Yeast Kinetochore Microtubule Dynamics Analyzed by High-Resolution Three-Dimensional Microscopy

Abstract

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.

Comparative Autoregressive Moving Average Analysis of Kinetochore Microtubule Dynamics in Yeast

“…From these positions and uncertainties, the SPB-CEN distance and its uncertainty were calculated at each time point. In the case of chromosome attachment, the SPB-CEN distance was approximately equal to the length of the corresponding kMT, and its variation over time reflected kMT dynamics (9).…”

“…This conclusion does not purport that dynamic instability is an inappropriate model of S. cerevisiae kMT dynamics-it simply means that, within the context of dynamic instability models, the average and distributions of growth and shrinkage speeds and rescue and catastrophe frequencies are not sufficient, and additional parameters are needed to obtain a complete characterization of kMT dynamics. An alternative approach for the analysis of MT dynamics that requires the estimation of only a small number of parameters is diffusion analysis (9), or diffusion-with-drift analysis (32,33). In this model, an MT end is assumed to undergo a one-dimensional, possibly confined, random walk with drift.…”

“…Not only are the observed kMT length series intrinsically stochastic due to the random switching of MTs between assembly and disassembly (1), but they also suffer from extrinsic stochasticity due to undersampling. As discussed in Dorn et al (9), the S. cerevisiae spindle requires three-dimensional imaging, currently limiting temporal sampling to 1 frame/s. However, the average time spent in either the growth phase or the shrinkage phase is observed to be ;1.5 s (9).…”

“…More advanced schemes have considered not only the averages of these descriptors but also their distributions (9,(14)(15)(16). However, even by including speed and frequency heterogeneity, differences between the three evidently dissimilar MT length series shown in Fig.…”

Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

“…Other methods utilize multiscale approaches, wavelets or Fourier space decomposition to identify the spots (4,(29)(30)(31)(32). Fluorescence spots can also be detected and localized based on finding local intensity maxima and subsequent model fitting (13,33,34).…”