Regulation of Chromosome Attachment and
Dynamics by Saccharomyces cerevisiae
Kinetochores
by
Jessica Dawn Tytell
B.S. Molecular Biology
University of California, San Diego
SUBMITTED TO THE DEPARTMENT OF BIOLOGY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN BIOLOGY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
FEBRUARY 2006
(0 Massachusetts Institute of Technology. All rights reserved.
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Thesis Supervisor
Stephen
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Professor of Biology
Chair, Committee for Graduate Students
MASSACHUSETTS INsTrW
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ARCHIVES
Regulation of Chromosome Attachment and Dynamics by
SaccharomycescerevisiaeKinetochores
by
Jessica Tytell
Submitted to the Department of Biology on October 31, 2005
In Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy in Biology
Abstract
Kinetochores are large, multi-protein complexes that bind centromeric DNA to
the microtubules of the mitotic spindle and mediate chromosome movement throughout
the cell cycle. The proteins that regulate both force generation at kinetochores as well as
and the cell-cycle-dependent changes in kinetochore architecture are largely unknown.
The relative simplicity of centromere specification and kinetochore-microtubule
attachment make Saccharomyces cerevisiae an attractive model organism for
investigations into kinetochore-microtubule attachment and regulation.
We used a combination of cell biology and biochemistry to study the roles of the
four nuclear kinesin motor proteins at budding yeast kinetochores. We discovered that
each of the four nuclear kinesins localizes to kinetochores. Three of these, Cin8p, Kiplp,
and Kip3p are present at mature chromosome-microtubule attachments in metaphase.
Cin8p and Kiplp align metaphase chromosomes into the characteristic bilobed
configuration that is analogous to the metaphase plate in higher eukaryotes. Kip3p
regulates microtubule dynamics throughout the cell cycle and regulates poleward
movemnentduring anaphase. Kar3p, the final nuclear kinesin, is recruited specifically to
detached kinetochores. In addition, we have discovered that kinetochore-microtubule
attachments alter during the cell cycle indicating that kinetochore function is temporally
regulated.
Thesis Supervisor: Peter K. Sorger
Title: Professor of Biology, Professor of Biological Engineering
2
Jessica Dawn Tytell
EDUCATION
1999--2006
Ph.D., Department of Biology, Massachusetts Institute of Technology (MIT)
Defense Date: 8-19-05
1994-- 1998
B.S., Molecular Biology with Honors, University of California, San Diego (UCSD)
Phi Beta Kappa, Magna Cum Laude
RESEARCH EXPERIENCE
2000 -- 2005
Graduate Studies
Massachusetts Institute of Technology
PrincipalInvestigator:Peter K. Sorger
19)98-- 1999
1997-- 1998
Research Associate
Undergraduate Studies
University of California, San Diego and
Ludwig Institute for Cancer Research
Principal Investigator:RichardD. Kolodner
1996-1997
Undergraduate Studies
University of California, San Diego Medical Center
Principal Investigator:Alice K Yu
PUBLICATIONS
Tytell J.D., and Sorger, P.K. "Analysis of kinesin motor function at budding yeast
kinetochores."J Cell Biol submitted.
McAinsh, A.D., J.D. Tytell, and P.K. Sorger. 2003. Structure, function, and regulation of
budding yeast kinetochores. Annu Rev Cell Dev Biol. 19:519-39
Kolodner, R.D., J.D. Tytell, J.L. Schmeits, M.F. Kane, R.D. Gupta, J. Weger, S.
Wahlberg, E.A. Fox, D. Peel, A. Ziogas, J.E. Garber, S. Syngal, H. Anton-Culver, and
F.P. Li. 1999. Germ-line msh6 mutations in colorectal cancer families. Cancer Res.
59:5068-74.
3
Acknowledgements
I would like to thank Peter for the opportunity to join his lab. Your enthusiasm and
excitement have always been contagious. I have also learned a great deal from your way of
looking at problems as mechanical systems and from your desire to embrace new technology. In
addition to the many thanks for scientific manners, I am forever grateful for your help and
support throughout what was a somewhat stressful pregnancy and during the transition back to
work with an infant. Your advice (and sense of humor) helped me transition from floundering
graduate student to more efficient and more confident scientist and mother.
I would like to thank Frank Solomon both for his help as a member of my Thesis Committee and
for his support during some serious frustrations in my graduate career. Thank you for reminding
me to follow my heart and for listening to my complaints, concerns, hopes, and fears. Knowing
that you were there to turn to made my problems more bearable.
I would like to thank Angelika Amon for her help on my Thesis Committee during most of my
graduate career. You incredible enthusiasm and insights were invaluable.
I would like to thank Steve Bell, Steve Harrison, Graham Walker, and Frank Solomon for their
time, comments, and insights at my Thesis Defense and in writing and preparing my thesis.
I would like to thank the more senior grad students in the Sorger lab during my time in the lab:
Max Dobles, Aurora Burds Connor, Emily Gillett, Chris Espelin, and Dan Rines. You were all so
wonderful at welcoming me to the lab, teaching me techniques, lending a hand (or a sympathetic
ear), and answering patiently the myriad of questions I had about everything under the sun.
Working with you and learning from you was a pleasure.
I would like to thank all members of the Sorger lab, past and present, for their support and
enthusiasm. You made coming to lab every day a pleasure and I will miss the camaraderie of the
lab very much.
I would like to thank my parents and siblings for their love and support over the years. You have
always encouraged me to follow my dreams and for that I am eternally grateful.
Finally, I would like to thank Dave and Ben for making everything in life more wonderful. Dave,
you believed in me even when I was losing faith and encouraged me in good times and in bad.
Thanks especially for your patience with me through the ups and downs. I know this degree was
hard work for you as well and I appreciate all that you have done. I could not have done this
without your love and support. Ben, you weren't born yet when I started this but you changed
my whole world when you were. Thank you for keeping me focused on what really matters. Our
family is more than I ever hoped for. I love you both so much.
4
Table of Contents
Abstract
2
Personal Statement
3
Acknowledgements
4
Chapter One
Structure, Function, and Regulation of
Budding Yeast Kinetochores
Chapter Two
Functional Analysis of Kinesinsat Budding
Yeast Kinetochores
Chapter Three Cell Cycle Regulation of Kinetochore-
6
57
94
Microtubule Attachment
Chapter Four
Conclusions and Future Directions
109
CHAPTER ONE
Structure, Function, and Regulation of
Budding Yeast Kinetochores
This chapter is adapted with permission from McAinsh et al 2003.
McAinsh, A.D., J.D. Tytell, and P.K. Sorger. 2003. Structure, function, and regulation of
budding yeast kinetochores. Annu Rev Cell Dev Biol. 19:519-39.
OUTLINE
1.1 Introduction ......................................................................................
8
1.2 Overview of Mitosis ............................................................................
9
1.2.1 Yeast Mitosis .........................................................................
11
1.2.2 The Dynamic Nature of Mitosis .................................................
13
1.3 Microtubule Attachment and Force Generation ........................................
15
1.3.1 Biochemistry of Microtubules ...................................................
15
1.3.2 Force Generation in the Mitotic Spindle .......................................
17
1.4 Kinetochore Assembly .........................................................................
19
1.4.1 Specifying Centromere Location ................................................
21
1.4.2 Linker Complexes...................................................................
24
1.4.3Microtubule Associated Proteins .................................................
28
1.4.4Kinetochore Architecture ..........................................................
33
1.4.5Complexity of MAP and Motor Localization .................................
38
1.5 Regulation of Microtubule Attachment ....................................................
39
1.5.1 Role of Aurora B/Ipllp .............................................................
40
1.5.2 The Spindle Checkpoint ............................................................
42
1.6 Summary..........................................................................................
45
7
1.1 Introduction
Nearly a century ago Boveri and Sutton postulated that chromosome segregation from
mother to daughter cells was the basis of heredity (Boveri, 1907). Since then, errors in
chromosome segregation have been shown to have dire consequences. Missegregation during
meiosis in gametogenesis can lead to chromosomal abnormalities such as Down, Klinefelter,
Prader-Willi and Angelman syndromes, and are a leading cause of infertility (Bittel and Butler,
2005; Jiang et al., 2004; Lowe et al., 2001; Tempest et al., 2004). Errors during mitosis can cause
chromosome instability and cancer (for review see (Draviam et al., 2004; Storchova and
Pellman, 2004). Therefore, a detailed knowledge of the mechanisms driving chromosome
segregation is vital for understanding and preventing these problems.
At the molecular level chromosome segregation is an extremely complex process. The
mitotic spindle, consisting of arrays of microtubule fibers emanating from two organizing
centers, called centrosomes in mammals (or spindle pole bodies (SPBs) in yeast), forms a
dynamic scaffold that organizes chromosomes and positions the axis of division. After DNA
replication, sister chromatids must establish attachments to microtubules from opposite SPBs.
Kinetochores are protein complexes that tether centromeric DNA to spindle microtubules,
forming a bridge between the sister chromatids and the spindle. Once all sister chromatid pairs
form mature bipolar attachments, the cell progresses into anaphase and the sisters irreversibly
separate and segregate into the new cells.
One major question in chromosome segregation is how the kinetochore regulates
chromosome movement. Throughout mitosis, the chromosomes oscillate, microtubules grow and
shrink, and the spindle elongates and matures. Microtubule dynamics are highly regulated; more
than 13 microtubule associated proteins (MAPs), including kinesin motors, bind to kinetochores
8
to regulate chromosome movement and spindle organization. However, the contributions of
many individual MAPs and motors are still unknown. I have used the budding yeast
Saccharomyces cerevisiae as a model system to investigate the role of kinesins at kinetochores
in regulating chromosome movement and attachment. I have also investigated the cell cycle
regulation of attachment. In this chapter, I present an overview of spindle architecture and
dynamics during mitosis, the mechanisms of chromosome movement, kinetochore composition
and assembly, and the role of kinetochores in responding to lesions in tension and attachment.
1.2 Overview of Mitosis
Mitosis consists of a series of stereotypical chromosome movements within the complex
mechanical system of microtubules, centrosomes, and other factors that constitute the mitotic
spindle. Live-cell analysis performed during the 1980s and 1990s in animal cells established the
critical features of chromosome and spindle dynamics during the three primary mitotic phases:
prometaphase, metaphase, and anaphase. During prometaphase, the chromosomes condense, the
nuclear envelope breaks down, and chromosomes attach to microtubules. Attachment occurs via
a "search and capture" process whereby microtubules nucleated at centrosomes grow and shrink
rapidly until they encounter and bind to a kinetochore (Kirschner and Mitchison, 1986). If the
sister kinetochore then captures a microtubule from the same pole, a syntelic attachment is
created; if the captured microtubule is from the opposite pole, the attachment is bipolar. Sister
chromatids are held together from S-phase through metaphase by cohesin. The cohesin complex
- composed of four subunits, Scclp/Mcdl, Scc3p, Smclp, and Smc3 - loads onto DNA during
replication (Carson and Christman, 2001; Uhlmann, 2004). In the absence of cohesion, such as in
temperature-sensitive alleles of Scclp, the spindle elongates and chromosomes segregate
9
prematurely. This early elongation demonstrates that cohesin exerts inward forces that oppose
the pulling from the kinetochore microtubules (k-MTs) and that this balance of force is vital to
proper spindle assembly and morphology (Figure 1.1;(Guacci et al., 1997b; Michaelis et al.,
1997).
During metaphase, sister chromatids with mature bipolar attachments congress toward
the center of the spindle to form the metaphase plate. When all chromosomes have made mature
bipolar attachments, tension is generated across the spindle. The number of microtubules
attached to mammalian kinetochores varies in response to tension and mature bipolar
attachments recruit 15-30 microtubules per kinetochore (Brinkley and Cartwright, 1971; Rieder,
1982). A surveillance mechanism, called the spindle checkpoint, monitors the state of
chromosome-mnicrotubuleattachment, delaying cell cycle progression until all pairs of sister
chromatids have formed bipolar attachments (reviewed in (Amon, 1999). It is not yet known how
bipolar attachments are distinguished from monopolar and syntelic attachments, but only bipolar
attachments are stable and give rise to tension across paired sister kinetochores. When all pairs of
sister chromatids have made stable bipolar attachments to microtubules, the mitotic checkpoint is
silenced, and the Anaphase Promoting Complex (APC) is activated, irreversibly driving the cells
into anaphase (Peters, 2002). APC then targets the securin Pds lp for degradation (Yamamoto et
al., 1996a; Yamamoto et al., 1996b). Pdslp degradation activates the separase Esplp, a protease
that cleaves the Scclp subunit of cohesin, allowing the sisters to separate (Ciosk et al., 1998).
