transferred into 4ml of protein-free medium
at around 6, 15, 11, and 10 hours after NEBD,
respectively. Then, 12ml of 1.333× NuPAGE
LDS sample buffer (Thermo Fisher Scien-
tific) with 100 mM dithiothreitol (DTT) was
added, and the mixture was immediately snap-
frozen in liquid nitrogen. For a positive con-
trol, 0.125 to 2.5mgofasychronizedHeLawhole
cell lysate (NBP2-10274; Novus Biologicals)
was used.
Samples were thawed at 37°C and snap-
frozen in liquid nitrogen twice more before
beingheatedat95°Cfor5min.Sampleswere
resolved on a 17-well NuPAGE 4 to 12% Bis-
Tris protein gel of 1.0 mm thickness (Thermo
Fisher Scientific) with NuPAGE MOPS SDS
Running Buffer (Thermo Fisher Scientific).
Proteins were transferred onto a 0.45-mm poly-
vinylidene difluoride (PVDF) membrane with
SDS-free Towbin buffer at 200 mA for 2 hours
on ice. Blots were stained with No-Stain Protein
Labeling Reagent (Thermo Fisher Scientific)
before blocking. Blocking and antibody incu-
bations were performed in Tris-buffered saline
(TBS) with 5% skim milk and 0.1% tween-20.
Primary antibodies used were rat anti–a-tubulin
(MCA78G; Bio-Rad), mouse anti–b-actin (ab8226;
Abcam), mouse anti–b-tubulin (T8328; Sigma-
Aldrich), rabbit anti–g-tubulin (T3559; Sigma-
Aldrich), rabbit anti-GAPDH (ab181602; Abcam),
mouse anti-NDC80 (sc-515550; Santa Cruz Bio-
technology), rabbit anti-KIFC1-N (ab172620;
Abcam), rabbit anti-KIFC1-C (20790-1-AP;
Proteintech), mouse anti-LIS1 (H00005048-
M03; Abnova), rabbit anti-NUMA (ab97585;
Abcam), and rabbit anti-PCNT (ab4448; Abcam).
Secondary antibodies used were horseradish
peroxidase (HRP)–conjugated anti-mouse
(P0447; Dako), anti-rabbit (A9169; Sigma-
Aldrich), and anti-rat (sc-2032; Santa Cruz
Biotechnology). Blots were developed with
SuperSignal West Femto Maximum Sensitiv-
ity Substrate (Thermo Fisher Scientific) and
documented with Amersham Imager 600 (GE
Healthcare). Care was taken that the exposure
time did not cause saturation.
General quantification
Time-lapse movies of live oocytes and images
of fixed spindles were analyzed in 3D using
Imaris (Bitplane). To determine the timings of
meiotic progression, different stages of meiosis
were quantified relative to the time of NEBD
(for mouse, bovine, and porcine oocytes) or to
the time of onset of microtubule nucleation
(for human oocytes). NEBD was defined as the
time point when the sharp boundary between
the nucleus and cytoplasm disappeared in the
differential interference contrast image (for
mouse oocytes) or when the nucleus started
collapsing in the H2B channel (for bovine and
porcine oocytes). Onset of microtubule nucle-
ation was defined as the time point when
microtubules were first detected outside the
chromosome aggregate. Anaphase onset was
defined as the time point before chromosome
separation was first observed. To score for
chromosome misalignment, chromosomes
that failed to congress to the metaphase plate
at anaphase onset were classified as mis-
aligned chromosomes. To score for chro-
mosome missegregation, chromosomes that
failed to clear the central spindle within 10 or
20 min after anaphase onset were classified
as mildly and severely lagging chromosomes,
respectively.
InFigs.1Iand3Eandfigs.S6(E,H,andK),
S7H, S8 (C, G, K, and O), and S9 (H and L),
bipolar spindles, in which ends of all micro-
tubules converged at the poles, were defined
as having focused poles. Spindles that had
a detectable spindle axis but failed to con-
verge ends of microtubules at the poles were
defined as having unfocused poles. For mouse
oocytes, unfocused poles were further clas-
sified into mildly unfocused poles (with par-
tially separated microtubule ends) and severely
unfocused poles (with fully separated micro-
tubule ends).
