Supporting Online Material


Histone H3 Thr-3 Phosphorylation by Haspin Positions Aurora B at Centromeres in Mitosis
Fangwei Wang, Jun Dai, John R. Daum, Ewa Niedzialkowska, Budhaditya Banerjee, P. Todd Stukenberg, Gary J. Gorbsky, Jonathan M. G. Higgins

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S12
References

This file is in Adobe Acrobat PDF format.

Other Supporting Online Material for this manuscript includes the following:

Movie s1
Control Xenopus S3 cells complete mitosis normally. An S3 cell was injected in prophase with rabbit IgG. The microinjection needle can be seen between 4 and 6 s. Seven of 7 injected cells progressed normally through mitosis. Time is shown in min:s.

Movie s2
Microinjection of H3T3ph-specific antibody compromises chromosome alignment and anaphase but not cytokinesis in Xenopus S3 cells. An S3 cell was microinjected in prophase with H3T3ph-specific antibody. The microinjection needle can be seen between 1 and 3 s (stills are shown in Fig. 4D). Chromosome alignment is compromised (yellow arrows) and chromosome decondensation and exit from mitosis occurs without anaphase, although there is clear formation of a cleavage furrow (red arrows). At first, these results are similar to the effects of anti-Aurora B or anti-INCENP injection, where chromosome alignment is compromised and chromosome decondensation and exit from mitosis occurs without obvious anaphase (S19, S20). In contrast, unlike anti-H3T3phinjected cells, anti-Aurora B and anti-INCENP injected cells fail to undergo cytokinesis. These results are consistent with the idea that anti-H3T3ph injection compromises centromere but not central spindle functions of the CPC. Three of 3 injected cells showed chromosome congression defects and failed to disjoin chromosomes at anaphase.

Movie s3
Control pig LLC-PK cells complete mitosis normally. An LLC-PK cell was injected in prophase with rabbit IgG. The microinjection needle can be seen between 12 and 16 s. Five of 5 injected cells progressed normally through mitosis.

Movie s4
Microinjection of H3T3ph-specific antibody compromises chromosome alignment and anaphase but not cytokinesis in LLC-PK cells. An LLC-PK cell microinjected in prophase with H3T3ph-specific antibody shows evidence of chromosome congression problems (white arrows) and exits mitosis in the absence of anaphase with clear formation of a cleavage furrow. The microinjection needle can be seen between 2 and 5 s. Five of 6 injected cells showed chromosome congression defects and all 6 cells failed to disjoin chromosomes at anaphase.

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