# Human kinetochores are swivel joints that mediate microtubule attachments

# Human kinetochores are swivel joints that mediate microtubule attachments

- Chris A Smith
- Andrew D McAinsh
- Nigel J Burroughs

University of Warwick, United Kingdom

Published September 3, 2016

Cite as eLife 2016;5:e16159

Download figureOpen in new tabFigure 1. 3D dual colour kinetochore tracking assay demonstrates rigid intra-kinetochore structure.(**A**) Schematic of imaging setup for tracking of fluorescently marked inner kinetochore (green circles) and outer kinetochore domains (red circles) within a 12 µm z-stack to measure intra-kinetochore distance, Δ, in HeLa-K cells. Schematic showing approximate architecture of the mammalian kinetochore including CENP-A nucleosomes (grey circle), CCAN (pink), MIS12 complex (dark blue) and the NDC80 complex (light blue with the Ndc80 subunit highlighted in grey). (**B**) Live cell imaging of eGFP-CENP-A and Ndc80-tagRFP in microscope *x’z’* plane demonstrates imaging of tagRFP in the central 3 µm of the *z'*-stack. Images in microscope *x’y’* plane demonstrate Gaussian-fitted spot centres. Schematic shows metaphase plate coordinate system, [*x,y,z] *(*x* is normal to the metaphase plate, *y* is the line intersection of the metaphase plate and the *x'y'* plane, and *z* is orthogonal to both to make a right-handed coordinate system), and measurements of intra- (Δ) and inter- (*d*) kinetochore distances. Scale bars 500 nm. (**C**) Trajectory of a kinetochore pair’s eGFP-CENP-A (green lines) and Ndc80-tagRFP signals (red lines) in *x*, with measurements of Δ 3D for each sister, where dashed lines represent time points removed in quality control (see Materials and methods). Regions of poleward (P) and away-from-the-pole (AP) movement are labelled. (**D**) Distribution of measurements of Δ in 3D (blue; n = 4291), and 1D measurement of Δ (gold; n = 1002) used in previous studies. (**E**) Distribution of measurements of Δ 3D in AP- (blue; n = 503) and P-moving kinetochores (gold; n = 458), demonstrating no significant difference (p = 0.211). Values given in **D** and **E** are medians ± standard error. (**F**) 2D histogram of Δ 3D against *d*, demonstrating no correlation (r = –0.019, p = 0.214; n = 4291).

**DOI:** http://dx.doi.org/10.7554/eLife.16159.002

Download figureOpen in new tabFigure 1—figure supplement 1. Inter- and intra-kinetochore distances during directed chromosome motion.(**A**) Distributions of inter-kinetochore distance, d, for kinetochores expressing no (blue; n = 155,100), low (gold; n = 155,015) and high levels (green; n = 154,626) of Ndc80-tagRFP. Values given are medians ± standard error. (**B**) Autocorrelation in kinetochore *x*-displacement between consecutive time points for kinetochores expressing no (blue), low (gold) and high levels (green) of Ndc80-tagRFP. A and B demonstrate no significant perturbation to kinetochore dynamics when expressing exogenous Ndc80-tagRFP. (**C**) Distributions of Δ 2D for kinetochores moving poleward (P; gold; n = 335) or away-from-the-pole (AP; blue; n = 362), demonstrating no significant difference (p = 0.589). Values given are medians ± standard error.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.004

Download figureOpen in new tabFigure 1—figure supplement 2. Intra-kinetochore distances with and without microtubule attachment.Schematic of the 3D mammalian kinetochore with (top) and without (bottom) attachment to a kinetochore-microtubule (kMT). The difference between endogenous CENP-A and eGFP-CENP-A has been attributed to limitations of antibody accessibility to CENP-A that is deep in centromeric chromatin (Magidson et al., 2016). As previously found (Suzuki et al., 2015) the majority of CENP-C amino-termini are 'free' and in close proximity of the centromere, rather than directly bound to the 20 nm long MIS12 complex (Petrovic et al., 2014). The orientation of the NDC80 complex is suggested based on the 60 nm length of the complex and the flexibility in the hinge region (Wei et al., 2005; Wang et al., 2008). Bub3 binds to kinetochores through the phospho-MELT repeats in KNL1 suggesting that this protein extends beyond the NDC80 complex – at least during metaphase.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.005