To score for spindle instability, spindle poles
were defined as regions of MAP4 or MAP4-
MTBD ora-tubulin intensity that prominently
protruded from the main microtubule mass.
In Fig. 3F, spindles with two well-defined poles
were scored as bipolar. Round spindles without
a detectable spindle axis were scored as apolar.
Spindles with more than two well-defined
poles were scored as multipolar. In Figs. 3I, 5
(C and H), and 8I and fig. S14D, spindles with
two well-defined poles were scored as bipolar.
Round spindles with two poorly defined poles
were scored as with broad poles. Spindles with
more than two well-defined poles were scored
as multipolar. Spindles without a barrel shape
but that contained several weakly associated
microtubule bundles were scored as disorga-
nized. In Figs. 6B and 7E and fig. S11D, spin-
dles that maintained a barrel-shaped bipolar
morphology were defined as having no spin-
dle instability. Spindles that lost their initial
bipolarity and underwent dynamic remodel-
ing of the poles were defined as having spindle
instability.Quantification of protein enrichment at the
spindle pole over the cytoplasm
Line profiles across half spindles were gen-
erated along the spindle axis in ZEN, and
mean fluorescence intensities were exported
into Excel (Microsoft). The intensity of an
equivalent ROI in the cytoplasm of the same
oocyte was subtracted to correct for cytoplasmic
background. Background-corrected data were
then normalized by the minimum intensity
of each line profile (cytoplasmic intensity).
Enrichment at the spindle pole over the cyto-
plasm was represented by the maximum in-
tensity of each line profile.Automatic quantification of standard deviation
of fluorescence intensity within spindle
isosurface and microtubule packing index
To reproducibly reconstruct spindles (labeled
with anti–a-tubulin) from different immu-
nofluorescence experiments, a previously
described MATLAB script ( 31 ) was used for
automatic surface creation in Imaris. Spe-
cific parameters used were: 0.1mm (for micro-
tubule packing index) or 1.0mm [for standard
deviation (SD) of fluorescence intensity within
spindle isosurface] for smoothing size, no back-
ground subtraction, 500 to 750mm^3 and 1000
to 1750mm^3 for minimum and maximum ex-
pected total volume of surface object, respec-
tively. SD of fluorescence intensity, volume,
and volume of the object-oriented minimum
bounding box for the spindle isosurface were
exported into Excel. The microtubule packing
index was calculated by dividing the volume of
the spindle isosurface by the volume of the
object-oriented minimum bounding box.Manual quantification of volume by 3D
reconstruction of NUMA clusters
Manual segmentations were performed using
the Surface function of Imaris. For KIFC1 de-
pletion experiments, NUMA clusters (labeled
with anti-NUMA) were smoothed with a sur-
face detail of 0.5mm and thresholded after
background subtraction with 0.5mmasthe
diameter of largest sphere that fits into the
object. Depending on the cytoplasmic back-
ground in each oocyte, a suitable threshold
value was selected for segmentation. The vol-
umes of the NUMA clusters’isosurfaces were
exported into Excel.Quantification of photoactivation experiments
Minor temporal drift was corrected using Rigid
registration in Icy (Institut Pasteur). Mean
intensities of photoactivated areas over time
were exported from Fiji into Excel for further
processing. Data were first corrected for cyto-
plasmic background by subtracting the in-
tensity of the photoactivated area before
photoactivation. Background-corrected data
were then normalized to the intensity of the
first postactivation time point. Plots of inten-
sity against time were fitted to linear functions
(y=mx+c, wheremis the slope andcis the
yintercept) or one-component exponential
functions [y=Ae(c−x)/t, wherecis the offset,
Ais the fraction of the component, andtis
the time constant] in OriginPro (OriginLab).
Half-times of decay (t1/2) were calculated by
−0.5/m ort× ln(2).Directionality analysis
Directionality of microtubules within spindles
were analyzed using OrientationJ in Fiji. The
specific parameters used were 9 pixels for local
window, Gaussian for gradient, HSB for color
survey, orientation for Hue, coherency forSoet al.,Science 375 , eabj3944 (2022) 11 February 2022 16 of 19
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