Science - USA (2022-02-11)

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.

RESEARCH | RESEARCH ARTICLE