During anaphase, chromatids move poleward while maintaining end-on microtubule
attachments. Chromosome-to-pole movements during anaphase A, and separation of the poles
during anaphase B, create two equal and separated sets of sister chromatids.
10
1.2.1 Yeast Mitosis
In contrast to higher eukaryotes, S. cerevisiae undergo a closed mitosis and possess a
relatively simple spindle in which the spindle pole bodies (SPBs) remain embedded in the
nuclear envelope and chromosomes are bound to microtubules throughout the cell cycle (Figure
1. A; and (Adams and Kilmartin, 2000). This results in close association between chromosomes
and SPBs from telophase through prometaphase in budding yeast (Guacci et al., 1997a; Jin et al.,
2000). In GI1,a short linear array of microtubules emanates from the unduplicated SPB.
Chromosomes are bound to these microtubules via their kinetochores (Figure 1.1A). During Sphase, the SPB duplicates and a short bipolar spindle forms (<1.O0tm).Chromosomes replicate
and the new sister must attach to the opposite SPB. It is believed that early in spindle assembly,
sister chromatids frequently bind microtubules emanating from the same SPB, creating a syntelic
attachment (Figure 1. I1B)that must be dissolved for mature bipolar attachments to form (Tanaka
et al., 2002).
The budding yeast spindle elongates steadily throughout metaphase, achieving a length of
about 2.5 pm prior to the onset of anaphase. The metaphase spindle contains two classes of
intranuclear microtubules: those that bind to kinetochores (k-MTs) and those that project into the
spindle midzone to overlap with microtubules from the opposite pole (polar; p-MTs) (Figure
1lC).
.1 In contrast to animal cells, each S. cerevisiae centromere binds to a single k-MT (Winey
et al., 1995). A fully assembled metaphase spindle contains approximately 32 k-MTs and 8 pMTs (O'Toole et al., 1999; Winey et al., 1995). A small number of SPB-nucleated astral
microtubules (a-MTs) project from the SPB toward the plasma membrane where they bind to
cortical attachment sites to generate forces that position the nucleus to the bud neck at anaphase
(Pearson and Bloom, 2004). The k-MTs, p-MTs, and a-MTs form a coupled mechanical system
11
E.
B.
,..
,
,
,,
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Fk-MT
Syntelic
attachment
Fk-MT
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....
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Bilobed Distribution \ \
Anaphase
• Cortical attachment site
• SPB
0 Kinetochore
U
Cohesin
Metaphase
Figure 1.1: Cell cycle in budding yeast. (A) Chromosomes have end-on attachments to microtubules
during G1. (B) After duplication of chromosomes and SPBs, sister chromatids can form syntelic attachments, where both sisters are attached to the same pole. (C) During metaphase, the nucleus is
positioned into the bud neck by the a-MTs. The spindle elongates via the crosslin king of the p-MTs at
the spindle midzone. Bipolar attachments are formed and tension is generated across the kinetochores.
(0) Once all chromosomes make bipolar attachments the cell proceeds into anaphase and the chromosomes are pulled to the SPBs. (E) Cohesin opposes the force applied to kinetochores by the k-MTs
resulting in tension at kinetochores and transient separations. (F) Representative image of the bilobed
distribution of kinetochores in metaphase in cells coexpressing the kinetochore protein Ndc80p-GFP
with the SPB protein Spc42p-CFP.
12
linked by SPBs, sister kinetochores, kinesins, microtubule associated proteins (MAPs), and
cohesin (Figure 1.1C).
Unlike metazoans, budding yeast chromosomes do not undergo congression per se.
However, S. cerevisiae kinetochores do adopt a highly characteristic biblobedmetaphase
configuration analogous to the metaphase plate (Figure 1. F; (Goshima and Yanagida, 2000; He
et al., 2000). Typically, kinetochores form two lobes that lie on either side of the spindle
midzone that are separated by roughly half the distance between the SPBs (Figure 1..1F). This
unexpected localization pattern, similar to the two foci of separated SPBs, initially caused some
confusion in the literature and several proteins originally designated as SPB components were
subsequently shown to localize to kinetochores. The bibobed localization pattern is the result of
chromosome oscillation and transient sister separations caused by tension across centromeres
(described below) and it changes subtly on a time scale of seconds as the extent of overlap
among kinetochores varies (He et al., 2000). However, once metaphase connections are
established, chromosomes cross between lobes only rarely (Pearson et al., 2004).
1.2.2 Dynamics
One striking feature of mitosis in all eukaryotes is that chromosomes and microtubules
are continuously in motion both before and after bipolar attachment. Throughout prometaphase
and metaphase sister chromatid pairs move back and forth along the spindle axis, a behavior
know as directional instability (Rieder and Salmon, 1998). Electron microscopy (EM) reveals
that metaphase microtubule attachments are "end-on" with the extreme plus-ends of spindle
microtubules embedded in kinetochores (McEwen et al., 1997). Thus, chromosome movement is
coupled to microtubule polymerization at one kinetochore and microtubule depolymerization at
13
its sister. The rates of polymerization and depolymerization are not always matched, leading to
tension across sister kinetochores that can transiently separate them (Goshima and Yanagida,
2000; He et al., 2001; Tanaka et al., 2000).The amount of tension varies on a time scale of
seconds and is sufficient to physically stretch centromeric chromatin (Shelby et al., 1996). The
coupling of directional instability to microtubule dynamics suggests that kinetochores regulate
both microtubule binding and regulate plus-end dynamics to control chromosome attachment and
movement along the spindle.
Examination of the movement of individual centromeres demonstrated that paired sister
chromatids in 5'. cerevisiae separate transiently toward opposite ends of the spindle prior to the
onset of anaphase (Goshima and Yanagida, 2000; He et al., 2000; Tanaka et al., 2000). These
transient separations pull the sisters apart for up to several minutes and cause centromeric
chromatin to become stretched, but do not involve cohesin degradation. Transient sister
separation is observed in chromatin up to 10kb away from the centromere and appears to involve
a towards-the-pole pulling force mediated by kinetochores, and a countervailing adhesive force
dependent on cohesin (Figure 1.1B). Similar transient separation of centromeric chromatin
occurs in animal cells, but in yeast, the magnitude of these separations (up to 1 m) is much
greater relative to the small size of the spindle (about 2 ~tm)(He et al., 2000; Shelby et al., 1996;
Tanaka et al., 2000). The existence of transient sister separation raises a number of interesting
issues about sister chromatid cohesion. Centromeres contain the highest levels of cohesin of any
region in the genome (Blat and Kleckner, 1999; Tanaka et al., 1999) and it is not obvious how
stretching and sister cohesion coexist. Nor is it clear how the 20kb domain of elastic chromatin
surrounding centromeres is defined. Changes in the pulling forces applied to pairs of sister
chromatids that have achieved bipolar attachment would be expected to cause centromeric
14
chromatin to undergo cycles of stretching and compaction. There is evidence suggesting that the
cohesin complex forms a ring around DNA rather than binding as rungs on a ladder (Gruber et
al., 2003). If this is the case, it is tempting to imagine the cohesin ring sliding along the DNA
away from centromeres in response to tension, allowing for the transient separation of sister
centromeres (Figure 1.1E; (Nasmyth, 2005). Thus, it seems very likely that transient sister
separation in yeast is a consequence of directional instability in chromosome movement and can
serve as a readout of the forces at the kinetochore-microtubule interface.
1.3 Microtubule Attachment and Force Generation
Chromosome movement is dependent on attachment to the spindle and on microtubule
dynamics. Depolymerizing microtubules can drive chromosome movement in vitro (Coue et al.,
1991). Addition of kinetochores to microtubules in vitro alters their dynamics (Hunt and
McIntosh, 1998). Therefore, kinetochores can drive chromosome movement by regulating the
dynamic instability of microtubules. In the next section, I describe the basic biochemistry of
microtubules and the way it is harnessed to generate forces within the mitotic spindle. In Section
1.4.3, I also describe many of the kinetochore components that regulate microtubule dynamics.
1.3.1 Biochemistry of Microtubules
Microtubules are polymers of ccand 3 tubulin heterodimers.
3 dimers self-assemble into
long hollow tubes 25nm in diameter, composed of 12-15 protofilaments (Amos and Klug, 1974).
Microtubules are asymmetrical, with at-tubulin exposed at the more stable minus-end and 3tubulin at the more dynamic plus-ends. Microtubules intermittently switch between growth and
shrinkage, a process known as dynamic instability (Desai and Mitchison, 1997). Microtubule
15
motion is characterized by four different types of movement: growth, shrinkage, catastrophe, and
rescue. "Growth" and "shrinkage" refer to steady addition or loss of tubulin subunits,
respectively. "Catastrophe" describes the rapid depolymerization of the microtubule and
"rescue" describes the subsequent switch back to growth. Both a and [3-tubulinbind GTP,
however, only 3-tubulin hydrolyzes and exchanges GTP and GDP (Mitchison and Kirschner,
1984). Only GTP-bound a3-tubulin dimers incorporate into the growing microtubule lattice.
Therefore, newly added subunits are GTP bound while previously added subunits are GDP
bound. The GDP-tubulin has a >1000 fold higher off rate from microtubule ends than GTPtubulin (Walker et al., 1988). Therefore, the GTP-tubulin stabilizes the growing ends and loss of
the GTP "cap" could explain the rapid depolymerization of microtubules undergoing catastrophe.
GDP-microtubule protofilaments have inherent curvature that is constrained in
microtubules, storing potential energy in the microtubule lattice in the form of steric strain. This
energy is released upon depolymerization where the protofilaments curve away from the
microtubule fiber. The energy released upon microtubule depolymerization can be harnessed to
exert force on other systems including transporting kinetochores and their attached chromosomes
toward the poles (Coue et al., 1991; Koshland et al., 1988).
The rates of growth, shrinkage, rescue, and catastrophe are much different in vivo than in
vitro indicating that microtubule dynamics are highly regulated in the cell (Heald and Nogales,
2002). Many proteins can alter microtubule dynamics, including the +TIP family of MAPs, and
kinesins (described in Section 1.4.3). In vitro studies of microtubule biochemistry show that GTP
state and plus-end status can be recognized and selectively bound by these proteins (Akhmanova
and Hoogenraad, 2005). Therefore, microtubule dynamics are highly regulated in vivo by a
number of MAPs and motors that fine tune and harness their energy.
16
1.3.2 Force Generation in the Mitotic Spindle
An important goal of mitosis research is determining the origins and magnitudes of the
forces that move sister chromatids. Experiments in animal cells show that both ATP-dependent
sliding of kinesins along microtubules and GTP-dependent turnover of microtubules are capable
of generating sufficient force to move chromosomes (Koshland et al., 1988; Maney et al., 2000).
In vitro, kinesins can couple the energy of ATP hydrolysis and microtubule depolymerization to
cause movement of chromosomes (Heald and Walczak, 1999). The relative importance of these
two processes is unknown, but is currently a subject of significant debate.
Four principles of general significance are emerging from studies of microtubule
attachment and force generation in yeast. First, multiple proteins mediate microtubule attachment
to kinetochores. Second, mutations in different microtubule attachment factors give rise to
diverse defects in chromosome movement, suggesting that the process of forming a mature
attachment to a kinetochore is quite complex. Third, many of the proteins involved in
kinetochore-microtubule attachment also localize to other microtubule-based structures in the
cell. Finally, chromosome-microtubule attachment must be regulated so that errors such as
syntelic attachment can be corrected.
Motor- and polymerization-dependent processes have the potential to generate force in at
least four spindle locations: the plus-ends of k-MTs embedded in kinetochores, the minus-ends
of k-MTs embedded in spindle poles (traction fiber forces; (Hays and Salmon, 1990), along
chromosome arms via interactions with non-kinetochore microtubules (polar ejection forces;
(Rieder et al., 1986) and at cortical tips. In higher eukaryotes, all of these mechanisms are
utilized. After nuclear envelope break down, the chromosomes must form new attachments to the
17
spindle. Initially, the chromosomes form lateral attachments and are carried by dynein to the
poles (Echeverri et al., 1996; Rieder and Alexander, 1990; Sharp et al., 2000; Vaisberg et al.,
1993). Dynein is highly concentrated at unattached kinetochores, but relocalizes to the spindle as
kinetochores develop a full complement of k-MTs (Banks and Heald, 2001; Hoffman et al.,
2001; King et al., 2000).
