The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis

Abstract

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the "tail." Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore-microtubule attachment in distinct ways. The most severe defects in kinetochore-microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore-microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.

FIGURE 1:

Stable kinetochore–microtubule attachment depends on the charged face of the Hec1 CH domain. (A) Immunofluorescence images showing HeLa cells depleted of Hec1 and rescued with mutant versions. The upper panels show a control cell and a cell depleted of endogenous Hec1. The lower panels show cells depleted of endogenous Hec1 and rescued with either WT Hec1 or a Hec1 mutant fused to GFP. (B) Quantification of chromosome alignment phenotypes in cells depleted of Hec1 or Nuf2 and cells rescued with Hec1 or Nuf2 GFP fusions. Cells with mostly aligned chromosomes (red) exhibited <5 chromosomes off of a well-formed metaphase plate, cells with partially aligned chromosomes (gray) exhibited 5–10 chromosomes off of a metaphase plate, and cells with mostly unaligned chromosomes (black) exhibited either no chromosome alignment or >10 chromosomes off of a metaphase plate. For each condition, at least 90 cells were scored. (C) Images of cells subjected to a cold-induced microtubule-depolymerization assay and immunostained with tubulin and ACA antibodies (recognizing CENP-A, -B, and -C). Cells depleted of Hec1 and cells depleted of Hec1 and rescued with either WT or mutant GFP fusions are shown. (D) Quantification of microtubule fluorescence intensity after cold-induced microtubule depolymerization. For each condition, spindles from at least 10 cells were measured.

FIGURE 2:

Cells rescued with a Hec1 CH domain charge reversal mutant arrest in mitosis with unaligned chromosomes. (A) Still images from time-lapse acquisitions of HeLa cells depleted of endogenous Hec1 and rescued with either WT Hec1-GFP or Hec1 mutants fused to GFP. Time is shown in minutes, and anaphase onset (AO) is indicated for the WT Hec1-GFP-rescued cell that entered anaphase. The DIC image shows the time point corresponding to the final mCherry image. (B) Quantification of metaphase plate formation during live cell imaging. Bar graph represents the percent of cells that attained metaphase alignment at some point during time-lapse imaging. (C) Quantification of anaphase entry during live cell imaging. Bar graph represents the percent of cells that entered anaphase during time-lapse imaging. All cells were filmed for 5 h except those rescued with WT Hec1, which were filmed for 2 h. Error bars in (B) and (C) represent SD across at least 2 independent experiments. The n values for each experiment are as follows: WT Hec1, 12 cells; Hec1^K166D, 18 cells; 9D Hec1, 17 cells.

FIGURE 3:

The charged face of the Nuf2 CH domain is not required for the formation of stable kinetochore–microtubule attachments. (A) Immunofluorescence images showing HeLa cells depleted of Nuf2 and rescued with various mutant versions. The upper panels show a control cell and a cell depleted of endogenous Nuf2 stained with Hec1 and tubulin antibodies. The lower panels show cells depleted of endogenous Nuf2 and rescued with either WT Nuf2 or a Nuf2 mutant fused to GFP. For the 2D Nuf2 mutant, two Lys-to-Asp substitutions were made at amino acid positions 33 and 41; the 3D Nuf2 mutant contains three Lys-to-Asp substitutions at amino acid positions 33, 41, and 115. (B) Images of cells subjected to a cold-induced microtubule-depolymerization assay and immunostained with tubulin antibodies, DAPI, and, in Nuf2-depleted cells, Hec1 antibodies. Cells depleted of Nuf2 alone and cells depleted of Nuf2 and rescued with either WT or mutant GFP fusions are shown. (C) Quantification of microtubule fluorescence intensity after cold-induced microtubule depolymerization. For each condition, spindles from at least 10 cells were measured. (D) Quantification of interkinetochore distances, which were measured from GFP-centroid to GFP-centroid in cells rescued with Nuf2-GFP fusion proteins. In cells depleted of Nuf2, interkinetochore distances were measured from ACA-centroid to ACA-centroid. For cells depleted of endogenous Nuf2 and rescued with GFP-fusions, kinetochores from bioriented chromosomes were measured.

FIGURE 4:

Cells rescued with Nuf2 CH domain charge reversal mutants arrest in mitosis with at least partially aligned chromosomes. (A) Still images from time-lapse acquisitions of HeLa cells depleted of endogenous Nuf2 and rescued with either WT Nuf2-GFP or Nuf2 mutants fused to GFP. Time is shown in minutes, and anaphase onset (AO) is indicated for the cell expressing WT Nuf2-GFP that entered anaphase. The DIC image shows the time-point corresponding to the final mCherry image. (B) Quantification of metaphase plate formation during live cell imaging. Bar graph represents the percent of cells that attained metaphase alignment at some point during time-lapse imaging. (C) Quantification of anaphase entry during live cell imaging. Bar graph represents the percent of cells that entered anaphase during time-lapse imaging. All cells were filmed for 5 h, except those rescued with WT Nuf2, which were filmed for 2 h. Error bars in (B) and (C) represent SD across at least 2 independent experiments. The n values for each experiment are as follows: WT Nuf2, 10 cells; Δ1–142 Nuf2, 8 cells; Nuf2^K41D, 19 cells; Nuf2^K33D, 15 cells; Nuf2^K115D, 12 cells; 2D Nuf2, 25 cells; 3D Nuf2, 34 cells.

FIGURE 5:

Charge reversal mutations within the Nuf2 CH domain do not affect recruitment of outer kinetochore proteins. (A) Images showing single kinetochore pairs from control cells, Nuf2-depleted cells, cells rescued with WT Nuf2-GFP, and cells rescued with 3D Nuf2-GFP. For each series, the first panel shows immunostaining for the antibody listed on the far left. ACA staining is shown as well a merge of ACA staining with each test antibody. (B) Chart summarizing immunofluorescence data shown in (A).

FIGURE 6:

Expression of a nonphosphorylatable Hec1 tail domain overcomes the mitotic arrest observed in cells rescued with a Nuf2 CH domain charge reversal mutant, but not a Hec1 CH domain charge reversal mutant. (A) Still images from time-lapse acquisitions of HeLa cells depleted of endogenous Hec1 and rescued with either 9A Hec1-GFP or 9A Hec1^K166D-GFP. The DIC image shows the time point corresponding to the final mCherry image. (B) Still images from time-lapse acquisitions of HeLa cells depleted of endogenous Nuf2 and Hec1 using siRNAs targeted to both Nuf2 and Hec1 genes, and rescued with either WT Nuf2-GFP or a mutant Nuf2-GFP containing Lys-to-Asp mutations in the CH domain, and either WT Hec1- or 9A Hec1-GFP. Time is shown in minutes, and anaphase onset (AO) is indicated for cells that enter anaphase in (A) and (B). (C) Quantification of anaphase entry during live cell imaging. Bar graph represents the percent of cells that entered anaphase during time-lapse imaging. All cells were filmed for 5 h. Error bars represent SD across at least 2 independent experiments. The n values for each experiment are as follows: 9A Hec1, 20 cells; 9A Hec1^K166D, 18 cells; WT Nuf2/9A Hec1, 28 cells; Nuf2^K115D/ WT Hec1, 20 cells; Nuf2^K115D/9A Hec1, 19 cells; 3D Nuf2/WT Hec1, 29 cells; 3D Nuf2/9A Hec1, 39 cells.