Automated 4D live cell assay for systematic probing of HeLa kinetochore dynamics
To dissect the mechanisms of sister oscillation and breathing and to deduce their functional implications on metaphase plate alignment, the full range of sister kinetochore dynamics must be sampled and the statistical distributions of motion behaviors must be analyzed under a variety of well-defined molecular perturbations. To this end, we developed a HeLa cell line stably expressing the inner kinetochore protein CENP-A fused to EGFP (Fig. S1
). We established a standardized 3D live cell imaging protocol by maximizing signal to noise ratio and spatiotemporal sampling while minimizing phototoxicity (). The acquisition of time-lapse images of EGFP–CENP-A fluorescence at a rate of one 3D stack (20 optical sections separated by 0.5 µm in focal position) every 7.5 s for 5 min yielded >90% kinetochore detectability and unaliased time courses of kinetochore movements while preserving normal progression through mitosis to anaphase in unperturbed cells (see Materials and methods; ). Under these imaging conditions, approximately two thirds of the kinetochore pairs were visible in the 3D volume (Video 1
Figure 1. Live cell assay that maximizes signal-to-noise ratio while minimizing phototoxicity. To define a standardized protocol for our assay, we established a range of imaging conditions that maintained cell viability, i.e., 80% of cells progressed through mitosis (more ...)
Contributions of MT dynamics and components of the kinetochore and sister cohesion were probed pharmacologically and by siRNA-mediated depletion. For each perturbation, we used reagents and protocols known to fully perturb the system or provide quantitative knockdown, and we validated knockdown levels in individual cells (Fig. S2
and Table S1
). We compared each perturbation with untreated wild-type (WT) cells or with WT cells synchronized by thymidine/aphidicolin block and release as appropriate.
Each time-lapse dataset was subjected to an automated image analysis pipeline that identified and tracked sister kinetochore pairs to yield their trajectories over time (see Materials and methods; and Videos 2–5
). Under experimental conditions in which kinetochores formed a clear metaphase plate, we tracked the plate position over the course of a video by fitting a plane to the detected kinetochore positions. This defined a metaphase plate–centered coordinate system for kinetochore positions, allowing each kinetochore to be classified as aligned or unaligned by determining whether it was an inlier or an outlier with respect to the distribution of kinetochore positions along the normal to the metaphase plate (, step 2; and Video 3). The metaphase plate–centered coordinate system was also a translation- and rotation-free coordinate system that assisted in the tracking of kinetochores over the course of a video (Jaqaman et al., 2008
). If anaphase onset occurred during the time window of a video, the software identified it as the time point at which the average separation between all sisters in the video started to monotonically increase.
Figure 2. Automated image analysis and quantitative analysis of sister kinetochore dynamics. (a) The four steps of image analysis to measure sister kinetochore dynamics. White lines show boundaries of the 3D dataset, and the black rectangle indicates the magnified (more ...)
For each condition, we analyzed a minimum of 17 cells (44 cells on average), displaying an average of 32 sister kinetochore pairs per cell (Table S2
). Although most conditions could be analyzed using a default set of image analysis parameters, some conditions changed kinetochore behavior so drastically that they required an adjustment of critical parameters (Table S3
). In all cases, the goal was to use parameters that yielded image analysis results of the same quality across conditions, as discussed in more detail in Materials and methods (Fig. S3
In our analysis of chromosome dynamics, we pooled trajectories of all aligned sisters (or all sisters if there was no metaphase plate) from each condition. Consequently, rare or outlier events were masked by the dynamic properties of the majority. Moreover, we have ignored potential systematic differences between sisters at different locations in the metaphase plate (e.g., at the periphery vs. in the middle) as well as differences between aligned and unaligned sisters. Future analyses of this image dataset may focus on the heterogeneity in sister dynamics to extract further spatial information about the process of chromosome alignment and spindle forces.
Aligned sister kinetochores oscillate and breathe with characteristic speeds
We characterized the dynamics of individual sister kinetochores over the time course of a video using the following parameters: (a) sister separation d (), (b) sister center position x along the normal to the metaphase plate if there was a metaphase plate (), and (c) the projections of the sister and center 3D displacements onto the vector connecting sister pairs ΔprL, ΔprR, and ΔprC (). The center normal position (x) indicated motion relative to the metaphase plate, whereas the displacement projection parameters were independent of the plate, thus allowing the characterization of kinetochore dynamics in conditions without a metaphase plate. Future versions of this assay may be combined with centrosome labeling, allowing us to monitor kinetochore motion relative to the spindle poles as well.
