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How the kinetochore couples microtubule force and centromere stretch to move chromosomes

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ABSTRACT

The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes.

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The tension at Ndc80 MTBDs is dependent on Dam1 drag force(a) Computer simulations of mechanical model for metaphase budding yeast for the condition when Dam1 drag force was increased 10-fold over control. Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles. (b) Representative Ndc80 FRET, mECFP, and mYpet images for a control cell and a dam1-765 cell (left). The average Emission Ratio at metaphase for control (2.12 ± 0.54, n = 117) and dam1-765 cells (3.20 ± 0.88, n = 80) (right). n values represent number of kinetochores clusters. *** Unpaired Student t-test (two-tailed), p < 0.01. Error bars are SD from the means. Scale bars are 5 μm. The mean values were calculated using data pooled from 2 independent experiments. The mean values of Emission Ratio, FRET efficiency, K-K distance are listed in Supplementary Table 1.
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Figure 6: The tension at Ndc80 MTBDs is dependent on Dam1 drag force(a) Computer simulations of mechanical model for metaphase budding yeast for the condition when Dam1 drag force was increased 10-fold over control. Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles. (b) Representative Ndc80 FRET, mECFP, and mYpet images for a control cell and a dam1-765 cell (left). The average Emission Ratio at metaphase for control (2.12 ± 0.54, n = 117) and dam1-765 cells (3.20 ± 0.88, n = 80) (right). n values represent number of kinetochores clusters. *** Unpaired Student t-test (two-tailed), p < 0.01. Error bars are SD from the means. Scale bars are 5 μm. The mean values were calculated using data pooled from 2 independent experiments. The mean values of Emission Ratio, FRET efficiency, K-K distance are listed in Supplementary Table 1.

Mentions: The above data suggest that the distribution of force within the kinetochore might be shifted depending on the relative affinities of MTBDs of individual sub-complexes. To test this hypothesis, we increased the Dam1 drag coefficient (10-fold over control) in the mechanical model without changing other parameters. Surprisingly, Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles (Figs. 6a and 5b). To ascertain whether an in vivo condition recapitulates the simulation, we measured Ndc80 tension by the FRET biosensor in dam1-765 mutants. Dam1-765 was isolated based on its lethality with defects in binding of MT minus-ends to spindle poles42. These mutants achieved metaphase with kinetochores closer to their spindle poles compared to controls and then exhibited normal anaphase segregation, (Supplementary Figs. 6c–d)42. In addition, mean kinetochore tension at metaphase was higher than for controls because centromere-linked LacO/LacI-GFP markers were significantly more stretched apart (Supplementary Fig. 6e). In contrast, the mean Emission Ratio from the Ndc80 FRET sensor for the dam1-765 cells was significantly higher compared to controls (Fig. 6b). These in vivo results and in silico predictions indicate that the average kinetochore force at metaphase depends critically on the relative binding affinities of individual kinetochore complexes. In addition, kinetochores that remain bound to their kMTs can become uncoupled from polymerizing MT ends at metaphase (Supplementary Fig. 7a and Video 4).


How the kinetochore couples microtubule force and centromere stretch to move chromosomes
The tension at Ndc80 MTBDs is dependent on Dam1 drag force(a) Computer simulations of mechanical model for metaphase budding yeast for the condition when Dam1 drag force was increased 10-fold over control. Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles. (b) Representative Ndc80 FRET, mECFP, and mYpet images for a control cell and a dam1-765 cell (left). The average Emission Ratio at metaphase for control (2.12 ± 0.54, n = 117) and dam1-765 cells (3.20 ± 0.88, n = 80) (right). n values represent number of kinetochores clusters. *** Unpaired Student t-test (two-tailed), p < 0.01. Error bars are SD from the means. Scale bars are 5 μm. The mean values were calculated using data pooled from 2 independent experiments. The mean values of Emission Ratio, FRET efficiency, K-K distance are listed in Supplementary Table 1.
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Figure 6: The tension at Ndc80 MTBDs is dependent on Dam1 drag force(a) Computer simulations of mechanical model for metaphase budding yeast for the condition when Dam1 drag force was increased 10-fold over control. Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles. (b) Representative Ndc80 FRET, mECFP, and mYpet images for a control cell and a dam1-765 cell (left). The average Emission Ratio at metaphase for control (2.12 ± 0.54, n = 117) and dam1-765 cells (3.20 ± 0.88, n = 80) (right). n values represent number of kinetochores clusters. *** Unpaired Student t-test (two-tailed), p < 0.01. Error bars are SD from the means. Scale bars are 5 μm. The mean values were calculated using data pooled from 2 independent experiments. The mean values of Emission Ratio, FRET efficiency, K-K distance are listed in Supplementary Table 1.
Mentions: The above data suggest that the distribution of force within the kinetochore might be shifted depending on the relative affinities of MTBDs of individual sub-complexes. To test this hypothesis, we increased the Dam1 drag coefficient (10-fold over control) in the mechanical model without changing other parameters. Surprisingly, Ndc80 tension was significantly reduced and the mean length of kMTs became longer than the mean distance of kinetochores to their poles (Figs. 6a and 5b). To ascertain whether an in vivo condition recapitulates the simulation, we measured Ndc80 tension by the FRET biosensor in dam1-765 mutants. Dam1-765 was isolated based on its lethality with defects in binding of MT minus-ends to spindle poles42. These mutants achieved metaphase with kinetochores closer to their spindle poles compared to controls and then exhibited normal anaphase segregation, (Supplementary Figs. 6c–d)42. In addition, mean kinetochore tension at metaphase was higher than for controls because centromere-linked LacO/LacI-GFP markers were significantly more stretched apart (Supplementary Fig. 6e). In contrast, the mean Emission Ratio from the Ndc80 FRET sensor for the dam1-765 cells was significantly higher compared to controls (Fig. 6b). These in vivo results and in silico predictions indicate that the average kinetochore force at metaphase depends critically on the relative binding affinities of individual kinetochore complexes. In addition, kinetochores that remain bound to their kMTs can become uncoupled from polymerizing MT ends at metaphase (Supplementary Fig. 7a and Video 4).

View Article: PubMed Central - PubMed

ABSTRACT

The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes.

No MeSH data available.


Related in: MedlinePlus