Science - USA (2022-02-11)

Unlike the other two kinesin-14 family mem-
bers KIFC2 and KIFC3, KIFC1 distinctively
possesses a microtubule-binding domain at
its tail ( 105 ). This domain, together with the
conserved, microtubule-binding motor domain,

allows KIFC1 to organize microtubules through

static cross-linking and/or sliding ( 106 ). To

investigate the requirement of different do-

mains in KIFC1, we expressed a series of trun-

cation mutants in aMTOC-free mouse oocytes

(fig. S14, H and I). The spindle localization of

KIFC1 in aMTOC-free mouse oocytes depended

predominantly on the tail domain and the stalk

but not on its motor domain (fig. S14J). To test

whether the motor domain is dispensable for

Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 10 of 19

Fig. 7. Introduction of
exogenous KIFC1 rescues
spindle instability and
reduces the risk of
aneuploidy in human
oocytes.(A) Coomassie
staining for the SDS–
polyacrylamide gel
electrophoresis (PAGE)
of Strep2-mClover3-KIFC1.
The black arrow marks the
band corresponding to
the protein. (B) Still images
from time-lapse movies of
noninjected and KIFC1-
injected human oocytes.
Green, microtubules
(5-SiR-CTX); magenta,
chromosomes (SPY555-
DNA). Time is given
as hours:minutes after
onset of microtubule
nucleation. (C) Still images
of metaphase II spindles
from live noninjected
and KIFC1-injected
human oocytes. Green,
mClover3-KIFC1; magenta,
microtubules (a-tubulin).
(D) Immunofluorescence
images of metaphase II
spindles from noninjected
human oocytes stained
with nonpreincubated
and KIFC1 peptide–
preincubated anti-KIFC1-N
+C and from KIFC1-injected
human oocyte. Green,
KIFC1; magenta, microtu-
bules (a-tubulin). Note
that metaphase II spindles
in KIFC1-injected human
oocytes have thicker
microtubule bundles than
those in noninjected
human oocytes in (C) and
(D). (E) Quantification of
the duration of spindle
instability in non-injected
and KIFC1-injected human
oocytes. ****P< 0.0001.
(F) Quantification of the
time of early spindle bipolarization in noninjected and KIFC1-injected human oocytes. N.S., not significant. (G) Quantification of the time of anaphase onset in noninjected
and KIFC1-injected human oocytes. N.S., not significant. Note that the onset of microtubule nucleation was chosen as the reference time point because the time gap in
between NEBD and the onset of microtubule nucleation varies between oocytes. (H) Quantification of chromosome misalignment at anaphase onset in noninjected
and KIFC1-injected human oocytes. (I) Quantification of chromosome missegregation during anaphase in noninjected and KIFC1-injected human oocytes. Yellow
arrowheads highlight spindle poles. The number of oocytes analyzed is specified in italics. See Materials and methods for box plot specifications. Scale bars are 5mm.

D

Microtubules

Merge

Microtubules

Merge

Chromosomes

Non-injected

KIFC1-injected

4:00 9:50 15:30 17:10 17:20 17:40

4:30 7:20 10:20 13:00 13:10 14:00

Human oocytes

B

E

Non-

injected ( 9 )

KIFC1-

injected ( 8 )

0

20

40

60

80

100

Perc

entag

e o

f ooc

yte

s

Misaligned chromosomes

at anaphase onset

Aligned chromosomes

at anaphase onset

H

Non-

injected ( 9 )

KIFC1-

injected ( 8 )

0

20

40

60

80

100

Pe

rce

ntage

of

oo

cytes

With severely lagging

chromosomes

With mildly lagging

chromosomes

No lagging chromosomes

I

Non-

injected ( 10 )

KIFC1-

injected ( 8 )

0

2

4

6

8

Time

of

early spindle bi

pol

ariz

ati

on

(h after microtubule nucleation onset)

N.S.

F

Non-

injected ( 9 )

KIFC1-

injected ( 8 )

0

6

12

18

24

Time o

f a

na

ph

as

e on

se

t

(h

a

fte

r microtubule nucleation onset)

N.S.

G

A

Strep2-

mClover3

-KIFC1

185

115

80

65

50

30

25

15

10

C

Non-

injected ( 10 )

KIFC1-

injected ( 10 )

0

4

8

16

12

Dur

ation

of spindle instabi

lit

y (h

)

****

Microtubules

Merge

KIFC1-injected

Peptide preincubation

anti-KIFC1-N+C

-peptide +peptide

Non-injected

Metaphase II

Non-injected KIFC1-injected

mClover3

Microtubules

Merge

Metaphase II

Expected

molecular

weight:

104 kDa

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