assembled multipolar spindles and spindles
with broad poles, respectively (Fig. 5, F to H;
and fig. S14, A and B). By contrast, codepletion
of other members in the kinesin-14 family,
KIFC2 and KIFC3, did not result in multipolar
spindles and round spindles with broad poles
in aMTOC-free mouse oocytes (fig. S14, C and
D). Thus, depletion of KIFC1 recapitulates the
spindle instability of human oocytes.
To further investigate spindle assembly in
the absence of KIFC1, we performed live
imaging in KIFC1-depleted bovine oocytes.
Depletion of KIFC1 resulted in spindle insta-
bility in more than 95% of bovine oocytes and
an increase in tridirectional division (Fig. 6, A
to C; and movies S13 to S15). In human oocytes,
7 to 26% of oocytes underwent tridirectional
division ( 2 , 3 ) (Fig. 6C). By contrast, around
Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 7 of 19
siKIF11/EG5
siKIF18A siCENPE siCLASP1+2
siPRC1 siKIF4A
siKIF14 siKIF23/MKLP1
siPCNT + siNUMA
A
2.5 1.25 0.5 0.25 0.125 12 10 20 30 40 50
Human
HeLa lysate (μg) oocytes Mouse oocytes
On-blot No-Stain protein staining
B
Microtubules
Anti-TUBA IB
Anti-KIFC1-N IB
siControl ( 30 ) siKIFC1( 30 )
Mouse oocytes
mKIFC1
D
mClover3
-hKIFC1- +
Anti-KIFC1-N IB
mKIFC1
mClover3
-hKIFC1
2.5 1.25 0.5 0.25 0.125 1.12 0.26 0.66 0.94 1.14 1.27
μg of
protein
loaded:
siControl siASPM
siKIF15/HKLP2 siKIFC1/HSET
siKIF2A
siRACGAP1/CYK4 siKIF12
siKIF20A/MKLP2
Directionality
siDLG5/HURP siTPX2
siHAUS6
Directionality color-code
+90o -45o 0 o +45o -90o
siKIF20B/MPP1
Chromosomes
Metaphase I
2.5 1.25 0.5 0.25 0.125 12 10 20 30 40 50
Human
HeLa lysate (μg) oocytes Mouse oocytes
NUMA
Anti-NUMA IB
hKIFC1
mKIFC1
Anti-KIFC1-N IB
hKIFC1
mKIFC1
Anti-KIFC1-N IB (Overexposed)
C
Fig. 4. Human oocytes are deficient in the molecular motor KIFC1/HSET.
(A) Immunofluorescence images of MI spindles from aMTOC-free mouse oocytes
codepleted of NUMA and one of the 20 candidate proteins, fixed at 7 hours
after release. Gray, microtubules (a-tubulin); magenta, chromosomes (Hoechst);
pseudocolor, directionality. For oocytes that did not assemble a spindle, chromosomes
are outlined by yellow dashed lines. Scale bars are 5mm. (B) No-Stain protein
staining for the blot of HeLa cell, human oocyte, and mouse oocyte lysates.
The amount of HeLa cell lysate or the number of oocytes loaded is indicated
above each lane. The corresponding amount of protein loaded is indicated
under each lane. (C) Immunoblots of HeLa cell, human oocyte, and mouse
oocyte lysates. The two arrows in anti-NUMA IB mark NUMA with and without
posttranslational modifications. (D) Immunoblots of control, KIFC1-depleted,
and mClover3-KIFC1Ðexpressing mouse oocytes. Human and mouse oocytes
were lysed at 15 and 6 hours after NEBD, respectively. Black arrows mark
the band corresponding to each protein. The number of oocytes loaded is
specified in italics.
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