The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement

Abstract

In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.

FIGURE 1:

The CHD of Hec1/Ndc80 is essential for congression of chromosomes to the metaphase plate. (A) Scheme used to visualize the first mitotic division after endogenous Hec1/Ndc80 is replaced by exogenous Hec1/Ndc80 in synchronized HeLa cells. (B) Ribbon diagram depicting the N-terminal CHD regions of Hec1/Ndc80 (light blue, amino acids 80–285) and Nuf2 (yellow, amino acids 4–169). Lysine residues altered in mutants are shown in dark blue. The predicted exit point of a tubulin E-hook is indicated in red. Diagram was constructed based on the crystal structure of Ndc80^bonsai (Cifferi et al., 2008) bound to a microtubule (Alushin et al., 2010). (C) Representative images of the predominant mitotic figures from siHec1 knockdown and rescued cells that have been stained for Hec1/Ndc80 (green), tubulin (red), and DNA (blue). WT and CHD mutant cells are identified by an expressed EGFP, and are costained for tubulin and DNA. For mock treated, Hec1^WT, Hec1^K89E, and Hec1^K115E cells, representative metaphase images are shown. For siHec1(siOnly), Hec1^K166E, Hec1^K89E/K115E, and Hec1^K89E/K166E, prometaphase cells are shown. (D) 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 one to two unaligned chromosomes (gray), and cells with three to five unaligned chromosomes (white). N = 3; 100 cells per experiment. Error bars = SD of total metaphase cell population. (E) Cells were incubated in ice-cold medium prior to fixation and immunostaining. Ten sister kinetochores from 5–10 cells (N = 3) were scored for kinetochore-embedded microtubules, and the mean percentage was plotted. Error bars = SD.

FIGURE 2:

Hec1/Ndc80 CHD mutant phenotypes cannot be rescued by preventing Aurora phosphorylation of the Hec1/Ndc80 tail. (A) Sequence of the 80-amino-acid Hec1/Ndc80 tail region. Nine WT residues were mutated to alanine (Hec1^9A) and are indicated in red. (B) Representative images of predominant mitotic figures from siHec1 knockdown and rescued cells that have been stained for Hec1/Ndc80, tubulin, and DNA. For Hec1^WT, Hec1^9A, Hec1^9A/K89E, and Hec1^9A/K166E, metaphase images are shown. For Hec1^{9A/K89E/K166E}, only prometaphase cells are observed. (C) Rescued mitotic cells were stained for tubulin and DNA, identified by an EGFP kinetochore signal, and scored for chromosome alignment. The percentage of metaphase-aligned cells was plotted in comparison to mock transfected cells. N = 3; ≥100 cells per experiment. Metaphase cells were further subdivided to indicate cells with all chromosomes aligned (black), cells with one to two unaligned chromosomes (gray), and cells with three to five unaligned chromosomes (white). Error bars = SD of total metaphase cell population. (D) Cells were incubated in ice-cold medium prior to fixation and immunostaining with tubulin and ACA antibodies. Ten kinetochores in 5–10 cells (N = 3) were scored for percentage of kinetochores containing embedded microtubules, and the mean percentage was plotted. Error bars = SD.

FIGURE 3:

The interaction between the Hec1/Ndc80 CHD and a microtubule requires charge. (A) Representative images of the predominant phenotype of Hec1/Ndc80 CHD mutant cells. For Hec1^K89E/K166E mutant cells, only early prometaphase figures were found. Both early prometaphase and late prometaphase (shown) figures were found in Hec1^K89A/K166A mutant cells. For Hec1^K89R/K166R mutant cells, both prometaphase and metaphase (shown) figures were observed. (B) Mitotic cells were scored for chromosome alignment, and the percentage of metaphase-aligned cells was plotted. N = 3; ≥100 cells counted in each experiment. (C) Cells were incubated in ice-cold medium prior to fixation and immunostaining with tubulin and ACA antibodies. Mock treated and siHec1 knockdown cells also were stained for Hec1/Ndc80 (9G3). Ten kinetochores in 5–10 cells (N = 3) were scored for the percentage of kinetochores containing embedded microtubules, and the mean percentage was plotted. Error bars = SD.

FIGURE 4:

Positive charge in unstructured Hec1/Ndc80 tail is required for microtubule binding and chromosome congression. (A) Amino acid sequence of charge-neutral tail Hec1 mutant with mutated residues indicated in red. (B) WT and charge-neutral Hec1 tail Ndc80^bonsai were sedimented with the indicated concentrations of microtubules, and pellet samples (N = 3) were subjected to Western blotting with anti-hSpc25 antibody. Signal intensities were quantified, and the mean percentage of Ndc80^bonsai bound at each microtubule concentration was plotted on a log and linear scale (inset). For representative primary data, see Supplemental Figure 7. Error bars = SD. (C) Representative image of a knocked down cell that has been rescued with Hec1^NEU. The cell is stained for Hec1/Ndc80 (green), tubulin (red), and DNA (blue). In this condition only early prometaphase figures are found. (D) Mitotic cells were scored for kinetochore alignment (N ≥ 100), and the percentage of mitotic cells with aligned chromosomes was plotted. (E) Ten kinetochores from five or more cells (N > 50) were scored for kinetochore-associated microtubules (N = 2), and the mean percentage in early prometaphase, late prometaphase, and metaphase was plotted. (F) Ten sister kinetochores in at least five cells (N > 50) were identified by ACA staining between Hec1/Ndc80 signals, and the distance between those sister kinetochores was measured (N = 3). The mean distance is plotted for early prometaphase and metaphase cells. Error bars = SD.

FIGURE 5:

Model for vertebrate Hec1/Ndc80’s interaction with a microtubule. (A) The unstructured tail of Hec1/Ndc80 (dark blue line) binds tubulin tails (red lines) and also acts to pack adjacent Ndc80 complexes on the surface of a microtubule. Lys-166 of the Hec1/Ndc80 CHD is a critical residue in the toe region of the protein, whereas tubulin E-hooks also interact with Lys-89 and Lys-115 of the Hec1/Ndc80 CHD (Alushin et al., 2010). (B) Alternatively, the tail may function primarily to tether the Ndc80 complex to microtubules during periods when CHD binding is lost. The models depicted in (A) and (B) need not be mutually exclusive and could be regulated by Aurora phosphorylation of the Hec1/Ndc80 tail. For simplicity, only tubulin tails involved in binding are depicted.