xNdc80 and xNuf2 form a complex in vivo that localizes to kinetochores from prometaphase through anaphase. (A) Characterization of polyclonal antibodies generated against xNuf2 and xNdc80. Affinity-purified anti-xNuf2 antibodies recognize a single 50-kD protein in both Xenopus interphase egg extract (IE) and XTC cell lysate (XTC). Affinity-purified anti-xNdc80 antibodies recognize a doublet protein at 75 kD in Xenopus interphase egg extract and a single protein at 75 kD in XTC cell lysate. The small band identified in the xNdc80 lane is either a degradation product or a cross-reacting protein. (B) Coimmunoprecipitation of xNdc80 and xNuf2 from Xenopus egg extracts. xNdc80 and xNuf2 protein were immunoprecipitated (IP) from interphase extract and blotted for xNuf2 and xNdc80. Preimmune (Pre-I) sera controls did not precipitate either xNuf2 or xNdc80 protein. (C) xNdc80 and xNuf2 cosediment on a sucrose density gradient as a 4.3S complex. High-speed supernatants (HSS) of mitotic extracts (ME) were sedimented through 5%–30% sucrose density gradient and blotted for xNdc80 and xNuf2. (D) Localization of xNdc80 on a mitotic chromosome. XTC cells, arrested in mitosis with nocodazole, were fixed and stained with anti-xINCENP (green), biotinylated anti-xNdc80 antibody (red), and Hoeschst 33342 to visualize DNA (blue). (E) xNdc80 and xNuf2 are associated with the kinetochore from prometaphase through anaphase. Cycling XTC cells were fixed and stained with mouse anti-tubulin antibody (green) and either anti-xNuf2 or anti-xNdc80 antibody (red). DNA visualized with Hoechst 33342 (blue). Bars: D,E, 5 μm.

Anti-xNdc80- and anti-xNuf2-antibody-injected cells bypass the spindle checkpoint. Mitotic XTC cells, treated with taxol, were injected with either anti-xNdc80 or anti-xNuf2 antibodies. After an additional 2 h, the cells were fixed, immunolabeled with fluorescent anti-rabbit IgG secondary to locate injected cells, and stained with DAPI. (A) Examples of the two major phenotypes resulting from anti-xNuf2 antibody injection in taxol. There were either multinucleated polyploid cells or cells with a “cut” phenotype. The insets show the injected antibody detected by fluorescent anti-rabbit IgG secondary. (B) Quantification of injected cells. The numbers above the bars indicate the number of injected cells in each category.

Addition of anti-xNdc80 antibodies to preassembled kinetochores disrupts spindle checkpoint signaling, but does not dramatically affect kinetochore assembly. (A) Sperm nuclei and nocodazole were incubated with CSF-arrested extracts for 30 min to assemble kinetochores and establish the spindle checkpoint. Control IgG or anti-xNdc80 antibody was then added for an additional 30 min before release from CSF arrest with calcium. Samples were taken before (t = 0) the addition of calcium and every 15 min thereafter to monitor histone H1 kinase activity. Autoradiograms of histone H1 kinase assays as well as photographs of nuclear morphology (t = 60) are shown. (B) Kinetochore structure is not severely disturbed following addition of anti-xNdc80 antibodies to a Xenopus extract containing preassembled kinetochores. Sperm nuclei and nocodazole were incubated with CSF-arrested extracts for 30 min. Control IgG or anti-xNdc80 antibody was then added for 30 min. Sperm chromatin was subsequently spun onto coverslips, fixed, and processed for immunofluorescence. Chromatin was stained for xNdc80 (by adding a CY-3-conjugated sheep anti-rabbit secondary antibody, “2° only”), xCenp-A, xP150 glued (Dynactin), or xRod (red). Chromatin was visualized with Hoechst 33342 (blue). Bars: A,B, 5 μm.

Characterization of degron-tagged ndc80 and nuf2 alleles. (A) ndc80^dt and nuf2^dt mutants. Cells were synchronized in G1 with α-factor and released into the cell cycle under degron-restrictive conditions. Cells were assayed for DNA content, budding index, and degron-tagged protein levels. Tubulin (Tub2) serves as a loading control. Squares represent small budded cells, triangles represent large budded cells, and circles represent multi-budded cells. (B) ndc80^dt and nuf2^dt cells arrest with short, broken, and often misaligned spindles that are slightly longer than the metaphase spindles of cdc23-1 cells, but distinct from the long anaphase spindles of cdc15-2 cells. Spindles were measured from ndc80^dt and nuf2^dt cells fixed at 120 min after release from α-factor into the cell cycle under degron-inducing conditions. Spindle lengths were measured in cdc23-1 (n = 68), cdc15-1 (n = 69), ndc80^dt (n = 101), and nuf2^dt (n = 91) cells. Error bars indicate standard error. (C) Systematic deletion of yeast kinetochore genes. Six deletion mutants lacking central kinetochore proteins and four mutants lacking outer kinetochore proteins were analyzed for checkpoint proficiency 6 h following nocodazole treatment. A summary of the data from these experiments and those described in Gardner et al. (2001) are shown. Red shapes indicate those proteins required for spindle checkpoint activity, and green shapes indicate proteins dispensable for checkpoint function. The black line represents centromeric DNA. Inner, central, and outer kinetochore designations are defined as described (Cheeseman et al. 2002). Shapes that are touching have been shown to interact biochemically.

