Mccleland Et Al. Supplemental Material

McCleland et al. Supplemental Material

Supplementary Figure Legends

Supplementary Figure 1. Identification of Xenopus homologs of xNdc80 and xNuf2. (A) Clustal W alignment of the amino acids encoding Xenopus Nuf2 (xNuf2), human Nuf2 (hNuf2), and Saccharomyces cerevisiae Nuf2 (scNuf2). Residues that are completely conserved are highlighted in black and residues that have conservative mutations are displayed in gray. xNuf2 is 63% and 40% similar to the human and yeast homologs, respectively. (B) Clustal W alignment of the amino acids encoding Xenopus Ndc80 (xNdc80), human Ndc80 (hNdc80), and S. cerevisiae Ndc80 (scNdc80). Conserved residues are displayed as described in A. xNdc80 is 72% and 40% similar to human and yeast homologs, respectively. (C) Structural evaluation of xNdc80 and xNuf2. Speckled boxes represent regions predicted to form -helical coiled coils and gray boxes display regions that have no significant homology to known domains.

Supplementary Figure 2. xNdc80 and xNuf2 cofractionate on both gel filtration and sucrose gradient and colocalize to mitotic kinetochores. (A) xNdc80 and xNuf2 cofractionate by gel filtration analysis as a 1-MDa complex that is not cell cycle regulated. Interphase and mitotic extracts were separated by Superose 6 gel filtration chromatography and equal volumes of alternate fractions were run on a 10% SDS-PAGE gel. The fractions were then immunoblotted for xNdc80 and xNuf2 to determine the molecular weight of the protein complex. (B) xNdc80 and xNuf2 cosediment on a sucrose density gradient as a 4S complex. Interphase extract was sedimented through 5%-30% sucrose density gradient. Equal volumes of each fraction were run on a 10% SDS-PAGE gel and immunoblotted for xNdc80 and xNuf2. XTC cells were arrested in mitosis with nocodazole, fixed, and stained with biotinylated anti-xNdc80 (red) and either anti-xIncenp (C) or anti-xNuf2 (D, green) affinity purified antibodies followed by the appropriate secondary reagent. Chromosomes were visualized by staining with Hoechst 33342. An enlargement of a subset of kinetochores from the merge (white box) is displayed on the far right of each panel.

Supplementary Figure 3. Systematic analysis of yeast kinetochore components for spindle checkpoint function. (A) ndc80-1 and nuf2-61 cells arrest in mitosis with a 2C DNA content, while spc24-1 and spc25-1 cells enter the next cell cycle and re-replicate their DNA. Cycling cells in rich media at 23°C were shifted to their restrictive temperature (37°C) and monitored by flow cytometry for DNA content. (B) The spindle checkpoint is still functional after deletion of several nonessential kinetochore components. Deletion alleles (from the haploid deletion set, S288C background) of bik1, ctf3, kip3, mcm16, mcm17/chl4, mcm19, mcm22, and ydr383C were grown to mid-log, and then resuspended in rich media containing 15 ug/mL nocodazole, resulting in an arrest with a 2C DNA content. In contrast, mad2 cells (strain 2036-7-2), used as a control, fail to arrest and re-replicate their DNA. Similar results were obtained with bim1, cbf1, cin8, kar3, and slk19 mutants (Gardner et al. 2001).

Supplementary Movie Legends

Supplementary Movie 1. An XTC cell injected with anti-xNdc80 antibody showing a “cut” phenotype. The cell was injected at late prophase ~5 min before the initiation of the time-lapse sequence. Images were captured at 30-sec intervals. Total elapsed time is 65 min.

Supplementary Movie 2. An XTC cell injected with anti-xNuf2 antibody showing a “cut” phenotype. The cell was injected at late prophase ~8 min before initiation of the time-lapse sequence. Images were captured at 60-sec intervals. Total elapsed time is 65 min.

Supplementary Movie 3. A control noninjected XTC cell showing normal progression through mitosis. Images were captured at 30-sec intervals. Total elapsed time is 62 min.

Supplementary Movie 4. An XTC cell injected with anti-xNuf2 antibody exits mitosis in the presence of taxol. The cell escapes the M phase block within 2 h from the injection. Images were captured at 2-min intervals. Total elapsed time is 151 min.

References

Gardner, R.D., Poddar, A., Yellman, C., Tavormina, P.A., Monteagudo, M.C., and Burke, D.J. 2001. The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain. Genetics157: 1493-1502.