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Tension-dependent Regulation of Microtubule Dynamics at Kinetochores Can Explain Metaphase Congression in Yeast Molecular Biology of the Cell
Vol. 16, 3764 3775, August 2005
Tension-dependent Regulation of Microtubule Dynamics
at Kinetochores Can Explain Metaphase Congression in
Yeast D
Melissa K. Gardner,* Chad G. Pearson, Brian L. Sprague, Ted R. Zarzar, Kerry Bloom, E. D. Salmon, and David J. Odde*
*Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455; Department of
Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and Laboratory of Receptor
Biology and Gene Expression, National Cancer Institute, Bethesda, MD 20892
Submitted April 3, 2005; Revised May 20, 2005; Accepted May 23, 2005
Monitoring Editor: Orna Cohen-Fix
During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching
their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to
single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer
modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating exper-
iments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during
metaphase as measured by uorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of
kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack
pulling forces from a sister kinetochore. We rule out a number of possible models and nd the best t between theory and
experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the
poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.
INTRODUCTION
During mitosis, a dynamic array of kinetochore microtu-
bules (kMTs) serve to accurately segregate a duplicated
genome into two complete sets of chromosomes (Inoue and
Salmon, 1995; Rieder and Salmon, 1998; Nasmyth, 2002;
Howard and Hyman, 2003; Scholey et al., 2003). Budding
yeast offers an attractive system for answering fundamental
questions about the regulation of kMT dynamics, because
each kinetochore is thought to be attached to only one kMT
plus-end (Peterson and Ris, 1976; Winey et al., 1995; OToole
et al., 1999). The relative simplicity of the yeast spindle, with
16 kMT minus-ends anchored at each pole, makes this an
excellent system for computational modeling. Although the
dynamics of individual kMTs have not been directly ob-
served in vivo, kMT-plus ends seem to exhibit dynamic
instability, switching stochastically between extended peri-
ods of polymerization and depolymerization (Maddox et al.,
2000; Pearson et al., 2001). In general, regulation of microtu-
bule (MT) dynamic instability involves control of four pa-
rameters: the rates of polymerization and depolymerization,
and the frequencies of catastrophe (transition from growing
to shortening) and rescue (transition from shortening to
growing) events. In budding yeast, kinetochore movement
during metaphase is coupled to individual kMT growth and
shortening, which likely occurs solely by polymerization
and depolymerization at the kinetochore-attached kMT
plus-ends (Maddox et al., 2000; Pearson et al., 2003).
Labeling of single centromere proximal markers in yeast
indicates that sister centromeres separate toward opposite
sides of the spindle during metaphase and exhibit abrupt
transitions in their direction of movement, as would be
expected for dynamic instability of kMTs (He et al., 2000;
Tanaka et al., 2000; Goshima and Yanagida, 2001; Pearson et
al., 2001). Fluorescently labeled kinetochores persist in clus-
ters midway between each spindle pole body and the spin-
dle equator during yeast metaphase, and therefore the os-
cillations of uorescent probes on chromosome arms
suggest that dynamic kMT plus-ends coordinate congres-
sion of kinetochores to a steady-state, bilobed metaphase
conguration in yeast (Pearson et al., 2001; Krishnan et al.,
2004).
Green uorescent protein (GFP) kinetochore fusions, such
as Cse4-GFP, allow for live cell imaging of kinetochores in
yeast spindles (Meluh et al., 1998; Chen et al., 2000; Pearson
et al., 2001). In our previous work, a stochastic model of kMT
plus-end dynamics in the budding yeast metaphase spindle
was developed and then evaluated by simulating images of
kinetochore-associated uorescent probes (Sprague et al.,
2003). Although individual kMT dynamics cannot be re-
solved, computer simulations of kMT dynamics combined
with statistical measures of how well the simulation data
predict experimental uorescence kinetochore distributions
recorded by live cell imaging can be used to build an un-
derstanding of budding yeast mitotic spindle kMT dynamics
(Sprague et al., 2003). Through this analysis, it was demon-
strated that a model based on any set of constant dynamic
instability parameters was insufcient to explain how kinet-
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05 04 0275)
on June 1, 2005. D
The online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Address correspondence to: David J. Odde (oddex002@umn.edu).
3764
© 2005 by The American Society for Cell Biology ochores tend to cluster midway between the poles and the
equator in yeast metaphase spindles (Sprague et al., 2003).
However, reasonable agreement between simulated and ex-
perimental data for the distribution of kinetochores was
found using a model with a temporally stable spatial gradi-
ent between the spindle poles in either catastrophe or rescue
frequency combined with constant values for the other fre-
quency (Sprague et al., 2003). For example, in the spatial
gradient models, higher frequencies of catastrophe in the
middle of the spindle relative to the poles promoted kinet-
ochore movement poleward, or higher frequencies of rescue
near the poles relative to the middle of the spindle promoted
kinetochore movement away from the poles.
It has been proposed for higher eukaryotes that mechan-
ical tension on the kinetochore could modulate MT stability,
acting as a key regulator of kMT dynamics (Nicklas, 1988;
Skibbens et al., 1993, 1995; Rieder and Salmon, 1994, 1998;
Inoue and Salmon, 1995; Skibbens and Salmon, 1997). Recent
evidence in Xenopus extract spindles indicated that mechan-
ical stress regulates MT dynamics locally at the kineto-
choreMT attachment site, such that tension between sister
kinetochores may promote MT polymerization (Maddox et
al., 2003; Cimini et al., 2004). In addition, tension between
sister kinetochores is important for the stability of kMT
attachments and for turning off the spindle checkpoint that
regulates anaphase onset in yeast (Dewar et al., 2004). Due to
the signicant spacing between sister kinetochores in yeast
metaphase ( 700 nm), communication between sister kinet-
ochores is likely facilitated via mechanical tension through
the intervening chromatin, because chemical signaling over
such a distance would be improbable.
Here, we have used computer simulation to explore how
mechanical tension at the kinetochore might contribute to
metaphase chromosome alignment in budding yeast. First,
we established that spatial gradient models similar to those
described by Sprague et al. (2003) do not predict the low
incidence of kinetochores crossing the equator, as observed
experimentally by measurements of uorescence recovery
after photobleaching (FRAP) of the kinetochore-associated
protein Cse4-GFP (Pearson et al., 2004). We then tested four
various ways that kinetochore tension alone or in combina-
tion with catastrophe or rescue gradients between the poles
would predict the extent of kinetochore movements as mea-
sured by the Cse4-green uorescent protein (GFP) FRAP
data. The best t to the experimental data was achieved by
kinetochores sensing a stable gradient between the poles to
spatially control kMT plus-end catastrophe frequency and
by sensing tension generated via chromatin stretching be-
tween sister kinetochores to control kMT plus-end rescue
frequency. This model also quantitatively reproduces meta-
phase kinetochore distributions and kMT dynamics as mea-
sured by GFP-Tubulin FRAP experiments without parame-
ter value adjustment between different experimental data
sets. In addition, by eliminating simulated tension between
sister kinetochores, the model quantitatively reproduces the
kinetochore distribution in yeast mutants (cdc6) that enter
mitosis with unreplicated chromosomes. In these cells, chro-
mosom