From the time courses of individual sister pair movements, we derived population-level oscillation and breathing speed measurements: (a) we assembled distributions of the frame to frame changes in sister center normal position Δ x and sister separation Δd. These distributions had a 0 mean value because of the symmetry of sister oscillation and breathing; however, their SDs yielded measures for the average sister center normal speed (i.e., center speed along the normal to the metaphase plate) and the average sister breathing speed (both in micrometers/minute). (b) Sister center speed was also derived from the 3D center displacement projection ΔprC (). Because of motion symmetry, the distribution of ΔprC also had 0 mean, whereas its SD yielded a measure for the average sister center projection speed (in micrometers/minute). For conditions without a metaphase plate, this was the only measure of sister center speed.
Assembling the data of 6,605 aligned sisters from 212 3D time-lapse videos of late prometaphase and metaphase WT cells, we measured an average sister breathing speed of 1.31 ± 0.18 µm/min and average sister center normal speed and projection speed of 1.46 ± 0.25 µm/min and 1.56 ± 0.26 µm/min, respectively (for all parameters, the uncertainty estimate reflects the SD of the distribution of single cell averages; ). We also obtained an average separation between individual sisters of 0.98 ± 0.08 µm, which is in agreement with previous values (Gerlich et al., 2006b
). Importantly, all speed values were significantly larger than the apparent speed values measured for fixed cells (). These apparent movements of fixed kinetochores reflect the uncertainty of our measurements, mostly associated with the uncertainties in estimating kinetochore positions as a result of image noise but possibly also with contributions from mechanical vibrations of the mounted sample and microscope stage. These results demonstrate that our live cell assay has sufficient sensitivity to resolve sister oscillation and breathing movements over a sampling interval as short as 7.5 s.
Aligned sister kinetochores oscillate and breathe with characteristic periods
For a temporal characterization of kinetochore dynamics, we initially measured oscillation half-periods τx
and breathing half-periods τd
(in seconds), defined as the durations between consecutive turning points in the time courses of sister oscillation and breathing (, insets; Dorn et al., 2005
). We found that HeLa kinetochores exhibited a wide distribution of oscillation and breathing periods (, WT distributions). To achieve a metric of the mixture of periods that would allow the extraction of the characteristic behavior of sister oscillation and breathing within each condition and the comparison of dynamics between conditions, we used auto- and cross-correlation methods (see Materials and methods). For sister breathing, we calculated the autocorrelation of the time course of the frame to frame change in sister separation Δd
(sister breathing autocorrelation), whereas for sister oscillations about the metaphase plate, we calculated the autocorrelation function of the time course of the frame to frame change in center normal position Δx
(sister center autocorrelation). In addition, because some conditions did not form a metaphase plate, we characterized sister movement by calculating the cross-correlation between the time courses of the plate-independent sister displacement projections ΔprL
(; sister displacement cross-correlation).
For a regular oscillator with a narrow distribution of periods (, model 1), auto- and cross-correlations display a series of strong sinusoidal-shaped side lobes (, model 1). The minimum of the first negative side lobe indicates the oscillation half-period (, arrows); the maximum of the first positive side lobe denotes the full period (, arrowheads). For oscillators with mixed periods over wider distributions, the side lobes are increasingly attenuated at longer time lags (, models 2 and 3), and the minima and maxima of the side lobes might get slightly shifted. The value of the sister displacement cross-correlation at lag ΔT = 0 reflects the degree of motion coupling between sisters (, carets). Its value ranges between −1 and 1, where 1 indicates perfect coupling, −1 indicates perfect anticoupling, and 0 implies that the two sisters move independently. In contrast to a semiregular oscillator, purely random motion such as diffusion or jitter as a result of noise has no characteristic time scale: its autocorrelation exhibits one negative value, −0.5 at the first lag and 0 otherwise, whereas its cross-correlation is 0 everywhere ().
Combining the data of all WT cells, the sister displacement cross-correlation and the sister center autocorrelation functions exhibited a negative lobe with a minimum at ~35 s and a small positive lobe with a maximum at ~80 s (; arrows and arrowheads, respectively). The value of the cross-correlation at lag ΔT = 0 was 0.66 ± 0.07. These results indicate that sisters oscillate about the metaphase plate with fairly tight coupling and an average half-period of 35–40 s. The sister breathing autocorrelation function also showed a negative side lobe and one small positive lobe with a maximum at 40 s, implying that the breathing half-period was ~20 s on average (). The auto- and cross-correlation curves of WT cells were very different from those of fixed cells, which in turn looked similar to the correlation curves resulting from random motion (, compare brown lines). The result that significant correlation values in WT cells occurred at lags greater than the sampling interval confirmed that the temporal sampling of our videos was sufficient to capture the dynamics of kinetochore movement and that kinetochore oscillation and breathing contain elements of regularity.
