figure s1 : toya et al. - images.nature.com · supplementary information. 2 . figure s2. control...
TRANSCRIPT
S U P P L E M E N TA RY I N F O R M AT I O N
WWW.NATURE.COM/NATURECELLBIOLOGY 1
DOI: 10.1038/ncb2242
Figure S1 AIR-1 is localised around the condensed chromosomes. Before (Control) and after 8 min of ice-cold treatment (4 °C), embryos were fixed and stained with antibodies against α-tubulin and AIR-1. DNA was visualised with DAPI. AIR-1 was detected at the condensed chromosomes (arrows). Bar: 10 μm.
α-tubulin AIR-1 DNA Merged
Control
4 °C
Figure S1 : Toya et al.
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA RY I N F O R M AT I O N
2 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S2 Control experiments. (a) Control experiments for the phspho-specific AIR-1 antibody. Embryos were fixed and immunostained with anti-(P)-AIR-1. Microtubules were stained with FITC-conjugated α-tubulin antibody. DNA was stined with DAPI. Upper panels, wild type embryo with air-1(RNAi). Middle and lower panels, air-1N(RNAi) embryos expressing AIR-1T201A or AIR-1K73R, respectively (see Fig. 5a). Bar: 10 μm. (b, c) Control experiments for the RNAi-resistant air-1 transgenes. (b) Embryos from a gfp::air-1cDNA or gfp::air-1R (see Fig. 5a) integrated strains with and without air-1N(RNAi). Selected images from live observation are shown.
Expression of endogenous air-1 and gfp::air-1cDNA was both impaired by air-1N(RNAi) (upper panels), whereas gfp::air-1R expression was not (lower panels). GFP::AIR-1R was confirmed to be functional because it could rescue air-1N(RNAi) embryos; whereas the embryonic lethality of GFP::AIR-1;air-1N(RNAi) was 99% (83/84), that of GFP::AIR-1R;air-1N(RNAi) was reduced to 7.7% (10/130). (c) Embryos from a gfp::air-1RT201A and a gfp::air-1RK73R integrated strains without RNAi. Localisation of AIR-1T201A and AIR-1K73R were indistinguishable from the wild type AIR-1. Times are relative to NEBD. Bar: 10 μm.
Figure S2 : Toya et al.
AIR-1RT201A air-1N(RNAi)
air-1(RNAi)
AIR-1RK73R air-1N(RNAi)
(P)-AIR-1 DNA Mergedα-tubulin
without RNAi
gfp::air-1cDNA
gfp::air-1R
integratedtransgene
air-1N(RNAi)
a
b
c
GFP::AIR-1RK73R
0 s 100 s 210 s 310 s
0 s 100 s 200 s 310 s
GFP::AIR-1RT201A
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA RY I N F O R M AT I O N
WWW.NATURE.COM/NATURECELLBIOLOGY 3
Figure S3 Live imaging of embryos expressing kinase-inactive forms of AIR-1. (a) Live imaging of GFP::AIR-1 and GFP::AIR-1RT201A. Upper panels correspond to Fig. 5c. Lower panels are magnified views of the selected images from time-lapse recordings. (b, c) Live imaging of GFP::AIR-1RK73R. (b) Images of air-1RK73R transgene–expressing air-1N(RNAi) embryos. Lower panels are magnified views of the selected images from time-lapse recordings.
Some (3 of 9) GFP::AIR-1RK73R-expressing embryos assembled a monopolar spindle (left column). Majority of them (6 of 9) assembled a bipolar spindle albeit abrogated formation of kinetochore microtubules (right column). (c) Time-lapse images of the GFP::AIR-1RK73R-expressing air-1N(RNAi);spd-5(RNAi) and air-1N(RNAi);tbg-1(RNAi) embryos. In both embryos, microtubules were formed and GFP::AIR-1RK73R was detected on them. Bar: 10 μm.