Injection of anti-xNdc80 and anti-xNuf2 antibodies into mitotic XTC cells induces a “cut” phenotype, defects in chromosome alignment, and diminution of kinetochore fiber microtubule bundles. (A) An XTC cell was injected with anti-xNuf2 antibody at early prometaphase, incubated for ∼60 min, fixed, immunolabeled to visualize the injected antibody, and stained with DAPI to visualize DNA. Cell cleavage resulted in a “cut” phenotype with unequally sized daughter nuclei connected by a chromatin bridge (arrow). (B) XTC cells injected with anti-xNuf2 antibody at early mitosis were incubated for 2 h in the presence of MG132, fixed, immunolabeled to visualize the injected antibody, and stained with DAPI. Many of the antibody-injected cells contained several unaligned chromosomes (arrowheads), whereas the majority of noninjected, mitotic cells (yellow arrow) in the same population had arrested with all chromosomes aligned at the metaphase plate. The inset shows a higher magnification of an anti-xNuf2-antibody-injected cell to reveal that the injected antibody concentrates at presumptive kinetochores. (C) Microinjection of anti-xNdc80 antibody also blocked chromosome alignment in cells maintained in the M phase with MG132. Labeling with anti-tubulin antibody reveals changes in spindle structure compared with an uninjected control cell on the same coverslip. Note the decrease in organized bundles of kinetochore microtubules extending from the spindle poles to the chromosomes. The inset shows the injected antibody labeled with secondary anti-rabbit IgG. Bar, 10 μm.

xNdc80 and xNuf2 are required for spindle checkpoint signaling in Xenopus egg extracts and localization of xMad1, xMad2, xBub1, xBub3, xRod, xZw10, and xP150 glued (dynactin) to the kinetochore. (A) xNdc80 and xNuf2 are required for spindle checkpoint establishment in Xenopus extracts. CSF-arrested extracts were preincubated for 30 min with control IgG, anti-xNuf2, or anti-xNdc80 antibodies. Sperm nuclei and nocodazole were added for 30 min, followed by the addition of calcium chloride. Samples were taken before (t = 0) the addition of calcium and every 15 min thereafter to monitor histone H1 kinase activity. Autoradiograms of histone H1 kinase assays as well as photographs of nuclear morphology (t = 60) are shown. (B) Anti-xNdc80 antibodies deplete both xNdc80 and xNuf2 proteins. An immunoblot of 1 μL each of Xenopus egg extract depleted with preimmune sera (Pre-I Δ) or anti-xNdc80 (xNdc80 Δ) antibodies was probed with anti-xNdc80 and anti-xNuf2 antibodies. (C) Depletion of the xNdc80 complex inhibits xMad1, xMad2, xBub1, xBub3, xRod, xZw10, and xP150 glued (dynactin) localization to kinetochores. CSF-arrested extracts were preimmune-sera-depleted or xNdc80-depleted, and sperm and nocodazole were added. Following 30 min, sperm chromatin was spun onto coverslips and fixed. Kinetochores were stained for xNdc80, xNuf2, xCenp-A, XKCM1, xP150 glued (dynactin), xRod, xZw10, xMad1, xMad2, xBub1, or xBub3 (red). Chromatin was visualized with Hoechst 33342 (blue). Representative pictures of xNdc80, xRod, xP150 glued, and xCenp-A are shown. (D) Quantification of chromatin with positive kinetochore staining. One hundred nuclei from C were scored for positive kinetochore staining of xNdc80, xNuf2, xCenp-A, XKCM1, xP150 glued (dynactin), xRod, xZw10, xMad1, xMad2, xBub1, or xBub3 from preimmune-sera- or xNdc80-depleted extracts. Black and gray bars represent preimmune sera and xNdc80 depletions, respectively. Bars: A,C, 5 μm.

ndc80-1 and nuf2-61 arrest at metaphase in an MAD2- and BUB2-dependent manner. Cells were synchronized in G1 with α-factor and released into the cell cycle at the restrictive temperature (37°C). Cells were assayed for DNA content, budding index, and Pds1 levels. Cycling cells (cyc) were also examined for Pds1 levels. Tubulin (Tub2) serves as a loading control. Wild-type (WT) cells (A), ndc80-1 and nuf2-61 mutants (B), and ndc80-1 and nuf2-61 mutants (C) containing deletions of either MAD2 or BUB2. Squares represent small budded cells, triangles represent large budded cells, and circles represent multibudded cells.