Oscillation and breathing require bipolar attachment to dynamic MT ends
To test whether MT plus end dynamics are the major driving force for kinetochore oscillations and breathing observed in HeLa cells, we analyzed sister kinetochore dynamics after (a) 10 nM taxol treatment, which keeps kinetochores attached to MTs but inhibits MT plus end dynamics, (b) 100 ng/ml nocodazole treatment, which depolymerizes MTs and leaves kinetochores detached, and (c) siRNA-mediated depletion of Hec1 or Nuf2R, two components of the Ndc80 complex that are essential for stable end-on attachment of kinetochores to MTs (DeLuca et al., 2005
As previously described (Jordan et al., 1993
; Waters et al., 1998
; DeLuca et al., 2005
), loss of tension as a result of taxol treatment, loss of attachment as a result of nocodazole treatment, or depletion of Nuf2R of Hec1 reduced the average sister separation (). Taxol treatment also reduced both breathing speed and sister center speed (), implying that a substantial component of kinetochore oscillations and breathing in HeLa cells is governed by MT plus end dynamics. As a further confirmation that after taxol treatment the observed kinetochore motion was reduced to random jitter on stationary MT plus ends, the auto- and cross-correlation curves after taxol treatment were similar to those of fixed cells (; compare with ).
Figure 3. Nonrandom sister oscillation and breathing require bipolar attachment of kinetochores to dynamic MT plus ends. (a) Sister separation. (b) Sister center normal and projection speeds and sister breathing speed. (c–f) Sister displacement cross-correlation (more ...)
Under nocodazole treatment or Hec1 or Nuf2R depletion, the auto- and cross-correlation curves were also indicative of random motion (). However, the sister center speed after Hec1 or Nuf2R depletion was significantly higher than after nocodazole treatment (). This implies that although the motion after nocodazole treatment is purely diffusive, kinetochore dynamics are at least partially motor driven along the sides of MTs after Hec1 or Nuf2R depletion, e.g., by CENP-E or dynein (Kapoor et al., 2006
; Gassmann et al., 2008
; Vorozhko et al., 2008
). Collectively, these results indicate that the oscillation and breathing of sister kinetochores observed in HeLa cells requires end-on attachment to dynamic MTs and that oscillation and breathing are driven by MT plus end dynamics.
Kinetochore-associated depolymerases set the oscillation and breathing speeds
Although kinetochore oscillations may be related to the intrinsic dynamics of MT plus ends, MT motor proteins may play an important role in sliding the kinetochore on MT ends and/or in modulating the growth and shrinkage of MTs. Therefore, we analyzed the effects of CENP-E, MCAK, and Kif18A depletions on sister kinetochore oscillation and breathing.
Our assay revealed that although the depletion of CENP-E resulted in alignment failure of some chromosomes (Fig. S2) in agreement with previous studies (McEwen et al., 2001
; Putkey et al., 2002
), it did not have any significant effect on the oscillation or breathing of aligned sisters in speed () or periodicity (). Thus, our data show that once kinetochores are aligned at the metaphase plate, their oscillation and breathing are independent of CENP-E. To confirm this conclusion, we treated CENP-E–depleted cells with 10 nM taxol. In contrast to cells with only CENP-E depletion or only taxol treatment, these cells did not form metaphase plates, indicating that chromosome alignment into a metaphase plate required at least the presence of CENP-E or dynamic MTs. However, the speeds and correlation curves for this condition were indistinguishable from those produced under taxol treatment of WT cells (). These results provide strong additional evidence that CENP-E does not contribute to kinetochore sliding on MT plus ends after end-on attachment and alignment on the metaphase plate.
Figure 4. Depolymerases set oscillation and breathing speeds, whereas sister linkage modulates oscillation and breathing period. (a and h) Sister separation. (b and i) Sister center normal and projection speeds and sister breathing speed. (c–f, j, and k) (more ...)
