Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases.

Generation of stable EGFP–CENP-A HeLa cell line

To obtain a stable EGFP–CENP-A cell line, HeLa cells were transfected with pEGFP–CENP-A–IRESpuro vector (provided by D. Gerlich, ETH Zurich, Zurich, Switzerland) using Fugene 6 (Roche). Cells were grown in DME supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 500 ng/ml puromycin (Invitrogen) at 37°C with 5% CO 2 in a humidified incubator. To obtain a homogeneous population of cells with respect to the EGFP–CENP-A kinetochore signal, we performed two rounds of manual single-colony selection and screened the clonal populations by visual inspection (Fig. S1).

Fluorescence time-lapse imaging of EGFP–CENP-A HeLa cells

Cells were seeded in dishes (LabTekII; VWR) and imaged in L-15 medium (Invitrogen) supplemented with 10% FCS (Invitrogen). Time points comprised of 20 z sections 0.5 µm apart were acquired every 7.5 s for 5 min (i.e., 41 time points in a video) with a 100× 1.35 NA objective lens (Olympus) and a camera (CoolSnap HQ or HQ2; Photometrics) on an imaging system (DeltaVision Core, Personal, or Spectris; Applied Precision) fitted with a 37°C environmental chamber. Camera pixels were binned (2 × 2), yielding a pixel size of 129 nm in the lateral direction. The 10-µm z stack covered about two thirds of the metaphase plate (Video 1). The acquisition routines in softWoRx (Applied Precision) were used to control all aspects of image data acquisition. Imaging conditions were designed to balance three essential imaging requirements ( Dorn et al., 2008 ): (1) exposing cells to as little light as possible to minimize phototoxicity, (2) obtaining images of sufficiently high signal to noise ratio to allow reliable detection of the EGFP–CENP-A signal, and (3) sampling sufficiently fast to measure unaliased kinetochore motion. (1) Cell viability. Phototoxic effects on cells, as a result of frequent exposure to too much light, can manifest themselves as delayed anaphase timing, i.e., a delay in the time between nuclear envelope breakdown and anaphase onset, as the result of an activation of the spindle checkpoint ( Mikhailov et al., 2002 ). To assess the effect of various 3D imaging protocols on cell viability, we first determined the normal anaphase timing of WT HeLa cells measured by imaging fields of cells exposed to minimal amounts of light. Specifically, cells were imaged every 5 min over 6 h using a 60× 1.4 NA lens (Olympus) with 25-ms exposure, 32% neutral density filter, and seven z sections 1 µm apart. This yielded a reference anaphase timing of ∼34 min ( Fig. 1 , top). To assess the effect of fast 3D imaging on cell viability, we varied exposure time, neutral density filter, recovery times between exposures and a number of imaged time points, and compared the resulting anaphase timing distribution with the reference anaphase timing. We found that an imaging protocol of 15-ms exposure time, 32% neutral density filter, and a sampling rate of one z stack (20 z sections 0.5 µm apart) every 7.5 s for 41 time points (resulting in a video length of 5 min), i.e., a total of 820 exposures per video, ensured that ≥80% of the cells exhibited the normal anaphase timing of ∼34 min. (2) Detection quality. Automated kinetochore detection (see following paragraph) fails to properly distinguish spot signals from noise if the signal amplitude becomes too similar to the noise amplitude. In such a situation, both the number of false positives, i.e., noise peaks that are mistaken for true kinetochore signal, and the number of false negatives, i.e., kinetochore signals that are missed, become large. Thus, it was essential to determine imaging protocols that minimized both false positives and false negatives while maintaining cell viability. The rate of false positives and negatives was estimated for each imaging protocol by taking multichannel images of EGFP–CENP-A cells, in which in each channel a different imaging protocol was used, and comparing the detection results between the different channels. In one channel, high light exposure that led to flawless spot detection was used to provide the detection ground truth (although such high light exposure did not satisfy the cell viability conditions). In the other two channels, light exposures and neutral density filter combinations that were consistent with cell viability were used. The kinetochore signal was detected in all three channels and compared ( Fig. 1 , bottom). With this, we found that imaging with 15-ms exposure and 32% neutral density filter, yielding videos with an average signal-to-noise ratio of 3–4, resulted in