Chromokinesins, which bind directly to the chromosome arms, function in chromosome
condensation, metaphase alignment, spindle organization, and cytokinesis (Mazumdar and
Misteli, 2005). While motors are actively positioning the chromosomes, the spindle exerts force
on the chromosomes. Spindle microtubules undergo treadmilling, a process in which the
centrosomes embedded minus-ends depolymerize, while the kinetochore embedded plus-ends
polymerize. This pull on the microtubule makes up a substantial fraction of force generation at
kinetochores. Therefore, both the chromosomes and the spindles are constantly in motion, and a
careful balance of plus and minus-end microtubule regulatory elements is likely required for
proper spindle function during mitosis.
The relative importance of these processes varies among different organisms, but EM and
live-cell studies suggest that polar ejection forces are unlikely to play a major role in yeast
(O'Toole et al., 1999). Photobleaching experiments detect no recovery of microtubules at the
minus-ends but swift recovery at the plus-ends, suggesting that there is little or no traction fiber
force (Maddox et al., 2000). Thus, our working hypothesis is that chromosome movement in S.
cerevisiae is powered largely, if not exclusively, by kinetochores and processes occurring at the
plus-ends of k-MTs.
Although much remains to be learned about the directions and magnitudes of the forces
operating on chromosomes in S. cerevisiae and animal cells, it is helpful to make some
18
comparisons between simple and complex eukaryotes. First, it is clear that the generation and
stabilization of bipolar kinetochore-microtubule attachment is a complex process that occupies
much of mitosis. In animal cells, both sisters kinetochores must capture microtubules, whereas in
yeast, it is thought that kinetochores remain bound to microtubules throughout the cell cycle.
Capture is presumably necessary in yeast only after centromeres are duplicated and a new
kinetochore assembles. In both yeast and animal cells, syntelic and monopolar attachments are
eliminated and cell cycle progression is delayed by the spindle checkpoint until all sets of
chromatids have achieved bipolar attachment. Attached chromosomes exhibit directional
instability (stochastic movement along the spindle axis), and tension is generated across pairs of
sister kinetochores (He et al., 2000; McEwen et al., 1997). It is tempting to consider the
conserved features of chromosome segregation - bipolar attachment, error correction, tension
generation, checkpoint control, and directional instability -to
be the most fundamental aspects
of the process.
1.4 Kinetochore Assembly
In the last few years significant progress has been made in identifying the protein
components of yeast kinetochores and determining their overall architecture. In 1995 there were
five known and five suspected kinetochore proteins (Hyman and Sorger, 1995). Yeast
kinetochores were thought to comprise a set of DNA binding proteins and a kinesin: Kar3p
(Hyman et al., 1992). We now know that budding yeast kinetochores contain at least 50 proteins
that are organized in at least three layers: DNA binding, microtubule binding, and a "linker"
layer of proteins that bind to neither the DNA nor the microtubules (Figure 1.4). About half of
these proteins are essential and many have close mammalian homologs (Kitagawa and Hieter,
19
2001). The HEC1/Ndc80 complex, which has four subunits, exhibits strong sequence and
structural conservation and human HEC protein can complement the yeast Ndc80 deletion
(described below) (Ciferri et al., 2005; Wei et al., 2005; Wigge and Kilmartin, 2001; Zheng et
al.. 1999). Conversely, the specialized histone H3, Cse4p can functionally substitute for its
homolog CENP-A in human cells, overcoming the arrest caused by RNAi depletion of CENP-A
(Wieland et al., 2004). Thus, important aspects of kinetochore architecture have been conserved
from yeast to humans despite the significant differences in mechanisms of centromere
specification.
In the following sections, I provide a brief description of selected kinetochore proteins
complexes before proposing a model for kinetochore assembly. Many kinetochore proteins form
discrete subcomplexes that can be isolated from cells and are thought to represent assembly
intermediates (De Wulf et al., 2003). Whenever possible, I have grouped kinetochore subunits
together by multi-protein complex or by shared function (e.g. "kinesins"). In lieu of a better
convention, I name complexes after the best-described component (e.g. "The Ndc80 Complex").
Very few kinetochore subunits have been studied biochemically, but I have indicated those
whose functions can be inferred by sequence homology or phenotype. In many cases I suggest
that proteins may be "linkers" (blue in Figures 1.3& 1.4) between DNA binding (pink) and
microtubule binding components (green). However, this designation is a reflection of the
preliminary state of functional studies rather than a true assignment of function. Although the
exact order of assembly is still unknown I have ordered the complexes from those thought to be
more centromere proximal to the microtubule binding proteins.
20
1.4.1 Specifying Centromere Location.
The first step in kinetochore attachment is assembling the core kinetochore complex onto
centromeric DNA. S. cerevisiae was the first organism in which centromeres were cloned and
sequenced. A 125 bp centromeric DNA (CEN) sequence, present in a single copy on each
chromosome, is necessary and sufficient for accurate segregation during meiosis and mitosis
(Clarke and Carbon, 1980; Cottarel et al., 1989). Comparison of centromeres from different S.
cerevisiae chromosomes reveals that they have three conserved elements: CDEI, CDEII, and
CDEIII (Fitzgerald-Hayes et al., 1982). CDEI and CDEIII are imperfect palindromes and are
bound in a sequence-specific manner by the CBF 1 and CBF3 protein complexes, respectively
(Cai and Davis, 1990; Lechner and Carbon, 1991; Ng et al., 1986). The sequence of CDEII
varies but is always about 85 bp long and highly A-T rich (Clarke and Carbon, 1980). CBF is
not essential for viability, though its deletion increases chromosome loss (Cai and Davis, 1990).
CBF3 is composed of four essential proteins, NdclOp, Ctfl3p, Cep3p, and Skplp and has a
molecular weight of 240 kDa (Connelly and Hieter, 1996; Goh and Kilmartin, 1993; Lechner and
Carbon, 1991; Strunnikov et al., 1995). The assembly of CBF3 is reasonably well understood
and involves a series of regulated steps that are described in Figure 1.2. CBF3 is required for the
association of all other known kinetochore proteins with centromeric DNA and loss of function
mutations in CBF3 proteins disrupt kinetochore-microtubule attachments in vitro and in vivo (He
et al., 2001; Sorger et al., 1994). The CBF3 component Ndc lOp is the only protein known to bind
CDEII in vitro, but other proteins have been suggested to be CDEII-interactors in vivo (Espelin
et al., 2003).
Centromeres from closely related yeasts such as K. lactis are similar in size and
organization to those in S. cerevisiae (Heus et al., 1990), but centromeres in other organisms are
21
5gtlp dimer
5kplp
B
(qilP
A~
~HSP9~O
C
Ctf13p
~~
5kplp
..
p
Ctf13p
265
Proteosome
-
.... .... ,
I
ICtf13pl
,
,
E
"
,
,,
,
,
,,
CDEII'
.
,,' . ~ DNA
.
T A G.~~TTT~AT
T C 1\.~1\
~ \A
cff1~~r
I
Extended CBF3
contacts
Complex
Ctf13p
A~:
Tt~~
fA A~'T
A
T
cff1~~J
Cep3p contacts
Ctf13p contacts
o crosslin king
• crosslinking
e BrdC interference
Figure 1.2: Steps Involved in Activation and Assembly of the CBF3 Complex. (A) Skp1 P binds to
the F-box of Ctf13p, an event that requires the chaperone Hsp90p to correctly fold Ctf13p (B) Ctf13p is
activated by Skp1p-dependent phosphorylation and the transient interaction of Sgt1p with Skp1p. (C)
Activated Ctf13p associates with Cep3p to form a complex competent in Ndc10p association and CEN
binding. (D) Skp1 p-Ctf13p-Cep3p is in equilibrium with free Skp1 p-Ctf13p which can be degraded
following Skp1p mediated ubiquitination by SCF and degradation via the 265 proteosome. (E) The CBF3
core complex binds to conserved bases in CDEIII. Ctf13p, Cep3p and Ndc10p contact DNA in the major
groove. Inset shows the bases that can be cross-linked to CBF3 subunits. An extended CBF3 complex
containing an extra Ndc10p dimer also assembles centromeric DNA by making direct contact with bases
proximal to the CDEIII core. Ndc10p also appears to bind to CDEII. In vitro this binding occurs in the
absence of other CBF3 subunits. Model based on (Espelin et aI., 1997; Espelin et aI., 2003; Kaplan et
aI., 1997; Rodrigo-Brenni et aI., 2004; Russell et aI., 1999; 5temmann et aI., 2002)
much larger and contain long stretches of repetitive DNA. In fission yeast, for example, the
centromeric region spans 40-100 Kb of DNA, in humans, centromeres are 1-40 Mb long, and in
worms there is no specific centromeric region and kinetochores assemble along the length of
chromosomes (Albertson and Thomson, 1982; Bloom, 1993). Thus far, it has not been possible
to identify sequence-specific DNA binding proteins in complex kinetochores that are analogous
to budding yeast CBF1 and CBF3. Rather, specification of more complex centromeres appears to
be linked closely to the formation of chromatin that contains the specialized histone H3 CENP-A
(or its homologues). Moreover, it is widely thought that an epigenetic rather than a sequencespecific mechanismnis responsible for determining the location of CENP-A containing
heterochromatin in organisms with complex centromeres (Choo, 2001). The pathogenic yeast
Candida albicans have no detectable point centromeres and no similarity between centromeres
of different chromosomes. Despite this they recruit CENP-A to their centromeres (Sanyal et al.,
2004; Sanyal and Carbon, 2002).
Until recently it was understood that point centromeres evolutionarily preceeded their
more complex counterparts. However, sequence analysis from multiple families of fungi
demonstrates that point centromeres exist in only a small subset of yeast, suggesting that point
centromeres are a specialization of regional centromeres rather than a precursor (E. Rheinbay,
personal communication). The fungus E. cuniculi, which possesses the smallest number of
kinetochore components and is believed to contain the most primitive kinetochores, has no
detectable point centromeres supporting the notion that regional centromeres evolved first.
Like animal centromeres, S. cerevisiae centromeres contain heterochromatin. Budding
yeast have a CENP-A homologue, Cse4p, that is essential for chromosome segregation (Stoler et
al., 1995). One model suggests that CSE4-containing nucleosomes bind to CDEI and CDEII
23
DNA and form the scaffold onto which other kinetochore proteins assemble (Figure 1.3A; (Keith
et al., 1999; Meluh et al., 1998). However, it is clear that CBF3 and not Cse4p is the primary
determinant of kinetochore location in S. cerevisiae: centromere binding by Cse4p in vivo
requires functional CBF3 but the reverse is not true (Measday et al., 2002; Ortiz et al., 1999).
Whereas all known kinetochore proteins require CBF3 for centromere association, only subsets
are known to require Cse4p (Measday et al., 2002). Current models for Cse4p function are based
on in vivo crosslinking studies, which have limited resolution (Meluh et al., 1998). Thus, the data
are equally consistent with Cse4p-containing nucleosomes forming the phased chromatin array
that lies on either side of the core kinetochore complex (Figure 1.3B). In this speculative model,
Cse4p would perform an essential function in centromere specification but one that is secondary
to CBF3.
1.4.2 Linker Complexes
THE CTF 19 COMPLEX. The Ctfl 9 complex, also called the COMA complex, contains at least four
stably associated subunits, Ctfl9p, Okplp, Mcm2lp, and Amelp, though some studies suggest
that it might contain additional subunits (Cheeseman et al., 2002; De Wulf et al., 2003; Ortiz et
al., 1999). CTF19 and MCM21 are non-essential whereas OKP1 and AME] are required for cell
growth. The biochemical functions of the Ctfl 9 complex are not know, but Ctfl 9p has been
shown by immunoprecipitation and yeast two-hybrid experiments to interact with the CBF3
complex (Ortiz et al., 1999). It is unclear whether this interaction is direct or indirect, but Ctfl9p
may bind directly to CBF3.
24
A.
- Nucleosome-centric
-:--CD
cse4P~
CBF3-centric
B.
I
c.
c
ro
:p
E
o
L..
.!:
U
DNA binding
) DNA-binding components
)
Linker components
MT-binding components
Figure 1.3: Kinetochore Assembly and Architecture. (A)
Nucleosome-centric model and (B) CBF3-centric model of kinetochore
assembly (C) Logical organization of the DNA-microtubule bridge that
forms at centromeres.
THE CTF3 COMPLEX.The composition of the Ctf3 complex is not yet entirely defined, but
immunoprecipitation experiments suggest that it contains at least three subunits, Ctf3p, Mcm22p,
and Mcml 6p, all of which are non-essential and localize exclusively to kinetochores (Measday et
al., 2002). Ctf3p has been conserved through evolution and the S. pombe homolog of Ctf3p,
Mis6+ is required for the recruitment of the fission yeast Cse4p histone (Cnpl+ ) to centromeric
DNA (Takahashi et al., 2000). However, the reciprocal is found in budding yeast and human
cells where Ctf3p/hMis6 requires Cse4p/CENP-A for CEN association (Goshima et al., 2003;
Measday et al., 2002). The current thinking is that the Ctf3 complex may participate in some
aspect of centromeric chromatin assembly.
