Science - USA (2022-02-11)

KIFC1’s functions in aMTOC-free spindles, we
depleted endogenous KIFC1 and reexpressed
wild-type KIFC1 and KIFC1(N593K), which
cross-links microtubules but cannot slide
owing to the mutation (Asn^593 →Lys) in its
motor domain ( 107 ) (Fig. 8G). Whereas almost
all aMTOC-free mouse oocytes rescued with
wild-type KIFC1 assembled a bipolar spindle,
nearly all oocytes rescued with KIFC1(N593K)

assembled disorganized spindles consisting

of several misaligned microtubule bundles

(Fig. 8, H and I). Thus, static cross-linking is

insufficient for restoring bipolar spindle as-

sembly, and KIFC1’s sliding activity is required

to align cross-linked microtubules into a bi-

polar spindle. Furthermore, around 25% of

oocytes rescued with wild-type KIFC1 assem-

bled bipolar spindles but had poorly focused

poles that lack NUMA (Fig. 8H); quantifica-

tion of the KIFC1–to–a-tubulin intensity ratio

revealed that almost twofold more KIFC1 was

loaded onto these spindles (Fig. 8J). Thus,

excessive KIFC1 could interfere with NUMA-

mediated pole focusing, and an optimal lev-

el of KIFC1 activities is required for proper

bipolar spindle assembly in the absence of

aMTOCs.

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

Fig. 8. An optimal level of KIFC1Õs
cross-linking and sliding activities is
required for proper bipolar spindle
assembly in aMTOC-free mouse oocytes.
(A) Still images from time-lapse movies
of control and PCNT-depleted mouse
oocytes. Green, mClover3-KIFC1; magenta,
microtubules (mScarlet-MAP4-MTBD);
blue, chromosomes (H2B-miRFP). Time is
given as hours:minutes after NEBD. (B) Still
images (top) of MI spindles from live
control and PCNT-depleted mouse oocytes at
6 hours after NEBD. The graph (bottom) is
the fluorescence profile of mClover3-KIFC1
across the spindle along the direction of
the yellow arrow. (C) Quantification of
mClover3-KIFC1 enrichment at the spindle
pole over the cytoplasm in control and
PCNT-depleted mouse MI oocytes at 6 hours
after NEBD. P< 0.0001. (D) Recovery
of photobleached mClover3-KIFC1 at the
spindle poles in control and PCNT-depleted
mouse MI oocytes at 6 hours after NEBD.
(E) Recovery of photobleached mClover3-
KIFC1 in the central region of the spindle in
control and PCNT-depleted mouse MI
oocytes at 6 hours after NEBD. Error bars
(shaded areas) represent SD. (F) Half-life of
mClover3-KIFC1 at different regions of MI
spindles from control and PCNT-depleted
mouse oocytes. P< 0.0001; N.S.,
not significant. (G) Schematic diagram of the
experiment in (H). (H) Immunofluorescence
images of MI spindles from KIFC1-depleted
aMTOC-free mouse oocytes rescued with
KIFC1-WT and KIFC1(N593K), fixed at 7 hours
after release. Gray, GFP-KIFC1; green, NUMA;
magenta, microtubules (a-tubulin); blue,
chromosomes (Hoechst). (I) Manual scoring
of spindle polarity in aMTOC-free mouse
MI oocytes rescued with KIFC1-WT and
KIFC1(N593K) (FisherÕs exact test,P<
0.0001). (J) Quantification of KIFC1 to
a-tubulin intensity ratio in bipolar MI spindles
from aMTOC-free mouse oocytes rescued with
KIFC1-WT. ****P< 0.0001. (K) Mechanistic
model for the organization and instability of
aMTOC-free spindle poles in mammalian
oocytes. See text for details. The number of
oocytes or poles analyzed is specified in
italics. See Materials and methods for box plot
specifications. Scale bars are 5mm. KIFC1 Bivalent

Legend: NUMA Other dynein cargoes Dynein complex

Microtubule

KIFC1

Chromosomes

Microtubules

Merge

KIFC1

Merge

anti-PCNT

Control IgG

mTRIM21

A microtubuleOnset of

nucleation

Growing

microtubule

aster

Early bipolar

spindle Metaphase I Anaphase onsetMetaphase II

I

KIFC1-WT

( 32 )

KIFC1(N593K)

(40)

0

20

40

60

80

100

Per

ce

ntag

e of

sp

ind

le

s

Bipolar

Multipolar

Disorganized

J

With NUMA

at poles ( 22 )

Without NUMA

at poles ( 8 )

0

0.6

1.2

1.8

2.4

3.0

KIFC1/α-tubulin intensity ratio

Bipolar MI spindle from

rescue with KIFC1-WT

****

C

BControl IgG anti-PCNT

0 5 10 15 20 25

-0.5

0.0

0.5

1.0

1.5

2.0 (^) Control IgG
anti-PCNT
Spindle length (μm)
Enrichment of KIFC1relative to the cytoplasm
KIFC1
0 h:7 min 0:28 3:58 5:08 8:31 13:18
0 h:20 min 4:05 6:39 8:59 10:58 15:03
Control
IgG ( 38 )
anti-PCNT
( 38 )
0.0 0.6 1.2 1.82.4 3.0
KIFC1 enrichment at the pole
over the cytoplasm

---

0 1428425670
0
0.3
0.6
0.9
1.2
Normalized KIFC1
intensit
y
Time (s)
Control IgG ( 18 )
anti-PCNT ( 17 )
0 14 28 42 56 70
0
0.3
0.6
0.9
1.2
Normalized KIFC1 inensity
Time (s)
Control IgG ( 10 )
anti-PCNT ( 10 )
Mobile fraction=48.1±3.89%
Mobile fraction=50.2±4.85%
Contr
ol IgG( 18
)
ant
i-P
CNT( 17 )
Con
trol
IgG( 10 )
anti

• PC

NT

(^10

)

0

10

20

30

40

50

t1/2

of KIFC1 (

s)

Polar

region

Central

region

**** N.S.

DEF

Fix

G

+siRNAs

9 - 12 days 6 h

Harvest oocyte NEBD

3 h

+KIFC1 mRNAdbcAMP washout

~1 h

siPCNT + siKIFC1 rescue

KIFC1-WT

NUMA Microtubules

KIFC1(N593K)

Chromosomes

Merge

KIFC1-WT

GFP-KIFC1

(22/32)

(8/32)

Metaphase I

H

K Stable spindle Unstable spindle

Mobile fraction=51.5±11.3%

Mobile fraction=60.2±11.3%

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