Download figureOpen in new tabFigure 1—figure supplement 3. The experimental setup to measure intra-kinetochore distance from single time point *z*-stacks.Live cell imaging of eGFP-CENP-A and Nnf1-Alexa594 in microscope *x’z’* plane demonstrates imaging of both eGFP and Alexa594 across the entire *z'*-stack. Images in microscope *x’y’* plane demonstrate Gaussian-fitted spot centres. Schematic shows measurements of intra- (Δ) and inter- (*d*) kinetochore distances. Scale bars 500 nm.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.006

Download figureOpen in new tabFigure 1—figure supplement 4. Chromatic shift correction precision and accuracy in the dynamic live assay to analyse intra-kinetochore measurements.(**A**) Histograms representing the compilation of all measurements of *x*- ($\zeta}_{x$; light blue), *y*- ($\zeta}_{y$; purple), and *z*-directional ($\zeta}_{z$; pink) chromatic shift, defined as the distance between the diffraction-limited spot centres of the two fluorophores, in cells expressing eGFP-CENP-A and mCherry-CENP-A, for 18 imaging sessions. Each distribution of measurements of chromatic shift for a given imaging session was shifted by its median ($\overline{\zeta}}_{x$, *i.e.* the measured chromatic shift for that imaging session) to centre it at zero. Values given are means of the standard deviations for each of these distributions (n = 18). (**B**) Schematic demonstrating the expected orientation of a kinetochore’s inner (green circles) and outer domains (red circles) relative to its spindle pole (black crossed circles) in both the *xy*- (top) and *xz*-plane (bottom). Blue lines represent the k-fibres which bind the kinetochores and spindle poles. Thin dashed red circles represent examples of biological variance in outer domain localisation, thick red circles represent the expected outer domain localisation. Dark blue arrows represent measurements of *x*- (Δ_{x}), *y*- (Δ_{y}) and *z*-directional (Δ_{z}) intra-kinetochore distance. (**C**) Histograms of measurements of *x*- (Δ_{x}; light blue), *y*- (Δ_{y}; purple) and *z*-directional (Δ_{z}; pink) intra-kinetochore distance from dynamic live movies of cells expressing eGFP-CENP-A and Ndc80-tagRFP. Values given are medians ± standard error, which are close to zero as expected from the schematic in (**B**).

**DOI:** http://dx.doi.org/10.7554/eLife.16159.007

Download figureOpen in new tabFigure 2. Nocodazole treatment marginally decreases 3D intra-kinetochore distance.(**A**) Live cell imaging of eGFP-CENP-A and Ndc80-tagRFP after 3 µM nocodazole treatment (600 nm *z*-projection). Representative images of a kinetochore pair demonstrating Gaussian-fitted spot centres. Schematic of the kinetochore pair shows measurements of intra-, Δ, and inter-kinetochore distances, *d*. Scale bars 500 nm. (**B**) Distribution of measurements of inter-sister distance *d* in 3D (left panel) and 2D (right panel), in untreated (blue; n = 1002) and 3 µM nocodazole treated cells (gold; n = 138); both are significantly different (p < 10^{–4}). (**C**) Distribution of measurements of intra-kinetochore distance Δ in 3D (left panel) and 1D (right panel), in untreated (blue; n_{3D} = 4291, n_{1D} = 1002) and 3 µM nocodazole-treated cells (gold; n_{3D} = 649, n_{1D} = 138); both are significantly different to 95% confidence. In (**B**) and (**C**), schematics illustrate measurement method, statistical tests were Mann-Whitney U tests, and values given are medians ± standard error. (**D**) Example of a kinetochore pair demonstrating rotation of the kinetochore axis relative to the sister-sister axis. Schematic illustrates how 1D and 3D measurements of Δ are made, and the resulting underestimate of Δ 1D. Scale bars 500 nm.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.008

Figure 2—source data 1.Swivel increases under nocodazole treatment for multiple kinetochore markers.Table of measurements of Δ 1D, Δ 2D, $\vartheta$ 3D-swivel and standard deviation of $\vartheta$* y*-swivel distribution for all kinetochore markers imaged in untreated and 3 µM nocodazole-treated cells. n values correspond to all measurements in columns to the left of the n-value column. Values given are medians ± standard error.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.009