THE NDC80COMPLEX. The Ndc80 Complex contains four protein subunits, Ndc80p, Nuf2p,
Spc24p, and Spc25p, all of which are essential and localize exclusively to kinetochores (He et
al., 2001; Janke et al., 2001; Wigge and Kilmartin, 2001). Mutations in yeast Ndc80 components
result in complete detachment of chromosomes from microtubules. Loss of SPC24 and SPC25
function causes microtubule detachment and also inactivates the spindle assembly checkpoint
(He et al., 2001; Janke et al., 2001), suggesting a role for the Ndc80 Complex in the recruitment
of spindle checkpoint proteins to kinetochores. The Ndc80 Complex also appears to be required
for kinetochore binding by the Dam complex and the MAP Stu2p, suggesting a linker role for
the Ndc80 Complex in bridging DNA and microtubule-binding components of the kinetochore
(He et al., 2001; Janke et al., 2002). Budding yeast Ndc80 Complex forms a rod-like structure
with globular domains in each end (Wei et al., 2005). The human Ndc80 Complex also appears
to be extremely elongated (Ciferri et al., 2005). Ndc80p and Nuf2p are evolutionarily well
conserved and are required for chromosome segregation in human cells, budding yeast, Xenopus
26
and other organisms (DeLuca et al., 2002; Martin-Lluesma et al., 2002; McCleland et al., 2003;
Wigge and Kilmartin, 2001; Zheng et al., 1999). Spc24p and Spc25p homologs are also present
in higher eukaryotes but their sequences are less conserved (Bharadwaj et al., 2004; McCleland
et al., 2004). The Ndc80 Complex appears to be functionally conserved since RNAi depletion of
Spc25 leads to aberrant mitosis and chromosome detachment in humans and Xenopus, and
Hecl /Ndc80 and Nuf2 are required for microtubule attachment (Bharadwaj et al., 2004; DeLuca
et al., 2005; McCleland et al., 2004). This complex is unique as it is the only complex other than
the CBF3 complex whose mutation causes complete loss of chromosome-microtubule
attachment.
THEMTW1COMPIEX.The Mtwl Complex contains at least four essential protein subunits
including Mtwlp, a protein initially identified as a homolog of the S. pombe Mis 12+ kinetochore
protein (Goshima and Yanagida, 2000). Mtwlp-GFP localizes to kinetochores by imaging, and
mtwl mutations result in a loss of tension across sister kinetochores (Goshima and Yanagida,
2000). Yeast Mtwlp mutants have some unattached chromosomes and it has been proposed that
Mtwlp aids in establishing bipolar attachments of kinetochores (Pinsky et al., 2003), but Mtwl's
biochemical function remains unknown. DSN1, NNF1 and NSL1 exhibit genetic or two-hybrid
interactions with Mtwlp and are stably associated with Mtwlp in solution in 1:1:1:1
stoichiometry (De Wulf et al., 2003; Euskirchen, 2002; Nekrasov et al., 2003; Westermann et al.,
2003). The Mtwlp homologs in fission yeast and humans, Mis12, localize to kinetochores.
Depletion of these proteins causes detachment of chromosomes from the spindle (Goshima et al.,
2003; Obuse et al., 2004). This suggests that Mtwlp function is conserved through evolution.
While the Mtw p protein may act as a linker between chromosomes and microtubules, no
27
biochemical function is known. Intriguingly, human Mtw /Mis12 requires a heterochromatin
protein (HP 1) 1"orkinetochore recruitment, suggesting a larger interplay between
heterochromatin and kinetochore function (Obuse et al., 2004). This heterochromatin
dependency has yet to be addressed in budding yeast.
THE SPC105COMPLEX. The Spc 105 Complex contains two subunits: Spc 05p and YDR532p
(De Wulfet al..,2003; Nekrasov et al., 2003). Both Spc105 and YDR532p are essential, and
mutations in Spc 105 lead to defects in chromosome segregation (Nekrasov et al., 2003). Spc 105
was originally purified with Mtwlp and the two complexes are thought to be spatially close
within the kinetochore structure. An Spc 105 homolog is also present in fission yeast. The
biochemical activity of the Spc 105 complex is unknown but this complex is required for
recruitment of a subset of kinetochore MAPs, indicating that it fine tunes microtubule attachment
or regulation (P. De Wulf, personal communication).
1.4.3 Microtubule Associated Proteins
THE DAM1COMPLEX. The Daml Complex binds to microtubules in vitro and contains 12
essential subunits (Figure 1.4). Damlp localizes to both kinetochores and spindle microtubules in
vivo and dam] mutants arrest as large budded cells with shortened or broken spindles
(Cheeseman et al., 2002; Cheeseman et al., 2001; Enquist-Newman
et al., 2001; Janke et al.,
2002; Li et al., 2002). The Daml Complex is required for both the establishment and the
maintenance of bipolar microtubule attachment. By live-cell analysis, dam1-] and dam]-] 1
mutants exhibit monopolar attachment and directional instability centered on a single spindle
pole. Elegant experiments by Janke et al. (2002) showed that when cohesin has been inactivated
28
in cells bearing a mutation in the Daml Complex member Spc34p, individual sisters segregate
almost equally between the two spindle poles (as opposed to being monopolar). This finding
implies that both sets of sister kinetochores in spc34 cells can capture microtubules, but the
attachments are not strong enough to counteract the pulling forces generated by bipolar
attachment. Thus, the chromosomes can be pulled off the microtubules (Janke et al., 2002).
fConsistent with this idea, chromatid pairs in dam] cells are observed to jump occasionally from
one SPB to the other, establishing frequent but unstable monopolar attachments first with one
SPB and then another. Further live-cell analysis demonstrates that dam] mutants display a range
of attachment phenotypes -including
some that are unattached (Dom, 2005; He et al., 2001).
Copurification of 10 Dam 1 Complex subunits revealed that they form a 210 kDa
heterodecamer that is globular in free form but forms rings or helices when incubated with
microtubules (estimated 10-15 heterodecamers/ring) (Miranda et al., 2005). The Dam rings
appear to stabilize microtubules, aid in microtubule polymerization, and bind preferentially to
GTP-tubulin in vitro (Westermann et al., 2005). The Daml Complex requires microtubules for
association with kinetochores (Li et al., 2002). These data suggest that the Daml Complex ring
acts as a collar around the microtubule to which CEN assembled kinetochore proteins attach.
Interestingly, there are no known homologs of Dam p in higher eukaryotes. This suggests that
higher eukaryotes, which have multiple microtubule attachments per kinetochore, utilize a
different tethering mechanism. However, it is also possible that structural homologs exist that
have not yet been identified by sequence homology.
THE +TIPS. +TIPs are a class of microtubule associated protein, so named because they associate
specifically with the plus-ends of microtubules (Akhmanova and Hoogenraad, 2005; Maekawa
29
and Schiebel, 2004). There are many +TIP families that function in microtubule regulation
during mitosis; some differ in their method of plus-end association. There are at least eight
distinct +TIP proteins at vertebrate kinetochores, all of which have budding yeast homologs.
However, several of these MAPs are found only in the cytoplasm in S. cerevisiae and, therefore,
the number of-+TIPs at budding yeast kinetochores is likely much lower (Akhmanova and
Hoogenraad, 2005). Thus far there are only two +TIPs - Stu2p and Biklp -- that have confirmed
kinetochore localization, but there are three others that may localize to kinetochores.
STU2P.The +TIP Stu2 has homologs in all major kingdoms, including XMAP215 in Xenopus
and ch-TOG1 from humans (Gard et al., 2004; Wang and Huffaker, 1997). Stu2p is localized in
budding yeast to kinetochores and cortical tips, two sites of plus-end microtubule dynamics, and
perhaps to the spindle midzone as well (He et al., 2001). The TOG/XMAP215 family of proteins
appears to stabilize microtubules (Gard and Kirschner, 1987; Tournebize et al., 2000; Vasquez et
al., 1994). Surprisingly, Stu2p destabilizes microtubules in vitro through direct plus-end binding
(Van Breugel et al., 2003). However, live-cell microscopy shows that Stu2p is transported from
newly captured kinetochores to the plus-ends of the attached microtubules, and its arrival at the
tip coincides with microtubule rescue (Tanaka et al., 2005). Therefore, Stu2p may actually
stabilize microtubules in vivo. In stu2 cells, chromosomes make bipolar attachments, but no
transient sister separation is observed and the velocity of chromosome oscillations is reduced
compared to wildtype cells (He et al., 2001). Taken together these data demonstrate that Stu2p
regulates microtubule dynamics.
30
BIK1P.The +TIP protein Biklp localizes to kinetochores as well as to cortical attachments sites
where it may fnction with Kar9p to capture the plus-end of a-MTs (Berlin et al., 1990; He et al.,
2001). The deletion of Biklp has no obvious phenotype in haploid or diploid cells, but Biklp
becomes essential in polyploid cells where it contributes to the generation of tension across sister
kinetochores (Lin et al., 2001). The Biklp homolog CLIP-170 selectively binds and stabilizes the
tips of growing microtubules in vitro (Perez et al., 1999). Biklp, in contrast, is held at both
growing and shrinking a-MT plus-ends by the kinesin motor Kip2p (Carvalho et al., 2004). Since
Kip2p is present only in the cytosol, other components must tether Biklp to kinetochores. Biklp
does not localize to kinetochores that have become detached from microtubules (Tanaka et al.,
2005) Biklp and CLIP-170 are not as conserved as Cse4 and CENP-A as Biklp can localize to
kinetochores but cannot substitute for CLIP-170 function in human cells (Wieland et al., 2004).
OTHER POSSIBLE KINETOCHORE +TIPS. Bimlp is another +TIP protein whose homologs (EB 1
and Mal3) have been shown to associate with kinetochores in mammalian cells and in fission
yeast (Kerres et al., 2004; Tirnauer et al., 2002). Xenopus EB 1 stabilizes microtubules and
depletion of EB 1 in human cells leads to a decrease in tension at kinetochores (Draviam, 2005;
Tirnauer et al., 1999) but their role in budding yeast remains unknown.
Stulp and PacIlp are +TIP members of the MAST/Orbit and LIS1 families of MAPs,
respectively. Both MAST and LIS 1 function at kinetochores in higher eukaryotes (Akhmanova
and Hoogenraad, 2005; Sharp, 2002). Stulp is essential in budding yeast and localizes to the
spindle midzone; stuI mutants undergo spindle collapse (Yin et al., 2002). STU has genetic
interactions with DAMI and CIN8 (Jones et al., 1999; Yin et al., 2002). It is currently unknown
31
whether Stul p associates with budding yeast kinetochores. PAC1 has genetic interactions with
many members of the kinetochore and cortical tip including BIM and CIN8 (Tong et al., 2004).
KINESNS.
Kinesins are motor proteins that harness the chemical energy of ATP hydrolysis and
convert it to mechanical force, most often in transporting cargo along microtubules (Asbury,
2005). Most kinesins are dimers that consist of a coiled-coil stalk connecting a cargo-binding
domain to globular heads. Each head is a catalytically active ATPase whose affinity for
microtubules depends on its bound nucleotide. Kinesin moves in 8nm steps along microtubule
protofilaments hydrolyzing one ATP per step. The exact mechanism of movement is still under
intense debate, but it appears that kinesin may walk by alternately swinging one "leg" past the
other. The many different kinesins subfamilies have diverse activities. Some kinesins function as
true motors translocating cargo toward the plus or minus-ends of microtubules. Others
specifically destabilize either plus-or minus-ends (for table see (Lawrence et al., 2004)).
Differences in kinesin directionality are due to changes in the neck region which reorient the
motor heads (Goldstein and Philp, 1999). Microtubule destabilization is caused by changes in the
motor heads causing them to exert pulling force on the microtubule protofilaments and
encouraging them to curve away from the microtubule polymer (Desai et al., 1999). While the
roles for several kinesins in higher eukaryotes are known, the functions of kinesins at budding
yeast kinetochores are less well established. In Chapter 2, I describe my efforts to determine the
complement of kinesin motors at budding yeast kinetochores and elucidate their functions.
