 37 °C. Fixed
oocytes were permeabilized with 4% BSA and 0.1% Triton X-100 for 10 min and
incubated with anti-β-actin (A5441, Sigma, diluted 1:1000 in blocking buffer)
antibodies for 2 h at room temperature. DNA was stained with DAPI mounting medium (Vector Laboratories). After washing six times in blocking buffer,
the sample was incubated with secondary FluoNanogold anti-mouse Fab´-Alexa
Fluor 488 (Nanoprobes; 1:100 diluted in blocking buffer) for 2–3 h.
For pre-embed labelling immuno-electron microscopy26,27, oocytes were fixed
in 4% paraformeldehyde and 0.01% glutraradehyde, and washed with blocking
solution (PBS containing 1% normal goat serum, 50 mM glycine, 1 mg ml–1 BSA,
0.02% sodium azide, and 0.01% saponin) for 2 × 30 min. The sample was incubated with anti-β-actin (A5441, Sigma, diluted 1:1000 in blocking buffer) for
24–48 h at 4 °C and rinsed four times in blocking buffer (15 min each time) followed by incubation with secondary FluoNanogold anti-mouse Fab´-Alexa Fluor
488 (Nanoprobes; 1:100 diluted in blocking buffer) for 2–3 h. After washing with
blocking buffer, the sample was post-fixed in 1% glutaraldehyde/PBS (pH 7.4)
overnight at 4 °C, washed, enhanced with goldenhance-EM (Nanoprobes) and
visualized using a Tecnai TEM (FEI).
For on-section labelling immuno-electron microscopy, oocytes were fixed in 4%
paraformaldehyde and 0.1% glutaraldehyde in PBS (pH 7.4) for 4–6 h, and washed
3 times in 0.1 M PBS for 15 minutes each. The sample was dehydrated by washing
three times in a graded series of alcohol (in PBS) for 15 min each: 30% ethanol, 50%
ethanol, 70% ethanol, 90% ethanol and 100% ethanol, and then infiltrated with LR
White resin in two 1 h changes and embedded in LR White resin. The section was
stained with mouse anti-β-actin (Sigma) or anti-GFP (Rockland), and secondary
10 nm gold-conjugated goat anti-mouse antibodies (Sigma; 1:20 dilution).
For ultrastructural visualization of actin28, oocytes were washed three times
(2 min each) in warm HPSS (+NaHCO3). Samples were fixed with 2% glutaradehyde in 0.1 M HEPES (pH 7.3), 0.05% saponin and 0.2% tannic acid (freshly filtered,
0.2 µm filter pore) for 40 min. Fixative was removed by washes in 0.1 M HEPES.
Samples were post-fixed with 0.1% OsO4 (aqueous) for 10 min, washed in twice
in distilled water (10 min each). Oocytes were dehydrated through a graded series
of ethanol to 100%, infiltrated with resin (epon 812): ethanol (1:3 and 1:1, each for
1 h), then stored in 3:1 resin:ethanol overnight at 4 °C. The resin was polymerized
at 37 °C and 60 °C each for 24 h; ultrathin sections (approximately 50–70 nm) were
cut on a Leica ultramicrotome using diamond knives. Sections were stained with
2% uranyl acetate and lead citrate for 10 min and 5 min, respectively.
Pyrene actin assembly assay and protein purification. Wild-type and mutant
(FH1FH2IRK) FH1FH2 fragments were inserted into pET28C vector (Novagen)
and expressed in BL21 bacteria. The His6-labelled proteins were purified by
Ni-NTA agarose (Qiagen) according to the manufacturer’s protocol and then
dialysed into G-actin buffer containing 5 mM Tris (pH 7.5), 0.2 mM DTT and
0.2 mM ATP. Pyrene actin assembly assay using 5 µM G-actin containing 10%
pyrene-labelled actin was performed as described previously29.
mRNA injection. Full-length Fmn2 and mutant Fmn2IRS were inserted into pCS2+
(ref. 19). Genes encoding the FH1FH2 fragment, N-terminal (amino acids 1–734)
or C-terminal (amino acids 735–1578) region of Fmn2–GFP and Fmn2–GFP were
inserted into pBluescript RN3 (ref. 30). Capped mRNA was synthesized from a
linearized template using mMessage mMachine (Ambion), purified with an mEGAclear kit (Ambion) and dissolved in RNase-free water (Ambion). 1–5% oocyte
volume was injected at a constant flow rate with a microinjector (Eppendorf). The
oocytes were maintained in IBMX for 3 h before release for maturation.
Note: Supplementary Information is available on the Nature Cell Biology website.
Acknowledgements
We thank Roland Wedlich-Soldner (Max Plank Institute, Munich) for providing
F-Lifeact peptide; Philip Leder (Harvard Medical School, Boston)) for Fmn2–/– mice
and full-length Fmn2 cDNA; Marie-Helene Verlhac (Universite Paris VI, Paris) for
pBluescript RN3 vector; Eric Jessen (Stowers Institute, Kansa City, applying to all
persons mentioned hereafter) for help in site-directed mutagenesis; Rhonda Allen
for electron microscopy; Xiaoxue Fan for help with actin preparation; Praveen
Suraneni and Manqi Deng for help with the mouse work; Heather Marshall and
Michael Durnin for training in oocyte dissection and microinjection; and Stowers
Imaging Center for assistance with live imaging; Stacie Hughes for comments
on the manuscript. This work was supported by funds to R.L. from the Stowers
Institute for Medical Research.
Author contributions
H.L. and R.L. designed the experiments, analysed the data and wrote the
manuscript; H.L. performed all experiments except the electron microscopy and
immuno-electron microscopy experiments, which were performed by F.G.; B.R.
assisted with data and statistical analyses; R.L supervised the whole project.
Competing financial interests
The authors declare no competing financial interests.
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s u p p l e m e n ta r y i n f o r m at i o n
Figure S1 a,b) Disruption of microtubules did not abolish chromosome
movement to the cortex. The untreated (a) or nocodazole-treated (b)
oocytes were stained with Phalloidine (red), anti-tubulin antibody
(green) and DAPI (blue). c) As a negative control for FITC-Lifeact, FITCDextran 4 showed a diffuse distribution in oocyte cytosol. Scale bars:
20 µm.
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Figure S2 Visualization of cytoskeletal structures in the chromosome vicinity
by thin-section EM. a) untreated oocyte; b) boxed region in (a) at a higher
magnification; c) Jas-treated; d) boxed region in (c) at a higher magnification;
e) Lat A-treated; and f) Fmn2-/- oocyte. g) Numbers of actin bundles around
2
chromosome were quantified. An area of 2 µm distance from chromosome
was counted in each image at the same magnification. The average was
obtained from 4 oocytes (total 16 images) for each condition. h). On-section
labeling IEM showing the presence of actin in the observed filament bundles.
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Figure S3 Location of Fmn2 by on-section labeling IEM.
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Supplementary Movie Legends
Movie S1 4D live imaging of an untreated oocyte (Interval 5 min, 25 frames per second).
Movie S2 Tracking of chromosome movement in an untreated oocyte with Imaris software (Interval 5 min, 5 frames per second).
Movie S3 Chromosome tracking in a 10 µM Jas treated oocyte (Interval 5 min, 5 frames per second).
Movie S4 3D representation of Fig 3a. (25 frames per second).
Movie S5 4D live imaging of untreated oocyte containing FITC-lifeact (Interval 30min, 2.5 frames per second).
Movie S6 4D live imaging of Jas treated oocyte containing FITC-lifeact (Interval 30min, 2.5 frames per second).
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