Figure S3 : Toya et al.
a b
c
0 s49 s
112 s
203 s
112 s
266 s
182 s
182 s
371 s
245 s
spd-5(RNAi); air-1N(RNAi)
tbg-1(RNAi); air-1N(RNAi)
196 s
21 s
0 s
112 s
56 s
196 s
259 s
182 s
21 s
0 s
112 s
56 s
182 s
224 s
GFP::AIR-1RK73R
GFP::AIR-1RK73R air-1N(RNAi)air-1N(RNAi)
GFP::AIR-1RT201AGFP::AIR-1R
175 s210 s
98 s
0 s
133 s
189 s
210 s
0 s
35 s
91 s
210 s
259 s
266 s 357 s
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA RY I N F O R M AT I O N
4 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S4 Concurrent live observation of microtubules and a kinase-inactive form of AIR-1. (a) Control experiments. Embryos from a gfp::β-tubulin, mCherry::air-1R integrated strain (SA449) and a gfp::β-tubulin, mCherry::air-1RK73RT201A integrated strain (SA511). Note that the progression of mitosis was indistinguishable in SA449 and SA511 embryos. Microtubules may be more stabilized in SA511 by the kinase-inactive form of AIR-1 (AIR-1K73RT201A). (b) Time-lapse images of air-1N(RNAi);air-1R(RNAi) in an
SA449 embryo. Expression of endogenous air-1 and mCherry::air-1cDNA was both impared. (c) Time-lapse images of air-1N(RNAi) in an SA511embryo. AIR-1K73RT201A was expressed whereas endogenous air-1 expression was impared. (d) Number and length of the microtubules in (b) (795s), (c) (375s), and additional two embryos at the equivalent stages. Micrubutule lengths in AIR-1K73RT201A-expressing embryos were significantly longer than air-1- embryos. Times are relative to NEBD. Bar: 10 μm.
Figure S4 : Toya et al.
GFP::β-Tubulin
β-Tubulin
Merged
Merged
mCherry::AIR-1Rwithout RNAi
AIR-1R
AIR-1
RNAi K73RT201A
b
c
d
a
GFP::β-TubulinmCherry::
AIR-1RK73RT201A
GFP::β-TubulinmCherry::AIR-1R
AIR-1R
AIR-1RK73RT201A
270 s
270 s
air-1N(RNAi);air-1R(RNAi)
β-Tubulin MergedAIR-1R
K73RT201A
air-1N(RNAi)
375 s
795 s
990 s
0 s
195 s
375 s
510 s
0 s
Num
ber o
f ob
serv
able
mic
rotu
bule
s
0
10
20
30
40
50~30 μm~25 μm~20 μm~15 μm~10 μm
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA RY I N F O R M AT I O N
WWW.NATURE.COM/NATURECELLBIOLOGY 5
Figure S5 Functional relationship between AIR-1 and TPXL-1. (a) Co-localisation of (P)-AIR-1 and AIR-1 in tpxl-1(RNAi) embryos. tpxl-1(RNAi) embryos were fixed and immunostained with anti-(P)-AIR-1 and anti-AIR-1. Microtubules were stained with FITC-conjugated α-tubulin antibody. DNA was stained with DAPI. Both AIR-1 and (P)-AIR-1 were detected on
centrosomes, but not on microtubules. (b) Time-lapse images of an spd-5(RNAi);tbg-1(RNAi);tpxl-1(RNAi) embryo expressing GFP::β-tubulin and mCherry::AIR-1R (SA449). (c) Number and length of the microtubules in (b) (270s) and two additional embryos at the equivalent stage. Times are relative to NEBD. Bar: 10 μm.
DNA Merged
tpxl-1(RNAi)
spd-5(RNAi); tbg-1(RNAi); tpxl-1(RNAi)
AIR-1
(P)-AIR-1
AIR-1 (P)-AIR-1
α-tubulin
α-tubulin
Figure S5 : Toya et al.
a
b c
β-Tubulin MergedAIR-1R
spd-5(RNAi);tbg-1(RNAi);tpxl-1(RNAi)
0 s
15 s
270 s
510 s
390 s
Num
ber o
f ob
serv
able
mic
rotu
bule
s0
10
20
30
40
50~20 μm~15 μm~10 μm
© 2011 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA RY I N F O R M AT I O N
6 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S6 Full scans
Figure S6 : Toya et al.
Figure 4a Figure 4b
Figure 5b
(P)-AIR-1 AIR-1
AIR-1 TBG-1
32P
25
80
40
60
75
Histone H3
AIR-137
15
CBB
© 2011 Macmillan Publishers Limited. All rights reserved.