32
1.4.4 Kinetochore Architecture
The budding yeast kinetochore has been estimated to be at least 5MDa in mass and
between 85-10Onm in length (De Wulfet al., 2003; Dorn, 2005). The large size and complexity
of kinetochores leads to the question of how such a structure might assemble. They could
resemble highly structured organelles such as ribosomes or SPBs that are composed of one or
two large, stable, multi-protein complexes that assemble in solution and bind - intact - to
centromeric DNA. An alternative view is that kinetochores resemble eukaryotic transcriptional
enhancers in being composed of many discrete complexes that assemble on DNA either by
contacting DNA bases directly or by binding to proteins that are themselves in contact with
DNA. The available evidence strongly favors the latter possibility and the existence of a multilayer structure (Figure 1.3C). Yeast kinetochores appear to be composed of at least eight and
perhaps as many as twenty protein complexes each containing up to twelve different components
(Figure 1.4). These kinetochore complexes can be isolated as stable species by velocity
sedimentation and sizing chromatography (De Wulf et al., 2003; Nekrasov et al., 2003).
If kinetochores contain distinct "layers," then one would expect a hierarchical set of
interdependencies among different kinetochore complexes. This is exactly what is observed. In
the first step of kinetochore assembly, shortly after DNA replication, CBF3 binds to DNA and
presumably initiates the formation of Cse4-containing centromeric chromatin. DNA-bound
CBF3 is required for the Ndc80 complex to bind to centromeres in vivo, but the reciprocal is not
true (He et al., 2001). The microtubule-binding Daml Complex and Stu2p require both CBF3
and Ndc80p, but, once again, the reciprocal is not true (Enquist-Newman et al., 2001; He et al.,
2001; Janke et al., 2002; Jones et al., 1999). Thus, we can develop a preliminary model in which
CBF3 is bound to centromeric DNA, the Ndc80 Complex associates with DNA-bound CBF3,
33
10nm
.. - ISPC10~P'
\
/
" .....
,
---
Sds22p
/"r,
-E)
GIC7P
NkP2P~lP
MCm16P.,~
.Mcm22p
o DNA:"binding components o Nonessential genes
e
Unker components
Regulatory components
o MT-binding components
Figure 1.4
o
--.
()
Essential genes
No known function
Potential component
I
Figure 1.4: Speculative model for the organization of known kinetochore components.
Protein complexes are drawn approximately to scale based on hydrodynamic analysis. The
assignment of functions to various proteins is preliminary, except in the case of CBF3 and CBF1
proteins. For references see text, except for Plclp (Lin et al., 2000), Slil 5/Ipllp/Birlp
(Cheeseman et al., 2002), Hirl/Caclp (Sharp et al., 2002), Mif2p (Meluh and Koshland, 1997),
and Slkl9p (Zeng et al., 1999).
35
and the Dam Complex and Stu2p associate with Ndc80 Complex proteins. Significantly, this
model is based on a set of genetic interdependencies and we do not know whether CBF3, Ndc80,
and Daml complexes interact directly. However, if this model is correct, we might expect
proteins within a layer to exhibit independent association with centromeric DNA. Once again,
this seems to be true. The Ndc80 Complex requires CBF3 for centromere association, as does the
Ctfl 9 Complex, but neither appears to be dependent on the other (He et al., 2001; Janke et al.,
2001; Ortiz et al., 1999).
By way of comparison, the mammalian interferon beta promoter is a prototypical
eukaryotic enhancer in which activators (NF-KB, ATF-Jun, etc.) and architectural proteins (e.g.
HMG(I)) bind in a sequence specific manner to cis-acting regulatory sequences (Struhl, 2001).
The similarity between enhancers and kinetochores suggests possible models for the function of
Cse4p-containing centromeres in yeast. In enhancers, nucleosomes are excluded from the core of
the enhancer but bind on either side - a possible model for the organization of centromeric
chromatin. DNA-bound activators then mediate the stepwise recruitment of a set of multiprotein
complexes that remodel surrounding chromatin and recruit additional complexes involved in
transcriptional transactivation. Similarly, it appears that some, but not all, kinetochore assembly
is Cse4p dependent: the Ctfl 9 and Ctf3 complexes require CSE4 function for centromere
association in vivo but, based on functional evidence, the Ndc80 Complex does not (Gardner et
al., 2001; McCleland et al., 2003; Measday et al., 2002). Additional support for the ordering of
complexes comes from the observation that components of individual complexes appear to
copurify (albeit in sub-stoichiometric amounts) with neighboring complexes but not distant ones.
Therefore, copurifying proteins can give an indication of nearby complexes (De Wulf et al.,
2003; Nekrasov et al., 2003; Westermann et al., 2003).
36
Preliminary data suggest that the assembly of kinetochores, like the assembly of
enhancers, is temporally regulated. Ipl lp, Slil 5p and Ndc lOp relocalize from the kinetochores to
the spindle midzone at the metaphase-to-anaphase transition (Buvelot et al., 2003), as does
Slkl9p, which is cleaved at the metaphase-to-anaphase transition by Esplp. The cleaved Nterminal fragment of Slkl 9p is recruited from the kinetochore to the spindle midzone where it is
thought to stabilize the anaphase spindle (Sullivan et al., 2001). These so-called "passenger
proteins" are found in many organisms (Vagnarelli and Earnshaw, 2004). In addition,
subcomplex purification has shown that some of these complexes can be found in intermediate
forms, which indicates that their composition could change during the cell cycle (De Wulf et al.,
2003). Moreover, we do not know whether all centromeres assemble identical kinetochore
complexes, or if yeast kinetochores assemble and disassemble depending on the state of
microtubule attachment during cell cycle progression. It is also unknown how phosphorylation
by cyclin/CDKs and other cell cycle modulators affects kinetochore function.
Kinetochores probably do not fully assemble on centromeric DNA prior to the
establishment of microtubule attachment. Whereas Bik 1p and Ctfl 9p are recruited to
kinetochores independently of microtubules, the Dam l Complex is not kinetochore-bound
following microtubule depolymerization by nocodazole. In normally dividing cells, Damlp is
found on both spindle microtubules and kinetochores (Hyland et al., 1999; Li et al., 2002; Lin et
al., 2001). Stu2p is also present on spindles and kinetochores, and remains on intranuclear
microtubules even when kinetochores are inactivated using an ndcl 0Omutation (He et al., 2001).
Therefore, some proteins that associate with centromeres in vivo probably bind microtubule plusends in the absence of kinetochore attachment. It is interesting to consider the possibility that
unattached kinetochores may not capture microtubules directly, but may instead associate with a
37
preformed set of plus-end binding proteins. The Dam ring makes an appealing candidate for a
capture structure on microtubules. One can imagine how centromere assembled kinetochore
structures might grab and hold on to the ring.
1.4.5 Complexity of MAP and Motor Localization
A significant fraction of kinetochore MAPs and motors localize to structures other than
kinetochores and have functions distinct from chromosome-microtubule attachment. In
particular, several microtubule binding proteins present at kinetochores are also found at sites of
cortical attachment. In addition to binding to kinetochores, Biklp and Kip3p localize to cortical
attachment sites; Stu2p localizes to the spindle, SPBs, and cortical attachments, and Damlp
localizes along the spindle (He et al., 2001; Hofmann et al., 1998; Jones et al., 2001; Kosco et al.,
2001; Lin et al., 2001). Moreover, Stu2p and Kip3p are known to play roles in nuclear
positioning and Cin8p and Kip lp have important functions in spindle assembly (Cottingham and
Hoyt, 1997; Kosco et al., 2001). These findings suggest a "mix and match" reuse of microtubule
binding and motor proteins in the spindle and at kinetochores. One striking example of this is the
colocalization of many MAPs and motors at kinetochores and cortical attachments sites at the
plus-ends of a-MTs. Both involve the capture of microtubule plus-ends and they may have
important structural similarities.
One consequence of the localization of MAPs and motors to multiple structures in the
cell is that it introduces considerable complexity in determining protein function. Available
evidence suggests that the roles of spindle components are determined as much by their location
as by their intrinsic biochemical activity. To prove this point, it will probably be necessary to
generate specific mutant alleles that disrupt recruitment to one structure but not to another. A
38
second issue arises from the tight mechanical coupling within the spindle. The disruption of one
component in the spindle has the potential to affect, via microtubule linkages, other elements that
are spatially distant. Thus, even when proteins are present on only one structure, their
inactivation can affect multiple spindle activities. With 16 kinetochore-mediated pole-to-pole
connections in yeast cells and perhaps 4-8 p-MTs, it is easy to imagine that spindle stability is
quite dependent on kinetochore function. Thus the interdepence of kinetochore function and
other cell cycle processes is becoming ever more complicated. As previously mentioned, spindle
dynamics appear to be coregulated with kinetochores at the metaphase-anaphase transition
(Higuchi and Uhlmann, 2005). Genetic evidence also links kinetochore function to processes as
diverse as the Rac, Ras, and PKA pathways, and chromatid cohesion (Li et al., 2005; Mayer et
al.. 2004). Therefore, dissecting out specific mechanisms and functions of these proteins will be
a challenge.
1.5 Regulation of Microtubule Attachment
During metaphase, chromosomes must form attachments to microtubules emanating from
opposite poles. These mature, bipolar attachments have two main characteristics: the
kinetochores are bound by microtubules, and tension is generated across the kinetochores. Errors
in tension or attachment are sensed by the cell and regulated by two different mechanisms. The
Aurora B/Ipl lp kinase is required for the resolution of syntelic attachments and acts early in the
cell cycle to release inappropriate kinetochore-microtubule connections and allow the sisters to
establish bipolar attachments (Tanaka, 2002). The spindle checkpoint recognizes lesions in
attachment and generates a "wait anaphase" signal until the lesions are resolved (Gillett and
Sorger, 2001). Although the function of the spindle checkpoint is well established, it is still
39
unclear as to whether the checkpoint arrests only in response to detachment, or if it recognizes
lesions in tension as well.
1.5.1 Role of Aurora B/Ipllp
Syntelic attachments are common early in the cell cycle and must be corrected to allow
the formation of bipolar attachments. These lesions are recognized by the conserved Aurora-B
type kinase Ipllp, and its partner protein Slil5p (whose human homolog is INCENP). ipll and
sli15 mutants fail to make bipolar attachments and, instead, create monopolar links roughly
similar to those seen in dam] mutants (He et al., 2001; Tanaka et al., 2002). However, unlike
dam] mutants, if ipll mutants are allowed to establish bipolar attachments at the permissive
temperature, and then shifted to the restrictive temperature, bipolarity is maintained. This
suggests that Ipllp/Sli 5p is required for the establishment - but not the maintenance - of
bipolar attachment (Tanaka et al., 2002). Ipllp/Slil5p responds to the absence of tension in
monopolar or syntelic attachments and resolves them by stimulating microtubule detachment,
thereby freeing kinetochores to attach to the opposite SPB (Tanaka et al., 2002).
In higher eukaryotes, removal of improper attachments from kinetochores is mediated by
the kinesin MCAK, a homolog of budding yeast Kip3p. Aurora-B from Xenopus and mammals
regulates the localization of MCAK to kinetochores as well as its depolymerization activity.
Phosphorylation of MCAK by Aurora B decreases its destabilization activity in vitro and
mutation of the phosphorylation sites leads to defects in spindle structure and chromosome
attachment in vivo (Andrews et al., 2004; Gorbsky, 2004; Lan et al., 2004). The current model
suggests that tension regulates the interaction of Aurora B and MCAK by controlling their
colocalization (Li et al., 2005). In the absence of bipolar attachments, sister centromeres are
40
closely situated and MCAK and Aurora-B signals completely overlap. When chromosome
achieve bipolar attachments, Aurora-B remains at kinetochores while MCAK relocalizes to the
inner centromere, where it is believed to be more accessible to type I phosphatases that oppose
Aurora B function (Andrews et al., 2004; Hsu et al., 2000; Mumrnionet al., 2001). This suggests a
mechanism whereby unstable attachments cause microtubule depolymerization and aid in the
release of improper attachments. Once bipolar attachment occurs, the forces become more
balanced leading to oscillation rather than detachment. This mechanism reinforces the
importance of microtubule dynamics, and kinesins, in regulation of cell cycle events. Currently
the regulation of Kip3p by Ipllp is unknown; however, these data suggest that Iplp may
regulate Kip3p function in budding yeast.
Phosphorylation of other Ipllp substrates, including Ndc 10p, Cse4p, Ndc80p, the Daml
Complex proteins Damlp, Spc34p, and Asklp may also play a role in removing tensionless
attachments in budding yeast (Biggins et al., 1999; Buvelot et al., 2003; Cheeseman et al., 2002;
Shang et al., 2003; Tanaka, 2002). Damlp phosphorylation may be important for its regulation
since phosphorylation site mutations phenocopy the chromosome segregation defects in ipll-2
mutants. In addition, at semi-restrictive temperatures, the ipll-2 mutation can be suppressed by
serine-to-aspartate mutations that mimic Dam lp phosphorylation (Cheeseman et al., 2002).
