Nbl1p is part of the CPC. (A) Confirmation of NBL1 expression by RT-PCR using duplicate cDNA samples. gDNA, genomic DNA (B) Growth phenotype of nbl1-6. Serial 10-fold dilutions of NBL1 and nbl1-6 cells on synthetic minimal medium plates incubated at 25 or 37°C for 48 h. (C) Nbl1p copurifies with the other CPC components. Sli15-TAP and Nbl1-TAP purified complexes. Top, silver-stained 12% discontinuous gel. Note that Nbl1p is too small to be seen on a discontinuous gel that is able to resolve Sli15p and Bir1p. ^† Bands that always copurify with Sli15-TAP (Cheeseman et al., 2002). * Contaminants. Bottom, Western blot of 4–20% continuous gel.

Nbl1p colocalizes with the CPC. (A) Representative images of diploid cells homozygous for NBL1-GFP and NUP84-mRFP from G1 to telophase. Images were acquired every 5 min with seven Z-sections separated by 0.5 μm and deconvolved. Bar, 2 μm. (B) Representative time-lapse images of Nbl1-GFP from the above cells. Images were acquired and processed as above. Bar, 2 μm. (C) Nbl1p colocalizes with CPC components. Representative images from diploid cells homozygous for NBL1-mCherry and SLI15-, IPL1-, or BIR1-3GFP. mCherry and GFP were simultaneously imaged using an optical beam splitter. Bar, 2 μm.

Nbl1p interacts with the C-terminus of Bir1p and the N-terminus of Sli15p. Yeast two-hybrid analysis of Nbl1p and various regions of the other CPC components. Relative strength of interactions is indicated: −, none; −/+, weak; and +++, very strong. All interactions occurred reciprocally among “bait” and “prey” constructs containing these domains. The known domains are shown in color: IAP, inhibitor of apoptosis domain; HS, homologous to Survivin (Thomas and Kaplan, 2007); IN, IN-box (Ipl1p/Aurora B interacting).

Fundamental design of the CPC is conserved from Fungi to Animalia. (A) (top) A comparative protein structure model of Bir1p_889-941-Nbl1p_8-67-Sli15p_3-46 (green, purple, and orange, respectively) superimposed with the human Survivin–Borealin_10-109–INCENP_1-58 complex (pale brown; Jeyaprakash et al., 2007). The overall protein sequence identity between these regions is 13.4%. Information on model evaluation is in Materials and Methods. Middle, images of the structural model of Bir1p_889-941-Nbl1p_8-67-Sli15p_3-46 (left) and the template crystal structure of the human Survivin–Borealin_10-109–INCENP_1-58 complex (right). Bottom, enlarged view showing the residues with hydrophobic properties that are directed toward the core of the THB. (B) Protein sequence alignment of the regions of Survivin/Bir1p, Borealin/Dasra/CSC-1/Nbl1p, and INCENP/Sli15p used for homology-based structure modeling. Residues showing conservation, based on the BLOSSUM62 matrix, in the multispecies alignment are shown with a yellow background; the most similar (global score cutoff = 0.2) are shown in orange, less similar (global score cutoff = 0.1) are shown in black, and those only partially conserved (conserved in more than two thirds of sequences, global score cutoff = 0.1) are shown in gray (see Materials and Methods for details). Secondary structure elements are shown below the protein sequences. Hydrophobic residues facing toward the interface between the helices are seen both in the crystal structure of the human Survivin–Borealin_10-109–INCENP_1-58 and in the homology-based model of Bir1p_889-941–Nbl1p_8-67–Sli15p_3-46 (purple circles), only in the human Survivin–Borealin_10-109–INCENP_1-58 (blue circles), or only in the Bir1p_889-941–Nbl1p_8–67–Sli15p_3-46 model (red circles). Arginines (R) and threonines (T) that are potentially involved in hydrophobic interactions in the structural model (pale purple text) and correlate with hydrophobic residues in the human structure (pale purple circles). Residues in Bir1p and Nbl1p whose mutation disrupts the integrity of the CPC (green arrowheads). Residues at the N-terminus of Borealin that were shown to be required for relocalization of the human CPC to the central spindle (blue line). Protein sequences from representative species of fungi, vertebrates, and invertebrates are shown: Sacce, S. cerevisiae; Homsa, Homo sapiens; Xenla, Xenopus laevis; Drome, Drosophila melanogaster; Caeel, Caenorhabditis elegans; Nemve, Nematostella vectensis; Schpo, Schizosaccharomyces pombe; Lacbi, Laccaria bicolor. Protein name abbreviations: Sur, Survivin; SurR, Survivin-related protein; Bor, Borealin; BorR, Borealin-related protein; Nbl1, N-terminal-Borealin-like protein; INC, INCENP; INCR, INCENP-related protein. * Unknown sequence position because of incomplete cDNA sequence.

