Science - USA (2022-02-11)

to the organization of spindle poles in human
oocytes.
To test directly if NUMA is required for
spindle pole organization, we depleted NUMA
protein in human oocytes. Many proteins are

synthesized and deposited during oocyte

growth, rendering RNA interference (RNAi)

of proteins with low turnover inefficient in

fully grown oocytes ( 55 ). Therefore, we used

Trim-Away ( 56 ) to trigger acute degradation

of NUMA protein. NUMA was enriched in the

nucleus before nuclear envelope breakdown

(NEBD) in control oocytes, and this enrichment

was lost upon Trim-Away of NUMA (Fig. 1, E

and F). After NEBD, NUMA-depleted oocytes

Soet al.,Science 375 , eabj3944 (2022) 11 February 2022 2 of 19

NUMA

Microtubules

Prophase

Nuclear

envelope

breakdown

Chromosomes

Growing

microtubule

aster Prometaphase I

Metaphase I

(multipolar) Metaphase II

Merge

C

Chromosomes

NUMA

Bi-directional anaphase I

Microtubules

NUMA

Microtubules

D

G

0 4 8 121620

-0.5

0.0

0.5

1.0

1.5

2.0

Enrichment of NUMA

relative to the cytoplasm

NUMA Spindle length (μm)

Human MI spindle

A

NUMA Microtubules

Human MI spindle (vertical view)

B

Tri-directional anaphase I

I

Control IgG

( 5 )

anti-NUMA

( 5 )

0

20

40

60

80

100

Pe

rce

nta

ge

of sp

in

dles

Focused poles

Defocused poles

Nucleus

NUMA Microtubules

+hTRIM21+anti-NUMA Fix

3 h 15 h

NEBD

Variable

hTRIM21

Control IgG

anti-NUMA

NUMA Microtubules Merge

Chromosomes

Metaphase I

E

hTRIM21

Control IgG

anti-NUMA

NUMA Merge

Microtubules

Chromosomes

+hTRIM21+anti-NUMA Fix

3 h 24 h

F H

Fig. 1. NUMA is required for pole focusing in human oocytes.(A) Immuno-
fluorescence images of a human meiosis I (MI) spindle fixed at 15 hours after NEBD.
Green, NUMA; magenta, microtubules (a-tubulin). The graph is the fluorescence
profile of NUMA across the spindle along the direction of the yellow arrow.
(B) Immunofluorescence image of a human MI spindle fixed at 15 hours after NEBD,
imaged in a vertical orientation. Green, NUMA; magenta, microtubules (a-tubulin).
(C) Immunofluorescence images of human oocytes fixed at different stages of
meiosis. Green, NUMA; magenta, microtubules (a-tubulin), blue, chromosomes
(Hoechst). (D) Immunofluorescence images of human oocytes undergoing
bidirectional and tridirectional anaphase I. Green, NUMA; magenta, microtubules
(a-tubulin), blue, chromosomes (Hoechst). (E) Schematic diagram of the experiment
in (F). (F) Immunofluorescence images of control and NUMA-depleted immature

human oocytes. Green, NUMA; magenta, microtubules (a-tubulin); blue, chromo-

somes (Hoechst). Note that the higher cytoplasmic background in the oocyte injected

with anti-NUMA in (F) was caused by the injected antibody but not due to perturbation

of NUMA entry into the nucleus because the higher cytoplasmic background was

consistently detected in the oocyte injected with anti-NUMA after NEBD in (H).

(G) Schematic diagram of the experiment in (H). (H) Immunofluorescence images of

MI spindles from control and NUMA-depleted human oocytes, fixed at 15 hours

after NEBD. Green, NUMA; magenta, microtubules (a-tubulin); blue, chromosomes

(Hoechst). (I) Manual scoring of spindle pole defocusing in control and NUMA-

depleted human MI oocytes (FisherÕs exact test,P< 0.01). The number of oocytes

analyzed is specified in italics. Nuclei are outlined by yellow dashed line circles. Yellow

arrowheads highlight prominent NUMA clusters. Scale bars are 5mm.

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