We next examined the role of two MT depolymerases, MCAK and Kif18A, on kinetochore dynamics (Desai et al., 1999
; Varga et al., 2009
). As previously shown (Kline-Smith et al., 2004
; Stumpff et al., 2008
), depletion of MCAK increased chromosome segregation errors during anaphase, and Kif18A depletion elevated the rate of chromosome alignment failure and led to a mitotic arrest (Fig. S2). Depletion of MCAK also reduced the oscillation speed of aligned kinetochores, whereas depletion of Kif18A increased it (), which are both consistent with previous observations (Wordeman et al., 2007
; Stumpff et al., 2008
). Depletion of either motor led to the disappearance of the positive lobe from the oscillation and breathing correlation plots and to a slight shift of the negative lobe in the oscillation correlation plots (), indicating that both depletions tend to randomize kinetochore oscillation and breathing (a broadening of the half-period distribution shifts the negative peak; ). However, neither depletion significantly changed the average oscillation and breathing periods, which is partially in disagreement with previous results (Stumpff et al., 2008
). We surmise that these differences are the result of a manual selection of sisters as opposed to a comprehensive tracking of a large set of sister kinetochore pairs in 4D. Collectively, our data suggest that the MT depolymerases MCAK and Kif18A primarily set the speed of kinetochore oscillations and breathing.
The mechanical linkage between sisters regulates oscillation and breathing periods
Directional switches in kinetochore oscillations require a switch in the state of a kinetochore from leading to following and vice versa. Recent data suggested that these states may partially depend on the varying level of polar ejection forces transmitted to the kinetochore as a function of the sister pair position along the spindle (Ke et al., 2009
). However, force levels at the kinetochores may also be altered, as sisters breathe under the forces exerted by the depolymerases on the two sisters. Therefore, we hypothesized that the mechanical linkage between sisters may be a second key factor in establishing directional switches.
Sister linkage is mediated mainly by cohesin that cross-links chromatids (Nasmyth, 2002
) as well as by condensin, which compacts centromeric chromatin (Swedlow and Hirano, 2003
; Oliveira et al., 2005
; Gerlich et al., 2006a
; Ribeiro et al., 2009
). To investigate how these components might affect sister oscillation, we first depleted the condensin I subunit CAP-D2. The reduced compaction of chromatin upon CAP-D2 depletion caused a large decrease in the sister cross-correlation at lag 0 (), implying a reduced coupling between sister kinetochores. It also increased the average sister separation and the sister breathing speed (), which is in agreement with previous results (Oliveira et al., 2005
; Gerlich et al., 2006a
; Ribeiro et al., 2009
). Interestingly, CAP-D2 depletion increased not only the characteristic half-period of breathing () but also the half-period of oscillation () compared with WT. It also increased the oscillation speed (), although to a lesser extent than Kif18A depletion. These observations indicate that the mechanical linkage between sisters indeed plays a role in regulating the oscillation of aligned sister kinetochores, particularly their directional switching frequency.
We next sought to modulate the level of cohesion between sister kinetochores. We considered depleting cohesin, but the resulting premature sister disjunction would make our assay unusable. Therefore, we depleted instead the enzyme separase, which cleaves cohesin (Uhlmann et al., 2000
; Hauf et al., 2001
). Complementary to the depletion of CAP-D2, in the absence of separase, we observed a reduction in the oscillation period () and a small reduction in oscillation speed (). These changes in kinetochore dynamics were observed in aligned chromosomes before anaphase onset, suggesting that separase modulates the extent of cohesion between sisters already during late prometaphase and metaphase. This implies that there is a dynamic balance of assembly and disassembly of cohesin before anaphase, as has been observed in yeast (Ocampo-Hafalla et al., 2007
). In summary, our data suggest that centromere stiffness regulates kinetochore sister oscillations primarily through their directional switching frequency. Whether condensin and cohesin directly collaborate to regulate centromere stiffness and balance the dynamics of kinetochores will require further study.
The metaphase plate gets thinner as cells progress toward anaphase because of a reduction in oscillation speed
How do oscillations and breathing affect the overall architecture of the metaphase plate? Visually, our live cell data suggested that the metaphase plate becomes thinner as cells progress toward anaphase. By quantifying metaphase plate thickness as the SD of the distribution of aligned sister center positions along the normal to the metaphase plate (, x axis), we found that cells entering anaphase in the course of a video indeed had systematically thinner plates (). Cells that spent a long period in late prometaphase with one or a few faraway unaligned kinetochores near the spindle poles tended to have thinner plates as well (). This suggests that thinning is the result of a time-dependent ordering of the metaphase plate that occurs independently of whether cells have attached all their kinetochores or not.
