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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.

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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

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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

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266 s 357 s

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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

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f ob

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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

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bule

s0

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50~20 μm~15 μm~10 μm

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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

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