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Download figureOpen in new tabFigure 2—figure supplement 1. Quantification of spindle depolymerisation following nocodazole treatment.(**A**) Images of HeLa-K cells expressing mCherry-Ndc80 after paraformaldehyde fixation and staining of CENP-C with AlexaFluor-488 and αTubulin with AlexaFluor-647, in untreated cells and cells treated with 3 µM nocodazole for 1 hr or 2 hr (top to bottom, respectively). Each is a projection in *z* of 2 µm. Scale bars 2 µm. (**B**) Quantification of αTubulin intensity in untreated (n = 115), 1 hr nocodazole (n = 21) and 2 hr nocodazole (n = 87) treated cells (left to right, respectively), demonstrating the requirement for 2 hr incubation with nocodazole to ensure loss of the mitotic spindle. Horizontal black lines represent statistical tests between samples, where *** indicates significance with at least 99.95% confidence.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.010

Download figureOpen in new tabFigure 2—figure supplement 2. Effect of Taxol on inter- and intra-kinetochore distance and swivel.Distributions of inter-kinetochore distance (*d*; top row) in 3D (left) and 2D (right), intra-kinetochore distance (Δ; middle row) in 3D (left) and 1D (right), and intra-kinetochore swivel ($\vartheta$; bottom row) in 3D (left) and within the *xy*-plane ($\vartheta$*y*-swivel, right) for untreated cells compared with 10 µM taxol. Values given are medians ± standard error, except for $\vartheta$* y*-swivel where distribution standard deviations are given. All medians are statistically different between treated and untreated (p < 10^{–4}), except for Δ 3D (p = 0.302).

**DOI:** http://dx.doi.org/10.7554/eLife.16159.011

Download figureOpen in new tabFigure 2—figure supplement 3. Kinetochore swivel is also present between other pairs of kinetochore markers.Example images of other pairs of kinetochore proteins tagged by fluorescent markers with visible swivel, including (clockwise from top left): CENP-A-Alexa488 and Ndc80-tagRFP; eGFP-CENP-A and Nnf1-Alexa594; Bub3-eGFP and mCherry-CENP-A; GFP-CENP-O and Ndc80-tagRFP; GFP-CENP-C and Ndc80-tagRFP; GFP-CENP-C and mCherry-Ndc80; CENP-C-Alexa488 and mCherry-Ndc80; and eGFP-CENP-A and mCherry-Mis12. Green dashed lines are the sister-sister axes, white dashed lines are intra-kinetochore axes. Scale bars 500 nm.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.012

Download figureOpen in new tabFigure 3. Outer kinetochore components are capable of ‘swivel’ about the inner kinetochore, which increases upon microtubule depolymerisation.(**A**) Example images of kinetochore pairs in untreated (top) and 3 µM nocodazole (bottom) cells exhibiting swivel in each of its kinetochores. Green and white dashed lines represent sister-sister and intra-kinetochore axes, respectively. Associated schematic shows measurements of swivel ($\vartheta$ swivel, between the sister-sister and marker-marker axes), and twist ($\vartheta$ twist, between the sister-sister axis and the metaphase plate). Stated values given are $\vartheta$*y*-swivel at each kinetochore. Scale bars 500 nm. (**B**) Distributions of $\vartheta$ swivel in 3D (histograms in top row) and projected on the *y*-axis (rose plots in bottom row), in untreated (blue; n = 4291) cells compared to 3 µM nocodazole (gold; n = 649), each significantly different to untreated (p < 10^{–4}). Values given are medians ± standard error in $\vartheta$ swivel, and standard deviation, σ, for $\vartheta$*y*-swivel. Statistical tests were Mann-Whitney U for $\vartheta$ swivel, and F test for $\vartheta$*y*-swivel. (**C**) A kinetochore pair in untreated cells exhibiting temporally-changing swivel. Time is given at the top right of each frame. Values given are $\vartheta$*y*-swivel at each kinetochore for each time point. Scale bar 500 nm.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.013

Download figureOpen in new tabFigure 3—figure supplement 1. Measurement of axial swivel, and characterisation of 1D delta’s underestimate of 3D delta.(**A**) Schematic of a kinetochore’s inner (CENP-A; green circles) and outer domain (Ndc80; red circles) markers relative to its sister in the *xy*-plane. Green and black dashed lines represent sister-sister and marker-marker axes, respectively. Measurements shown are the inter-kinetochore distance (d_{y}; black), intra-kinetochore distance (Δ_{y,1}; light blue), and a third distance measurement, ε_{y,1}(defined as the distance between a kinetochore’s outer domain marker and its sister’s inner domain marker; purple), and kinetochore swivel ($\vartheta}_{\text{y},1$; dark blue). (**B**) The triangle formation used to measure axial swivel of a kinetochore. (**C**) Schematic of a kinetochore pair, as in (**A**), however both inner-inter-kinetochore (d_{y,1}; between inner domain markers) and outer-inter-kinetochore (d_{y,2}; between outer domain markers) are shown. (**D**) The triangle formation used to calculate the underestimate of 3D intra-kinetochore distance using 1D measurements.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.014