Interestingly, Dam lp is not recruited to kinetochores in an ipll-2 mutant, a phenotype that is also
rescued by mimicking Damlp phosphorylation. The current model is that the phosphorylation of
Damlp by Ipllp downregulates Damlp activity and promotes bipolar attachment early in the cell
cycle (Cheeseman et al., 2002). The role of phosphorylation in the regulation of the other Ipl lp
substrates is unknown. Thus, Damlp and other proteins may be phosphorylated to decrease their
affinity for microtubules, thereby favoring chromosome detachment.
41
1.5.2 The Spindle Checkpoint
Not all attachment errors are sensed and corrected by Aurora B/Ipl lp. Therefore, the
spindle checkpoint is required to block the metaphase-to-anaphase transition until all
chromosomes have attained bipolar attachment to the mitotic spindle. In budding yeast, this
arrest depends on the protein products of the MAD, BUB, and MPSI genes (Hoyt et al., 1991; Li
and Murray, 1991; Weiss and Winey, 1996). The spindle checkpoint is extremely sensitive.
Studies in PtKI cells demonstrate that a single unattached kinetochore is sufficient to engage the
spindle checkpoint (Ault et al., 1991; Ault and Rieder, 1992). The spindle checkpoint is essential
in higher eukaryotes where each chromosome must form a novel attachment in every cell cycle.
All metazoan checkpoint proteins are localized to kinetochores in prometaphase cells, and
several of them are released as chromosomes become attached to microtubules. In budding yeast
the checkpoint is non-essential but Bublp and Bub3p associate with kinetochores until
metaphase, when they are displaced (Gillett et al., 2004; Hoyt et al., 1991; Li and Murray, 1991).
Bublp is also recruited to unattached kinetochores later in the cell cycle. Mad2p, in contrast, is
recruited only to unattached kinetochores following microtubule depolymerization or
kinetochore disruption, but not to kinetochores in a normal cell cycle (Gillett et al., 2004; Iouk et
al., 2002).
Kinetochores are required for checkpoint function. The complete inactivation of CBF3
leads to an abrogation of checkpoint control (Gardner et al., 2001; Goh and Kilmartin, 1993) and
prevents Mad and Bub proteins from associating with kinetochores in vivo. However,
hypomorphic mutations in CBF3 subunits engage the checkpoint (Doheny et al., 1993). Thus, the
checkpoint is sensitive to partially inactive kinetochores but if no kinetochores are present, then
42
checkpoint signaling complexes cannot form. A similar phenomenon is observed in DNA
replication: hypomorphic mutations in replication factors allow origins to form and engage the
DNA replication checkpoint, but complete loss-of-function mutations cause cells to skip DNA
synthesis, bypass the checkpoint, and undergo a haploid mitosis (Kelly et al., 1993; Piatti et al.,
1995). It remains unknown how checkpoint proteins associate with kinetochores and sense
attachment problems, but two-hybrid interactions have been detected between yeast Spc25p and
Madlp and between human Ndc80 and Madlp (Martin-Lluesma et al., 2002; Newman et al.,
2000). Moreover, recruitment of Bublp and Bub3p to centromeres requires some but not all
members of the Ndc80p Complex, implicating this complex in checkpoint protein recruitment
(Gillett et al., 2004).
Kinesin motors also function in the spindle checkpoint. In mammalian cells, the BubRI
kinase, similar to Mad3p in budding yeast, binds the kinesin CENP-E (Chan et al., 1999; Yao et
al., 2000). Depletion of BubRI by RNAi decreases the amount of CENP-E at kinetochores
indicating that CENP-E is recruited to kinetochores by BubRI (Johnson et al., 2004). CENP-E
stimulates the kinase activity of BubRI, and ca-CENP-E antibodies that mimic MT binding lead
to downregulation of BubRI activity (Mao et al., 2003). Therefore kinesin activity regulates the
spindle checkpoint in mammalian cells through interaction with BubRl. CENP-E has no clear
homolog in budding yeast; however, these data indicate that kinesins can regulate checkpoint
activity in addition to their roles in mediating chromosome attachments and dynamics.
The loss of kinetochore-microtubule attachment is sufficient to activate the spindle
checkpoint, but it is unclear if loss of tension alone can activate the spindle checkpoint. In
praying mantid spermatocytes, application of tension to an unpaired X chromosome by pulling
with a glass needle leads to the initiation of anaphase approximately one hour later (Li and
43
Nicklas, 1995), indicating that establishment of tension can satisfy the checkpoint. Evidence that
lack of tension activates the checkpoint in budding yeast comes from cdc6 mutants, which enter
mitosis with unreplicated chromsomes. In these cells, kinetochores form monopolar attachments
but have a spindle-checkpoint dependent delay in anaphase onset indicating that lack of tension
is sufficient to engage the checkpoint (Stem and Murray, 2001). However, there are several
caveats to these tension experiments. First, the application of tension stabilizes microtubules and
it is possible that pulling on chromosomes leads to subsequent recruitment of microtubules (King
and Nicklas, 2000). Therefore, the checkpoint could be silenced by microtubules that attach to
kinetochores after the application of tension. Second, little is known about the cell cycle
regulation of kinetochores and it is possible that kinetochores mature after replication. Therefore,
the cdc6 mutants may have an abnormal metaphase kinetochore structure, which might activate
the spindle checkpoint regardless of tension. Distinguishing tension from attachment sensing is
also complicated by Ipllp, which detaches chromosomes not under tension (Tanaka et al., 2002).
These detached chromosomes may be required to signal the spindle checkpoint.
Furthermnore,evidence from human cells indicates that loss of tension alone does not
engage the checkpoint. Depletion of the MAPs EB 1 and Adenomos Polyposis Coli lead to
decreases in interkinetochore distance signifying loss of tension. However, these cells do not
signal the checkpoint, and instead, proceed into anaphase with segregation errors (Draviam,
2005). Studies fromnbudding yeast also suggest that tension may not signal the checkpoint.
Mutations in the +TIP Stu2p cannot exert tension on the spindles and engage the spindle
checkpoint. However, visualization of chromosomes in these cells demonstrates that detached
chromosomes are present and that they recruit high levels of checkpoint proteins. These data
indicate that detached chromosomes, rather than tensionless chromosomes, are signaling the
44
spindle checkpoint. Thus, tension and attachment are extremely interconnected and convincingly
discriminating between the two is currently a major challenge in the field.
1.6 Summary:
Kinetochores are large, complex structures whose function is vital to the fidelity of
chromosome segregation. Although much is known about kinetochore structure and function
much still remains obscure. Two fundamental aspects in kinetochore function are the
kinetochore-mediated regulation of k-MT dynamics and chromosome attachment and the
temporal regulation of kinetochore activity. Therefore, during my thesis research I have focused
on kinetochore regulation of microtubule dynamics and attachment. Kinesins function in many
diverse and important aspects of chromosome segregation from attachment and regulation of
movement to regulation of spindle checkpoint activity. In Chapter 2, I present the results of my
extensive investigation into the roles of the four nuclear budding yeast kinesins. In Chapter 3, I
present preliminary data exploring the alterations in chromosome-microtubule attachment during
the cell cycle. These studies have advanced the field of kinetochore biology and suggest even
more avenues for future investigation that are detailed in Chapter 4.
45
References
Adams, I.R., and J.V. Kilmartin. 2000. Spindle pole body duplication: a model for centrosome
duplication? Trends Cell Biol. 10:329-35.
Akhmanova, A., and C.C. Hoogenraad. 2005. Microtubule plus-end-tracking proteins:
mechanisms and functions. Curr Opin Cell Biol. 17:47-54.
Albertson, D.G., and J.N. Thomson. 1982. The kinetochores of Caenorhabditis elegans.
Chromosoma. 86:409-28.
Amon, A. 1999. The spindle checkpoint. Curr Opin Genet Dev. 9:69-75.
Amos, L., and A. Klug. 1974. Arrangement of subunits in flagellar microtubules. J Cell Sci.
14:523-49.
Andrews, P.D., Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Wordeman, and J.R.
Swedlow. 2004. Aurora B regulates MCAK at the mitotic centromere. Dev Cell. 6:25368.
Asbury, C.L. 2005. Kinesin: world's tiniest biped. Curr Opin Cell Biol. 17:89-97.
Ault, J.G., A.J. DeMarco, E.D. Salmon, and C.L. Rieder. 1991. Studies on the ejection properties
of asters: astral microtubule turnover influences the oscillatory behavior and positioning
of mono-oriented chromosomes. J Cell Sci. 99 ( Pt 4):701-10.
Ault, J.G., and C.L. Rieder. 1992. Chromosome mal-orientation and reorientation during mitosis.
Cell Motil Cytoskeleton. 22:155-9.
Banks, J.D., and R. Heald. 2001. Chromosome movement: dynein-out at the kinetochore. Curr
Biol. 11 :R128-31.
Berlin, V., C.A. Styles, and G.R. Fink. 1990. BIK1, a protein required for microtubule function
during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell
Biol. 111:2573-86.
Bharadwaj, R., W. Qi, and H. Yu. 2004. Identification of two novel components of the human
NDC80 kinetochore complex. JBiol Chem. 279:13076-85.
Biggins, S., F.F. Severin, N. Bhalla, I. Sassoon, A.A. Hyman, and A.W. Murray. 1999. The
conserved protein kinase Ipl 1 regulates microtubule binding to kinetochores in budding
yeast. Genes Dev. 13:532-44.
Bittel, D.C., and M.G. Butler. 2005. Prader-Willi syndrome: clinical genetics, cytogenetics and
molecular biology. Expert Rev Mol Med. 7:1-20.
Blat, Y., and N. Kleckner. 1999. Cohesins bind to preferential sites along yeast chromosome III,
with differential regulation along arms versus the centric region. Cell. 98:249-59.
Bloom, K. 1993. The centromere frontier: kinetochore components, microtubule-based motility,
and the CEN-value paradox. Cell. 73:621-4.
Boveri, T. 1907. Zellenstudien VI: Die Entwicklung dispermer Seeigelier. Ein Beitrag zur
Befruchtungslehre und zur Theorie des Kernes. Naturw. 43:1-292.
Brinkley, B.R., and J. Cartwright, Jr. 1971. Ultrastructural analysis of mitotic spindle elongation
in mammalian cells in vitro. Direct microtubule counts. J Cell Biol. 50:416-31.
Buvelot, S., S.Y. Tatsutani, D. Vermaak, and S. Biggins. 2003. The budding yeast Ipll/Aurora
protein kinase regulates mitotic spindle disassembly. J Cell Biol. 160:329-39.
Cai, M., and R.W. Davis. 1990. Yeast centromere binding protein CBF , of the helix-loop-helix
protein family, is required for chromosome stability and methionine prototrophy. Cell.
61:437-46.
46
Carson, D.R., and M.F. Christman. 2001. Evidence that replication fork components catalyze
establishment of cohesion between sister chromatids. Proc Natl Acad Sci U S A. 98:82705.
Carvalho, P., M.L. Gupta, Jr., M.A. Hoyt, and D. Pellman. 2004. Cell cycle control of kinesinmediated transport of Bikl (CLIP-170) regulates microtubule stability and dynein
activation. Dev Cell. 6:815-29.
Chan, G.K., S.A. Jablonski, V. Sudakin, J.C. Hittle, and T.J. Yen. 1999. Human BUBR1 is a
mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the
cyclosome/APC. J Cell Biol. 146:941-54.
Cheeseman, I.M., S. Anderson, M. Jwa, E.M. Green, J. Kang, J.R. Yates, 3rd, C.S. Chan, D.G.
Drubin, and G. Barnes. 2002. Phospho-regulation of kinetochore-microtubule
attachments by the Aurora kinase Ipllp. Cell. 111:163-72.
Cheeseman, I.M., M. Enquist-Newman, T. Muller-Reichert, D.G. Drubin, and G. Barnes. 2001.
Mitotic spindle integrity and kinetochore function linked by the Duo Ip/Dam p complex.
JCellBiol. 152:197-212.
Choo, K.H. 2001. Domain organization at the centromere and neocentromere. Dev Cell. 1:16577.
Ciferri, C., J. De Luca, S. Monzani, K. Ferrari, D. Ristic, C. Wyman, H. Stark, J. Kilmartin, E.D.
Salmon, and A. Musacchio. 2005. Architecture of the human HEC 1/NDC80 complex, a
critical constitutent of the outer kinetochore. J Biol Chem.
