Mechanism of Ska Recruitment by Ndc80 Complexes to Kinetochores

Abstract

Yeast use the ring-shaped Dam1 complex to slide down depolymerizing microtubules to move chromosomes, but current models suggest that other eukaryotes do not have a sliding ring. We visualized Ndc80 and Ska complexes on microtubules by electron microscopic tomography to identify the structure of the human kinetochore-microtubule attachment. Ndc80 recruits the Ska complex so that the V shape of the Ska dimer interacts along protofilaments. We identify a mutant of the Ndc80 tail that is deficient in Ska recruitment to kinetochores and in orienting Ska along protofilaments in vitro. This mutant Ndc80 binds microtubules with normal affinity but is deficient in clustering along protofilaments. We propose that Ska is recruited to kinetochores by clusters of Ndc80 proteins and that our structure of Ndc80 and Ska complexes on microtubules suggests a mechanism for metazoan kinetochores to couple the depolymerization of microtubules to power the movement of chromosomes.

Keywords: Hec1; Ndc80 complex; Ska complex; cell division; kinetochore; microtubule; mitosis; mitotic spindle.

Figure 1. Ndc80 recruits Ska complex to microtubule protofilament

(A) Representative slices (thickness: 0.4427 nm) of tomographic reconstructions of taxol-stabilized microtubules (MT), microtubules decorated by Ndc80^Bonsai WT (red, Movie S1), 1x Ska complex (purple), Ndc80^Bonsai WT and 1x Ska complexes (green, 6 images), or saturating concentration of Ska (4x Ska, cyan). White arrowheads indicate positions of the tips of the V-shapes. Scale bar = 10 nm. (B) Model of the Ska complex based on structures of the Ska complex core (PDB: 4AJ5) and Ska1 CTD (C-terminal domain = MTBD, microtubule binding domain, PDB: 4CA0). Comparison between size of the Ska core complex dimer from crystal structure and average measured size of the V-shapes identified in tomographic reconstructions of microtubules incubated with both Ndc80^Bonsai WT and Ska is shown in the table below (mean ± SD). w = width; h = height; n = number of V-shapes measured. (C) Structure of Ska core complex (PDB: 4AJ5) fitted to EM map of representative V-shape. Thickness: 2.6568 nm. (D) Quantification of the V-shaped densities on microtubules identified in tomographic reconstructions (mean ± SD, average of 7 microtubules scored; WT = Ndc80^Bonsai WT; +4CT = Ndc80^Bonsai +4CT; * - p < 0.0002; Student’s t-test) (E) Distance (D, red) between centroid of tip of V-shape (dashed line circle) and line connecting centroids of tubulin subunits within a single protofilament (dashed straight line) was measured and represented in panel E. (F) Box and whisker plot of distribution of distances between tips of V-shapes and microtubule protofilaments measured as shown in panel D (Central line – median, Whiskers represent 10 – 90% range; n = 58, 18, 22, respectively. * - p < 0.000001; N/S – p > 0.99; Student’s t-test)

Figure 2. Ndc80 interacts with Ska3 in both prometaphase and metaphase

(A) Representative images of Ska3-Ndc80 Proximity Ligation Assay (red), with additional immunostaining of Borealin (cyan) and tubulin (green). Top – prometaphase cells, middle – metaphase cells, bottom – control metaphase cells. (B) Centromeres in prometaphase or metaphase cells, as identified by Borealin immunostaining, were scored based on the proximity to Ska3-Ndc80 PLA signal (mean ± SD; N > 100 centromeres from four cells). Cells with only Ska3 probe were used as a control. N/S – p > 0.05, * - p < 0.05, *** - p < 0.0001. (Student’s t-test) (C) Centromeres in prometaphase or metaphase cells, as identified by Borealin immunostaining, scored based on the number of proximal Ska3-Ndc80 PLA signals (mean ± SD; N > 100 centromeres from four cells). N/S - p > 0.05, * - p < 0.05, *** – p < 0.0001. (Student’s t-test)