Download figureOpen in new tabFigure 4. Spatial and temporal control of kinetochore swivel.(**A**) Kinetochore pairs in centre (centre) and periphery (right) from a metaphase plate (left) showing Gaussian-fitted spot centres, $\vartheta$*y*-swivel, sister-sister axis and marker-marker axis. (**B**) 2D histogram of $\vartheta$*y*-swivel vs. spindle position in *y* (n = 4291). White dashed lines divide spindle into the central and peripheral regions, separated at 4 µm. White curve is best-fit degree-3 polynomial, with coefficient of determination, R^{2} = 0.97. (**C**) Pairs of sister kinetochores expressing eGFP-CENP-A, Ndc80-tagRFP and stained with αTubulin antibodies. (**D**) Best-fit sheet through $\vartheta$*y*-swivel against spindle position in *y* (degree-3 polynomial) and metaphase plate thickness (degree-1 polynomial; n = 3987), with coefficient of determination, R^{2} = 0.95. (**E**) Absolute $\vartheta$*y*-swivel (blue, left axis), and Δ 1D and Δ 3D (gold, right axis), for central and peripheral kinetochores for early (> 0.65) and late (< 0.45) metaphase (E, L, respectively) as determined by plate thickness. Statistical tests were significant (p < 10^{–4}) except where n.s. (no significance) is stated. Values are given in Figure 4—source data 1. (**F**) Examples of kinetochore pairs at spindle periphery during early (left) and late (right) metaphase. (**G**) (**i**) Schematic of dependency of swivel on spatial position, attachment status, and progression through metaphase towards anaphase onset. Light blue box indicates thickness of the metaphase plate. (**ii**) Mechanical degrees of freedom in kinetochore include changes in intra-kinetochore distance (delta; ∆ – black arrow) and swivelling ($\vartheta$ – blue arrows), which may involve remodelling of the kinetochore domains and/or tilting of the entire kinetochore structure within the chromosome. All scale bars 500 nm.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.015

Download figureOpen in new tabFigure 4—figure supplement 1. Inner and outer kinetochore markers are generally co-linear with their attached k-fibre.(**A**) Kinetochores exhibiting swivel marked by eGFP-CENP-A, Nnf1-Alexa594 and αTubulin-Alexa647. Green and red crosses represent the centre of eGFP and Alexa594 spots, respectively; dashed blue lines represent kinetochore-microtubule (kMT) axes determined from k-fibre fluorescence close to the kinetochore. Scale bars 200 nm. The schematic demonstrates the measurement of the angle tended by the kMTs on the sister-sister axis, $\vartheta$ kMT. (**B**) Distributions of $\vartheta$*y*-swivel (gold; n = 2427) and $\vartheta$ kMT (blue; n = 610) in cells expressing eGFP-CENP-A and Nnf1-Alexa594. (**C**) 2D histogram of absolute $\vartheta$ kMT versus absolute $\vartheta$ *y*-swivel, which are significantly correlated (r = 0.371, p < 10^{–4}, n = 573; see supplementary material), demonstrating that kMTs are generally aligned along the intra-kinetochore axis. (**D**) Permutation test of significance of correlation between absolute $\vartheta$*y*-swivel and absolute $\vartheta$ kMT (blue), and the best fit Gaussian distribution to the data (red; µ = 2.1 × 10^{–6}, σ = 0.042), with Monte Carlo sampling of 10^{6} permutations. Red dashed line is the observed correlation between $\vartheta$*y*-swivel and $\vartheta$ kMT, which demonstrates the significance of correlation (p = 2.8 × 10^{–19}, z = 8.9).

**DOI:** http://dx.doi.org/10.7554/eLife.16159.017

Figure 2—source data 1.Swivel increases under nocodazole treatment for multiple kinetochore markers.Table of measurements of Δ 1D, Δ 2D, $\vartheta$ 3D-swivel and standard deviation of $\vartheta$* y*-swivel distribution for all kinetochore markers imaged in untreated and 3 µM nocodazole-treated cells. n values correspond to all measurements in columns to the left of the n-value column. Values given are medians ± standard error.

**DOI:** http://dx.doi.org/10.7554/eLife.16159.009

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