Supplement Figure 1.

GV GVBD MI MII

CapZ β2

Merge

DNA CapZ β2

A

DIC GFP-CapZ β2 Merge

250ng

500ng

B CapZ β2-GFP

Journal of Cell Science | Supplementary Material

Supplement Figure 2.

A B Control

early MI

late MI

RNAiCapZαβ

Control RNAiCapZαβ

n=10

n=20

N.S.

Pha

lloid

in In

tens

ity(A

.U.)

DNA Actin

Tubulin

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Journal of Cell Science | Supplementary Material

Supplementary Data

Supplement Figure 1. Localization of actin-capping protein (CP) during mouse

oocyte maturation. (A) Subcellular localization of CapZβ2 during mouse oocyte meiotic

maturation. Immunofluorescence staining was performed using an anti-CapZβ2 antibody

after methanol fixation. Blue: DNA; green: CapZβ2. Bar = 20 µm. (B) Localization of

CapZβ2 in germinal vesicle (GV)-stage oocytes. GFP-CapZβ2 mRNA was injected into

the oocyte cytoplasm. Two different concentrations (250 and 500 ng/µl) of. GFP-CapZβ2

mRNA were injected. Bar = 20 µm.

Supplement Figure 2. Knockdown of actin-capping protein (CP) have no effect

formation of the cortical actin and actin cap in maturing oocytes. (A) Cortical actin

and actin cap formation by CP knockdown. After dsRNA injection and maturation arrest

to ensure CP was knocked down, maturation was resumed and samples were collected 7

h and 9 h later. Representative oocytes were immunostained for actin (red), β-tubulin

(green), and DNA (blue). Bar = 20 μm. (B) Fluorescence intensity of Cortical actin

labeling after injection of CapZα1 and CapZβ2 dsRNA (CPKD) (Control: n = 10; RNAi:

n = 20). N.S.: not statistically significant (p > 0.05).

!

Supplement Figure 3. Relationship between GFP fluorescence intensity and the

polar body/oocyte diameter ratio in GFP-CapZβ2-overexpressing oocytes. GFP

fluorescence intensity was measured in single oocytes (Y axis) and plotted against the

ratio between the diameter of the polar body and that of the oocyte (X axis). Spindle

migration speed in control GFP-expressing oocytes (A) and GFP-CapZβ2-overexpressing

Journal of Cell Science | Supplementary Material

oocytes (B).Maturing oocytes injected with the actin probe mCherry-UtrCH (red) and

GFP-CapZβ2 (green) were imaged. DNA (blue) was stained with Hoechst 33342. The

number of hours after the resumption of maturation is indicated in each frame. Bar = 20

μm.

Supplementary Movies Movies 1 and 2. Time-lapse movie of an oocyte injected with α-Tubulin-GFP

(control). Images were taken 5–13 h after the resumption of maturation. The frame

interval is 300 s and the total length of the movie is 28,800 s. Left: differential

interference contrast (DIC), middle: spindle labeled with α-Tubulin-GFP (green), and

right: merged.

Movies 3 and 4. Impaired spindle migration in an oocyte injected with CapZα1 and

CapZβ2 double-stranded RNA (dsRNA) and α-Tubulin-eGFP complementary RNA

(cRNA). Images were taken 5.5–13.5 h after the resumption of maturation. The frame

interval is 300 s and the total length of the movie is 28,800 s. Left: differential

interference contrast (DIC), middle: CapZα1 and CapZβ2 dsRNA and spindle labeled

with α-Tubulin-eGFP (green), and right: merged.

Movie 5. Symmetric division of an oocyte overexpressing GFP-CapZβ2. Images were

taken 6–11 h after the resumption of maturation. Cytokinesis starts at 9 h and is

completed at 11 h, which is earlier than in double-stranded RNA (dsRNA)-injected

oocytes. The frame interval is 300 s and the total length of the movie is 18,000 s. Left:

Journal of Cell Science | Supplementary Material

differential interference contrast (DIC), middle: GFP-CapZβ2 and H2B-mCherry, which

labels chromatin, and right: merged.

Movie 6. Early spindle migration and abnormal polar body protrusion in an oocyte

overexpressing GFP-CapZβ2. Images were taken 6–11 h after the resumption of

maturation, which was initiated by the removal of milrinone from the medium. Polar

body extrusion is completed at 10 h, which is earlier than in control oocytes (α-Tubulin-

eGFP-injected) and double-stranded RNA (dsRNA)-injected oocytes. Segregation of the

polar body is observed at 10.5 h. The frame interval is 300 s and the total length of the

movie is 18,000 s. Left: differential interference contrast (DIC), middle: GFP-CapZβ2

and H2B-mCherry, which labels chromatin, and right: merged.

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Journal of Cell Science | Supplementary Material

Movie 1.

Movie 2.

Movie 3.

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Movie 4.

Movie 5.

Movie 6.

Journal of Cell Science | Supplementary Material

Table S1. Primers used in this study

Gene Accession no. Primer sequence Use of the primer

CapZ�1 NM_009797.2

5’-ATGGCCGACTTTGAGGATCG-3’ qPCR (Forward)

5’-CAGAGCACTGTCACACGAC-3’ qPCR (Reverse)

5’-TAATACGACTCACTATAGGGAGACCAC TGGTGACTTGGTAACAGCA-3’

dsRNA (Forward)

5’-TAATACGACTCACTATAGGGAGACCAC GATTTTGGTGCGGGTAACTG-3’

dsRNA (Reverse)

CapZ�2 NM_001271405.1

5’-AGGCAGCCTAACCAGACAGA-3’ qPCR (Forward)

5’-CCTCCACCAGGTCGTTCTTA-3’ qPCR (Reverse)

5’-TAATACGACTCACTATAGGGAGACCAC GGTGGGCAAGGATTACCTTT-3’

dsRNA (Forward)

5’-TAATACGACTCACTATAGGGAGACCAC CCTCCACCAGGTCGTTCTTA-3’

dsRNA (Reverse)

dsRNA; double-stranded RNA; qPCR, quantitative PCR.  

Journal of Cell Science | Supplementary Material