Figure 3. Ndc80⁺ mutant is deficient in recruitment of Ska3 to the kinetochore

(A) Left: Purple-Localization of electron density patch attributed to the C-terminal region of Ndc80 tail. Note that it sits between two adjacent calponin homology domains (CHD) (Adapted from Alushin, et al., 2012). Right: Electrostatic surface potential of two Ndc80 CHD bound to tubulin dimer with the tail densities removed bound to a microtubule (PDB: 3IZ0) to illustrate the negatively charged surface between the Ndc80 CHD molecules. Black ellipse represents the localization of the purple patch visualized on the left panel. We mutated positively charged residues in the C-terminal region of the patch assuming that they bridged the negatively charged surfaces on the Ndc80 CHD to generate cooperative binding. (B) Cartoon depicting the wild type Ndc80, Ndc80⁺ and Ndc80⁺ tail mutants to illustrate the change in charge that are created by the K/R to A changes in the Ndc80⁺ and Ndc80⁺ proteins. Although, it is likely that we are also affecting Aurora B phosphorylation sites since they are defined by K/RxS/T amino acids, we do not observe significant differences in in vitro phosphorylation by Aurora B between mutants and wild type protein (Fig. S3A–C). CHD = Calponin Homology Domain, CCD = coiled-coil domain. Drawing not to scale. (C) Immunofluorescence staining of Ndc80^WT and Ndc80^+4CT stable cell lines after depletion of endogenous Ndc80 shows reduced levels of Ska3, on the kinetochores of Ndc80^+4CT stable cell lines. Scale bar = 5 µm. (D) Box and whisker plots representing the quantification of Ska staining intensities on kinetochores represented in (C) (N > 100 kinetochores from at least 5 cells, Whiskers – 5–95% percentile). a.u. = arbitrary units. * - p < 0.0001 (Unpaired t-test with Welch’s correction). (E) Immunofluorescence staining of Ndc80^WT and Ndc80^+4CT stable cell lines treated with 3.3 µM nocodazole (Noc) and 2 µM ZM447439 (ZM) after depletion of endogenous Ndc80 shows reduced levels of Ska3 (red) on kinetochores. Scale bar = 5 µm. (F) Box and whisker plot representing the quantification of the Ska staining intensities on kinetochores in cells treated with nocodazole and ZM447439 represented in (E) (N > 100 kinetochores from at least 5 cells, Whiskers – 5–95% percentile). a.u. = arbitrary units. * - p < 0.0001 (Unpaired t-test with Welch’s correction).

Figure 4. Ndc80^+4CT tail mutant metaphase arrest does not satisfy spindle assembly checkpoint and is Aurora dependent

(A) Graph representing chromosome alignment among mitotic cells expressing Ndc80 mutants. Mitotic cells were scored for chromosome alignment into a metaphase plate, and the percentage of metaphase cells was plotted. Metaphase cells were further subdivided to indicate cells with all chromosomes aligned (black), cells with 1–2 unaligned chromosomes (gray) and cells with 3–5 unaligned chromosomes (white). Mitotic cells with 6 or more unaligned chromosomes are not shown in the graph. N > 100 mitotic cells counted per experiment, N = 3. Data is represented as mean ± SD (SD represents total of cells with 0–5 unaligned chromosomes). (B) Graph representing interkinetochore distance measurements in cells expressing Ndc80 mutants. Ten sister kinetochores in at least five cells (N > 50) were identified by ACA staining between Ndc80 signals, and the distance between those sister kinetochores was measured (N=3). The distance (mean ± SD) is plotted for early prometaphase (EPM) (gray), late prometaphase (LPM) (dark gray), and metaphase (black) cells, Noc = nocodazole-treated cells (white). * - p < 0.0002. N/S - p > 0.2. (Student’s t-test) (C) The percentage of mitotic cells in anaphase was plotted for Ndc80^WT, Ndc80 knockdown and indicated Ndc80 tail mutant cells (mean ± SD). For the Ndc80^+4CT mutant, cells were also co-transfected with siRNA targeting Mad2. For Ndc80 tail mutants, one hundred mitotic cells counted per experiment (n = 3). For Mad2 co-transfection with Ndc80^+4CT, twenty-five cells were counted. (D) Immunofluorescence staining of Ndc80^WT and Ndc80^+4CT (green) stable lines after depletion of endogenous Ndc80. Ndc80^+4CT cells arrest with at least one Mad2 positive kinetochore (red, upper row) with no loss of BubR1 localization (red, lower row). Scale bar = 5 µm.

Figure 5. Ndc80^+4CT tail mutant is deficient in clustering on microtubules

(A) Fluorescence anisotropy measurements of fluorescently labeled Ndc80^Bonsai WT (left panel, orange) or Ndc80^Bonsai +4CT (right panel, dark green) incubated with increasing concentrations of taxol-stabilized microtubules, plotted on log₁₀ scale (mean ± SD). Hill equation was used for fitment of the data. Small graphs represent the data in the linear scale. N = 3. (B) Representative projections of 5 consecutive Z-sections (1.107 nm) of the tomographic reconstructions show Ndc80^Bonsai +4CT form smaller clusters than Ndc80^Bonsai WT. Black lines indicate the positions of the Ndc80 molecules. (C) Quantification of cluster sizes represented in (B). N, number of quantified microtubules, n, total number of Ndc80^Bonsai molecules quantified.

Figure 6. Schematic models summarizing potential modes of mature kinetochore-microtubule attachment

“Sliding foot” model – Ska complex is localized and oriented along protofilament of the depolymerizing microtubule end by clusters of Ndc80. This mechanism likely allows for utilization of energy released by curving protofilaments. “Molecular lawn” model – current model for passive binding of multiple individual Ndc80 molecules to depolymerizing microtubule end.