Nbl1p is required for faithful chromosome segregation. (A) DNA morphology of NBL1 and nbl1-6 in anaphase. Left, representative images of haploid cells fixed and stained with anti-tubulin antibody and DAPI. The nbl1-6 image shows an example of lagging chromosomes. Bar, 2 μm. Right, mean percentage and SD for three replicates (μ+σ) of cells (n = 100) with abnormal DNA morphology, lagging chromosomes (blue), and unequal DAPI staining (yellow). (B) Segregation of centromere III proximal region (CEN3) in NBL1 and nbl1-6 in anaphase. Left, representative images showing cells with GFP marked CEN3. Bar, 2 μm. Right, percentage (μ+σ) of cells (n = 100) with CEN3 missegregation. (C) CEN3 positioning relative to the metaphase spindle in NBL1 and nbl1-6. Left, representative images showing GFP marked CEN3, DNA localization, and the spindle. Right, percentage (μ+σ) of cells (n > = 80) showing CEN3 within 0.2 μm of one spindle pole. At metaphase, pairs of CEN3 markers are unseparated because this marker is 23 kb away from the centromere (Straight et al., 1996).

Nbl1p is essential for CPC integrity, stability, and localization. (A) Localization of Sli15p and Bir1p depends on Nbl1p. Localization of Sli15-3GFP (left) and Bir1-3GFP (right) in NBL1 and nbl1-6 haploid strains in anaphase at 23 and 37°C. Bar, 2 μm. (B) CPC integrity and stability requires Nbl1p. Top, silver stain of a 12% discontinuous gel showing complexes purified using Sli15-TAP from NBL1 and nbl1-6 strains exposed to 23 or 37°C for 3 h. Bottom, Western blot of whole cell extracts used for the purification using anti-PGK antibody. (C) Protein levels of Sli15p and Bir1p in NBL1 and nbl1-6 cells. (top) Western blot of whole cell extracts from NBL1 and nbl1-6 cells bearing a single copy of BIR1-13myc at the BIR1 locus using an anti-myc antibody. The cells were treated as described in B. The same blot was reprobed with an anti-PGK antibody as a loading control. Bottom, Western blot of whole cell extracts from NBL1 and nbl1-6 cells bearing a single copy of SLI15-13myc at the SLI15 locus using an anti-myc antibody. The same blot was reprobed with an anti-PGK antibody as a loading control. (D) Interaction between Bir1p and the Sli15p-Ipl1p subcomplex requires Nbl1p. Top, colloidal blue–stained 4–20% continuous gel showing the CPC purified from insect cells transfected with virus expressing Ipl1p, Sli15p, and Bir1p in the presence (right lane) or absence (left lane) of virus expressing Nbl1p. Bottom, Western blot of whole cell extracts used for the purification (top) using anti-Bir1p antibody and purified rabbit IgG for Sli15-TAP detection.

Nbl1p is a highly diverged homolog of Borealin/Dasra/CSC-1. (A) Block diagram summarizing features of sequences similar to Borealin/Dasra/CSC-1 from various fungal and animal taxa. Frequency of amino acid occurrence in conserved regions from the protein sequence alignment are shown as sequence logos (WebLogo: http://weblogo.berkeley.edu/; Crooks et al., 2004). (B) Phylogeny of Borealin/Dasra/CSC-1–related sequences. Consensus Parsimony tree with branch lengths estimated by Maximum Likelihood using PHYLIP v. 3.68 (http://evolution.genetics.washington.edu/phylip.html). Default settings were used and the sequence order was jumbled 10 times for the 1000 bootstrap replicates. The tree was based on the THB-forming domains from proteins listed in Supplemental Table S2A. Protein sequences missing more than 10% of this region were excluded. Borealin/Dasra/CSC-1–related sequences that have been characterized are underlined.