Figure 5. Metaphase plate thinning in preparation for anaphase is induced by a reduction in oscillation speed. (a) Histogram of average plate thicknesses in individual videos. (b) Scatter plot of oscillation extent versus plate thickness. Black line, one-line fit (more ...)
Plate thickness is expected to be determined primarily by kinetochore oscillations. This means that plate thickness and oscillation extent, defined as the center normal speed multiplied by the oscillation half-period, should be coupled with a proportionality coefficient equal to one. A scatter plot of oscillation extent versus plate thickness averaged over the duration of an entire video indicated that the two variables were indeed proportional to each other (). The slope of a straight line fitted to the data using least-median squares was found to be 0.63 ± 0.05 (goodness of fit evaluation; Fig. S4
; Jaqaman and Danuser, 2006
). This value is significantly different from 0, confirming the coupling between plate thickness and oscillation extent. However, it is also significantly different from 1. We hypothesized that the slope was shifted toward smaller values because of the large data scatter for thicker metaphase plates and possible lack of coupling between the two variables in that regime. Thus, we fitted the scatter plot with two lines with an unknown transition point between them. The fit yielded a transition point at 0.59 ± 0.03 µm, with line slopes of 1.17 ± 0.13 and 0.04 ± 0.15 for the regimes below and above the transition point, respectively (). The two-line fit was only marginally better than the one-line fit (an F-test comparing the two fits’ residuals yielded P ≈ 0.22; Jaqaman and Danuser, 2006
); nevertheless, the line slope below the transition point was statistically indistinguishable from 1 (P = 0.1). This confirms that plate thickness is indeed tightly coupled to the extent of kinetochore oscillations, especially in the regimen of thinner, more organized plates.
We next asked whether speed, period, or both were altered to induce metaphase plate thinning. We approached this question in three ways. First, we generated scatter plots of half-period versus plate thickness and center normal speed versus plate thickness, fitted each with a straight line, and calculated how different the slope of each line was from zero. With this, we found weak coupling between plate thickness and oscillation half-period () but a strong coupling between plate thickness and center normal speed (). In a second approach, we used multivariable regression, where we investigated the dependence of plate thickness on center normal speed alone, oscillation half-period alone, or center normal speed and oscillation half-period together. We compared the residuals from the different regressions using an F-test (Jaqaman and Danuser, 2006
). The fit to speeds had much smaller residuals than the fit to periods, and the fit to both variables did not yield any statistically significant improvement beyond fitting to center normal speeds alone (). Third, in addition to the analyses based on whole-video averages, we monitored the variation of plate thickness and center normal speed within the 5-min window of each video. We calculated averages of plate thickness and center normal speed in the first 10 frames and the last 10 frames of each video and found that center normal speeds decreased in 78% of the cases where the plate got thinner within a video (). These three sets of independent analyses provide strong evidence that plate thinning during metaphase is primarily coupled to a reduction in kinetochore oscillation speed, with only a small change in oscillation period.
Metaphase plate thinning through oscillation speed reduction depends on the mechanical linkage between sisters
Next, we investigated which factors might be responsible for the gradual decrease in oscillation speed throughout mitotic progression toward anaphase. We fitted the scatter plot of oscillation speed versus plate thickness after depletion of MCAK, Kif18A, CAP-D2, or separase with a straight line and compared the resulting slopes with the slope in WT. To our surprise, depletion of MCAK only shifted the vertical position of the regression line without changing its slope (), indicating that MCAK sets the baseline speed of oscillation but is not essential for the mechanisms responsible for the progressive speed reduction necessary for plate thinning. Kif18A depletion changed the line’s slope (); however, this result needs to be interpreted with caution because the two conditions occupy very different, almost nonoverlapping plate thickness regimes.
The depletion of CAP-D2 or separase markedly reduced the slope of the regression line while maintaining a WT range of plate thicknesses (). Thus, the coupling between oscillation speed and plate thickness was reduced after these perturbations. Interestingly, despite the lack of oscillation speed reduction after perturbing sister linkage, plate thinning still occurred (). This may be the result of a compensatory mechanism that increases under these conditions the dependence of plate thickness on oscillation period. Indeed, we found that the slope of the line correlating plate thickness with oscillation period increased after CAP-D2 or separase depletion (unpublished data). Collectively, our observations suggest that in WT HeLa cells, the gradual reduction of oscillation speed of aligned kinetochores results from a gradual change in the mechanical linkage between sisters.