Ciosk, R., W. Zachariae, C. Michaelis, A. Shevchenko, M. Mann, and K. Nasmyth. 1998. An
ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to
anaphase transition in yeast. Cell. 93:1067-76.
Clarke, L., and J. Carbon. 1980. Isolation of a yeast centromere and construction of functional
small circular chromosomes. Nature. 287:504-9.
Connelly, C., and P. Hieter. 1996. Budding yeast SKP1 encodes an evolutionarily conserved
kinetochore protein required for cell cycle progression. Cell. 86:275-85.
Cottarel, G., J.H. Shero, P. Hieter, and J.H. Hegemann. 1989. A 125-base-pair CEN6 DNA
fragment is sufficient for complete meiotic and mitotic centromere functions in
Saccharomyces cerevisiae. Mol Cell Biol. 9:3342-9.
Cottingham, F.R., and M.A. Hoyt. 1997. Mitotic spindle positioning in Saccharomyces
cerevisiae is accomplished by antagonistically acting microtubule motor proteins. J Cell
Biol. 138:1041-53.
Coue, M., V.A. Lombillo, and J.R. McIntosh. 1991. Microtubule depolymerization promotes
particle and chromosome movement in vitro. J Cell Biol. 112:1165-75.
De Wulf, P., A.D. McAinsh, and P.K. Sorger. 2003. Hierarchical assembly of the budding yeast
kinetochore from multiple subcomplexes. Genes Dev. 17:2902-21.
DeLuca, J.G., Y. Dong, P. Hergert, J. Strauss, J.M. Hickey, E.D. Salmon, and B.F. McEwen.
2005. Hec and nuf2 are core components of the kinetochore outer plate essential for
organizing microtubule attachment sites. Mol Biol Cell. 16:519-31.
DeLuca, J.G., B. Moree, J.M. Hickey, J.V. Kilmartin, and E.D. Salmon. 2002. hNuf2 inhibition
blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa
cells. J Cell Biol. 159:549-55.
Desai, A., and T.J. Mitchison. 1997. Microtubule polymerization dynamics. Annu Rev Cell Dev
Biol. 13:83-117.
47
Desai, A., S. Verma, T.J. Mitchison, and C.E. Walczak. 1999. Kin I kinesins are microtubuledestabilizing enzymes. Cell. 96:69-78.
Doheny, K.F., P.K. Sorger, A.A. Hyman, S. Tugendreich, F. Spencer, and P. Hieter. 1993.
Identification of essential components of the S. cerevisiae kinetochore. Cell. 73:761-74.
Dorn, J.F., K. Jaqaman, D. R. Rines, G. S. Jelson, P. K. Sorger, and G. Danuser. 2005.
Interphase kinetochore microtubule dynamics in yeast analyzed by high-resolution
microscopy. Biophys. J. accepted.
Draviam, V.M. 2005. Misorientation and reduced centromeric stretching promote chromosome
missegregation. manuscript in preparation.
Draviam, V.M., S. Xie, and P.K. Sorger. 2004. Chromosome segregation and genomic stability.
Curr Opin GenetDev. 14:120-5.
Echeverri, C.J.. B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular characterization
of the 50-kD subunit of dynactin reveals function for the complex in chromosome
alignment and spindle organization during mitosis. J Cell Biol. 132:617-33.
Enquist-Newman, M., I.M. Cheeseman, D. Van Goor, D.G. Drubin, P.B. Meluh, and G. Barnes.
2001. Dad Ip, third component of the Duo 1p/Dam I p complex involved in kinetochore
function and mitotic spindle integrity. Mol Biol Cell. 12:2601-13.
Espelin, C.W., K.T. Simons, S.C. Harrison, and P.K. Sorger. 2003. Binding of the essential
Saccharomyces cerevisiae kinetochore protein Ndc lOp to CDEII. Mol Biol Cell. 14:455768.
Euskirchen, G.M. 2002. Nnflp, Dsnlp, Mtwlp, and Nsllp: a new group of proteins important
for chromosome segregation in Saccharomyces cerevisiae. Eukaryot Cell. 1:229-40.
Fitzgerald-Hayes, M., L. Clarke, and J. Carbon. 1982. Nucleotide sequence comparisons and
functional analysis of yeast centromere DNAs. Cell. 29:235-44.
Gard, D.L., B.E-.Becker, and S. Josh Romney. 2004. MAPping the eukaryotic tree of life:
structure, function, and evolution of the MAP215/Dis family of microtubule-associated
proteins. Int Rev Cytol. 239:179-272.
Gard, D.L., and M.W. Kirschner. 1987. A microtubule-associated protein from Xenopus eggs
that specifically promotes assembly at the plus-end. J Cell Biol. 105:2203-15.
Gardner, R.D., A. Poddar, C. Yellman, P.A. Tavormina, M.C. Monteagudo, and D.J. Burke.
2001. The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore
function and maps to the CBF3 domain. Genetics. 157:1493-502.
Gillett, E.S., C.W. Espelin, and P.K. Sorger. 2004. Spindle checkpoint proteins and
chromosome-microtubule attachment in budding yeast. J Cell Biol. 164:535-46.
Gillett, E.S., and P.K. Sorger. 2001. Tracing the pathway of spindle assembly checkpoint
signaling. Dev Cell. 1:162-4.
Goh, P.Y., and J.V. Kilmartin. 1993. NDC10: a gene involved in chromosome segregation in
Saccharomyces cerevisiae. J Cell Biol. 121:503-12.
Goldstein, L.S., and A.V. Philp. 1999. The road less traveled: emerging principles of kinesin
motor utilization. Annu Rev Cell Dev Biol. 15:141-83.
Gorbsky, G.J. 2004. Mitosis: MCAK under the aura of Aurora B. Curr Biol. 14:R346-8.
Goshima, G., T. Kiyomitsu, K. Yoda, and M. Yanagida. 2003. Human centromere chromatin
protein hMis 12, essential for equal segregation, is independent of CENP-A loading
pathway. J Cell Biol. 160:25-39.
48
Goshima, G., and M. Yanagida. 2000. Establishing biorientation occurs with precocious
separation of the sister kinetochores, but not the arms, in the early spindle of budding
yeast. Cell. 100:619-33.
Gruber, S., C.H. Haering, and K. Nasmyth. 2003. Chromosomal cohesin forms a ring. Cell.
112:765-77.
Guacci, V., E. Hogan, and D. Koshland. 1997a. Centromere position in budding yeast: evidence
for anaphase A. Mol Biol Cell. 8:957-72.
Guacci, V., D. Koshland, and A. Strunnikov. 1997b. A direct link between sister chromatid
cohesion and chromosome condensation revealed through the analysis of MCD1 in S.
cerevisiae. Cell. 91:47-57.
Hays, T.S., and E.D. Salmon. 1990. Poleward force at the kinetochore in metaphase depends on
the number of kinetochore microtubules. J Cell Biol. 110:391-404.
He, X., S. Asthana, and P.K. Sorger. 2000. Transient sister chromatid separation and elastic
deformation of chromosomes during mitosis in budding yeast. Cell. 101:763-75.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochoremicrotubule attachment in budding yeast. Cell. 106:195-206.
Heald, R., and E. Nogales. 2002. Microtubule dynamics. J Cell Sci. 115:3-4.
Heald, R., and C.E. Walczak. 1999. Microtubule-based motor function in mitosis. Curr Opin
Struct Biol. 9:268-74.
Heus, J.J., B.J. Zonneveld, H.Y. Steensma, and J.A. Van den Berg. 1990. Centromeric DNA of
Kluyveromyces lactis. Curr Genet. 18:517-22.
Higuchi, T., and F. Uhlmann. 2005. Stabilization of microtubule dynamics at anaphase onset
promotes chromosome segregation. Nature. 433:171-6.
Hoffman, D.B.. C .G. Pearson, T.J. Yen, B.J. Howell, and E.D. Salmon. 2001. Microtubuledependent changes in assembly of microtubule motor proteins and mitotic spindle
checkpoint, proteins at PtK1 kinetochores. Mol Biol Cell. 12:1995-2009.
Hofmann, C., I.M. Cheeseman, B.L. Goode, K.L. McDonald, G. Barnes, and D.G. Drubin. 1998.
Saccharomyces cerevisiae Duolp and Damlp, novel proteins involved in mitotic spindle
function. J Cell Biol. 143:1029-40.
Hoyt, M.A., L. Totis, and B.T. Roberts. 1991. S. cerevisiae genes required for cell cycle arrest in
response to loss of microtubule function. Cell. 66:507-17.
Hsu, J.Y., Z.W. Sun, X. Li, M. Reuben, K. Tatchell, D.K. Bishop, J.M. Grushcow, C.J. Brame,
J.A. Caldwell, D.F. Hunt, R. Lin, M.M. Smith, and C.D. Allis. 2000. Mitotic
phosphorylation of histone H3 is governed by Ipl /aurora kinase and Glc7/PP 1
phosphatase in budding yeast and nematodes. Cell. 102:279-91.
Hunt, A.J., and J.R. McIntosh. 1998. The dynamic behavior of individual microtubules
associated with chromosomes in vitro. Mol Biol Cell. 9:2857-71.
Hyland, K.M., J..Kingsbury, D. Koshland, and P. Hieter. 1999. Ctfl9p: A novel kinetochore
protein in Saccharomyces cerevisiae and a potential link between the kinetochore and
mitotic spindle. J Cell Biol. 145:15-28.
Hyman, A.A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubulemotor activity of a yeast centromere-binding protein complex. Nature. 359:533-6.
Hyman, A.A., and P.K. Sorger. 1995. Structure and function of kinetochores in budding yeast.
Annu Rev Cell Dev Biol. 11:471-95.
49
Iouk, T., O. Kerscher, R.J. Scott, M.A. Basrai, and R.W. Wozniak. 2002. The yeast nuclear pore
complex functionally interacts with components of the spindle assembly checkpoint. J
Cell Biol. 159:807-19.
Janke, C., J. Ortiz, J. Lechner, A. Shevchenko, M.M. Magiera, C. Schramm, and E. Schiebel.
2001. The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at
the kinetochore and are important for kinetochore clustering and checkpoint control.
Embo J. 20:777-91.
Janke, C., J. Ortiz, T.U. Tanaka, J. Lechner, and E. Schiebel. 2002. Four new subunits of the
Dam 1-Duo 1 complex reveal novel functions in sister kinetochore biorientation. Embo J.
21:181-93.
Jiang, Y.H., J. Bressler, and A.L. Beaudet. 2004. Epigenetics and human disease. Annu Rev
Genomics Hum Genet. 5:479-510.
Jin, Q.W., J. Fuchs, and J. Loidl. 2000. Centromere clustering is a major determinant of yeast
interphase nuclear organization. J Cell Sci. 113:1903-12.
Johnson, V.L., M.I. Scott, S.V. Holt, D. Hussein, and S.S. Taylor. 2004. Bubl is required for
kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome
congression. J Cell Sci. 117:1577-89.
Jones, M.H., J.B. Bachant, A.R. Castillo, T.H. Giddings, Jr., and M. Winey. 1999. Yeast Damlp
is required to maintain spindle integrity during mitosis and interacts with the Mps lp
kinase. Mol Biol Cell. 10:2377-91.
Jones, M.H., X. He, T.H. Giddings, and M. Winey. 2001. Yeast Damlp has a role at the
kinetochore in assembly of the mitotic spindle. Proc Natl Acad Sci U S A. 98:13675-80.
Keith, K.C., R.E. Baker, Y. Chen, K. Harris, S. Stoler, and M. Fitzgerald-Hayes. 1999. Analysis
of primary structural determinants that distinguish the centromere-specific function of
histone variant Cse4p from histone H3. Mol Cell Biol. 19:6130-9.
Kelly, T.J., G.S. Martin, S.L. Forsburg, R.J. Stephen, A. Russo, and P. Nurse. 1993. The fission
yeast cdc 18+ gene product couples S phase to START and mitosis. Cell. 74:371-82.
Kerres, A., C. Vietmeier-Decker,
J. Ortiz, I. Karig, C. Beuter, J. Hegemann, J. Lechner, and U.
Fleig. 2004. The fission yeast kinetochore component Spc7 associates with the EB 1
family member Mal3 and is required for kinetochore-spindle association. Mol Biol Cell.
15:5255-67.
King, J.M., T.S. Hays, and R.B. Nicklas. 2000. Dynein is a transient kinetochore component
whose binding is regulated by microtubule attachment, not tension. J Cell Biol. 151:73948.
King, J.M., and R.B. Nicklas. 2000. Tension on chromosomes increases the number of
kinetochore microtubules but only within limits. J Cell Sci. 113 Pt 21:3815-23.
Kirschner, M.W., and T. Mitchison. 1986. Microtubule dynamics. Nature. 324:621.
Kitagawa, K., and P. Hieter. 2001. Evolutionary conservation between budding yeast and human
kinetochores. Nat Rev Mol Cell Biol. 2:678-87.
Kosco, K.A., C.G. Pearson, P.S. Maddox, P.J. Wang, I.R. Adams, E.D. Salmon, K. Bloom, and
T.C. Huffaker. 2001. Control of microtubule dynamics by stu2p is essential for spindle
orientation and metaphase chromosome alignment in yeast. Mol Biol Cell. 12:2870-80.
Koshland, D.E.. T.J. Mitchison, and M.W. Kirschner. 1988. Polewards chromosome movement
driven by microtubule depolymerization in vitro. Nature. 331:499-504.
Lan, W., X. Zhang, S.L. Kline-Smith, S.E. Rosasco, G.A. Barrett-Wilt, J. Shabanowitz, D.F.
Hunt, C.E. Walczak, and P.T. Stukenberg. 2004. Aurora B phosphorylates centromeric
50
MCAK and regulates its localization and microtubule depolymerization activity. Curr
Biol. 14:273-86.
Lawrence, C.J., R.K. Dawe, K.R. Christie, D.W. Cleveland, S.C. Dawson, S.A. Endow, L.S.
Goldstein, H.V. Goodson, N. Hirokawa, J. Howard, R.L. Malmberg, J.R. McIntosh, H.
Miki, T.J. Mitchison, Y. Okada, A.S. Reddy, W.M. Saxton, M. Schliwa, J.M. Scholey,
R.D. Vale, C.E. Walczak, and L. Wordeman. 2004. A standardized kinesin nomenclature.
J Cell Biol. 167:19-22.
Lechner, J., and J. Carbon. 1991. A 240 kd multisubunit protein complex, CBF3, is a major
component of the budding yeast centromere. Cell. 64:717-25.
Li, J.M., Y. Li, and S.J. Elledge. 2005. Genetic analysis of the kinetochore DASH complex
reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel
subunit required for Askl association. Mol Cell Biol. 25:767-78.
Li, R., and A.W. Murray. 1991. Feedback control of mitosis in budding yeast. Cell. 66:519-31.
Li, X., and R.B. Nicklas. 1995. Mitotic forces control a cell-cycle checkpoint. Nature. 373:6302.
Li, Y., J. Bachant, A.A. Alcasabas, Y. Wang, J. Qin, and S.J. Elledge. 2002. The mitotic spindle
is required for loading of the DASH complex onto the kinetochore. Genes Dev. 16:18397.
Lin, H., J.H. Choi, J. Hasek, N. DeLillo, W. Lou, and A. Vancura. 2000. Phospholipase C is
involved in kinetochore function in Saccharomyces cerevisiae. Mol Cell Biol. 20:3597607.
Lin, H., P. de Carvalho, D. Kho, C.Y. Tai, P. Pierre, G.R. Fink, and D. Pellman. 2001.
Polyploids require Bikl for kinetochore-microtubule attachment. J Cell Biol. 155:117384.
Lowe, X., B. Eskenazi, D.O. Nelson, S. Kidd, A. Alme, and A.J. Wyrobek. 2001. Frequency of
XY sperm increases with age in fathers of boys with Klinefelter syndrome. Am J Hum
Genet. 69:1046-54.
Maddox, P.S., K.S. Bloom, and E.D. Salmon. 2000. The polarity and dynamics of microtubule
assembly in the budding yeast Saccharomyces cerevisiae. Nat Cell Biol. 2:36-41.
Maekawa, H., and E. Schiebel. 2004. CLIP-170 family members: a motor-driven ride to
microtubule plus ends. Dev Cell. 6:746-8.
Maney, T., L.M. Ginkel, A.W. Hunter, and L. Wordeman. 2000. The kinetochore of higher
eucaryotes: a molecular view. Int Rev Cytol. 194:67-131.
Mao, Y., A. Abrieu, and D.W. Cleveland. 2003. Activating and silencing the mitotic checkpoint
through CENP-E-dependent activation/inactivation of BubRl. Cell. 114:87-98.
Martin-Lluesma, S., V.M. Stucke, and E.A. Nigg. 2002. Role of Hecl in spindle checkpoint
signaling and kinetochore recruitment of Mad 1l/Mad2.Science. 297:2267-70.
Mayer, M.L., I. Pot, M. Chang, H. Xu, V. Aneliunas, T. Kwok, R. Newitt, R. Aebersold, C.
Boone, G.W. Brown, and P. Hieter. 2004. Identification of protein complexes required
for efficient sister chromatid cohesion. Mol Biol Cell. 15:1736-45.
Mazumdar, M., and T. Misteli. 2005. Chromokinesins: multitalented players in mitosis. Trends
Cell Biol. 15:349-55.
McCleland, M.L., R.D. Gardner, M.J. Kallio, J.R. Daum, G.J. Gorbsky, D.J. Burke, and P.T.
Stukenberg. 2003. The highly conserved Ndc80 complex is required for kinetochore
assembly, chromosome congression, and spindle checkpoint activity. Genes Dev. 17:10114.
51
McCleland, M.L., M.J. Kallio, G.A. Barrett-Wilt, C.A. Kestner, J. Shabanowitz, D.F. Hunt, G.J.
Gorbsky, and P.T. Stukenberg. 2004. The vertebrate Ndc80 complex contains Spc24 and
Spc25 homologs, which are required to establish and maintain kinetochore-microtubule
attachment. Curr Biol. 14:131-7.
McEwen, B.F., A.B. Heagle, G.O. Cassels, K.F. Buttle, and C.L. Rieder. 1997. Kinetochore fiber
maturation in PtK1 cells and its implications for the mechanisms of chromosome
congression and anaphase onset. J Cell Biol. 137:1567-80.
Measday, V., D.W. Hailey, I. Pot, S.A. Givan, K.M. Hyland, G. Cagney, S. Fields, T.N. Davis,
and P. Hieter. 2002. Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and
Mcml 6p at the yeast outer kinetochore. Genes Dev. 16:101-13.
Meluh, P.B., and D. Koshland. 1997. Budding yeast centromere composition and assembly as
revealed by in vivo cross-linking. Genes Dev. 11:3401-12.
Meluh, P.B., P.. Yang, L. Glowczewski, D. Koshland, and M.M. Smith. 1998. Cse4p is a
component of the core centromere of Saccharomyces cerevisiae. Cell. 94:607-13.
Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cohesins: chromosomal proteins that prevent
premature separation of sister chromatids. Cell. 91:35-45.
Miranda, J.J., P. De Wulf, P.K. Sorger, and S.C. Harrison. 2005. The yeast DASH complex
forms closed rings on microtubules. Nat Struct Mol Biol. 12:138-43.
Mitchison, T., and M. Kirschner. 1984. Dynamic instability of microtubule growth. Nature.
312:237-42.
Mumion, M.E., R.R. Adams, D.M. Callister, C.D. Allis, W.C. Earnshaw, and J.R. Swedlow.
2001. Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3
phosphorylation. J Biol Chem. 276:26656-65.
Nasmyth, K. 2005. How might cohesin hold sister chromatids together? Philos Trans R Soc Lond
B Biol Sci. 360:483-96.
Nekrasov, V.S., M.A. Smith, S. Peak-Chew, and J.V. Kilmartin. 2003. Interactions between
centromere complexes in Saccharomyces cerevisiae. Mol Biol Cell. 14:4931-46.
Newman, J.R., E. Wolf, and P.S. Kim. 2000. A computationally directed screen identifying
interacting coiled coils from Saccharomyces cerevisiae. Proc NatlAcadSci USA.
97:13203-8.
Ng, R., J. Ness, and J. Carbon. 1986. Structural studies on centromeres in the yeast
Saccharomyces cerevisiae. Basic Life Sci. 40:479-92.
Obuse, C., O. Iwasaki, T. Kiyomitsu, G. Goshima, Y. Toyoda, and M. Yanagida. 2004. A
conserved Mis 12 centromere complex is linked to heterochromatic HP1 and outer
kinetochore protein Zwint- 1. Nat Cell Biol. 6:1135-41.
Ortiz, J., O. Stemmann, S. Rank, and J. Lechner. 1999. A putative protein complex consisting of
Ctfl 9, Mcm21, and Okp 1 represents a missing link in the budding yeast kinetochore.
Genes L)ev. 13:1140-55.
O'Toole, E.T., M. Winey, and J.R. McIntosh. 1999. High-voltage electron tomography of spindle
pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol Biol
Cell. 10:2017-31.
Pearson, C.G., and K. Bloom. 2004. Dynamic microtubules lead the way for spindle positioning.
Nat Rev Mol Cell Biol. 5:481-92.
Pearson, C.G., E. Yeh, M. Gardner, D. Odde, E.D. Salmon, and K. Bloom. 2004. Stable
kinetochore-microtubule attachment constrains centromere positioning in metaphase.
Curr Biol. 14:1962-7.
52
Perez, F., G.S. Diamantopoulos, R. Stalder, and T.E. Kreis. 1999. CLIP-170 highlights growing
microtubule ends in vivo. Cell. 96:517-27.
Peters, J.M. 2002. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol
Cell. 9:931-43.
Piatti, S., C. Lengauer, and K. Nasmyth. 1995. Cdc6 is an unstable protein whose de novo
synthesis in G 1 is important for the onset of S phase and for preventing a 'reductional'
anaphase in the budding yeast Saccharomyces cerevisiae. Embo J. 14:3788-99.
Pinsky, B.A., S.Y. Tatsutani, K.A. Collins, and S. Biggins. 2003. An Mtwl complex promotes
kinetochore biorientation that is monitored by the IpI 1/Aurora protein kinase. Dev Cell.
5:735-45.
Rieder, C.L. 1982. The formation, structure, and composition of the mammalian kinetochore and
kinetochore fiber. Int Rev Cytol. 79:1-58.
Rieder, C.L., and S.P. Alexander. 1990. Kinetochores are transported poleward along a single
astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell
Biol. 110:81-95.
Rieder, C.L., E.A. Davison, L.C. Jensen, L. Cassimeris, and E.D. Salmon. 1986. Oscillatory
movements of monooriented chromosomes and their position relative to the spindle pole
result fiom the ejection properties of the aster and half-spindle. J Cell Biol. 103:581-91.
Rieder, C.L., and E.D. Salmon. 1998. The vertebrate cell kinetochore and its roles during
mitosis. Trends Cell Biol. 8:310-8.
Sanyal, K., M. Baum, and J. Carbon. 2004. Centromeric DNA sequences in the pathogenic yeast
Candida albicans are all different and unique. Proc Natl AcadSci U SA. 101:11374-9.
Sanyal, K., and J. Carbon. 2002. The CENP-A homolog CaCse4p in the pathogenic yeast
Candida albicans is a centromere protein essential for chromosome transmission. Proc
NatlAcadSci USA. 99:12969-74.
Shang, C., T.R. Hazbun, I.M. Cheeseman, J. Aranda, S. Fields, D.G. Drubin, and G. Barnes.
2003. Kinetochore protein interactions and their regulation by the Aurora kinase Ipl p.
Mol Biol Cell. 14:3342-55.
Sharp, D.J. 2002. Cell division: MAST sails through mitosis. Curr Biol. 12:R585-7.
Sharp, D.J., G.C. Rogers, and J.M. Scholey. 2000. Microtubule motors in mitosis. Nature.
407:41-7.
Sharp, J.A., A.A. Franco, M.A. Osley, and P.D. Kaufman. 2002. Chromatin assembly factor I
and Hir proteins contribute to building functional kinetochores in S. cerevisiae. Genes
Dev. 16:85-100.
Shelby, R.D., K.M. Hahn, and K.F. Sullivan. 1996. Dynamic elastic behavior of alpha-satellite
DNA domains visualized in situ in living human cells. J Cell Biol. 135:545-57.
Sorger, P.K., F.F. Severin, and A.A. Hyman. 1994. Factors required for the binding of
reassembled yeast kinetochores to microtubules in vitro. J Cell Biol. 127:995-1008.
Stern, B.M., and A.W. Murray. 2001. Lack of tension at kinetochores activates the spindle
checkpoint in budding yeast. Curr Biol. 11:1462-7.
Stoler, S., K.C. Keith, K.E. Curnick, and M. Fitzgerald-Hayes. 1995. A mutation in CSE4, an
essential gene encoding a novel chromatin-associated protein in yeast, causes
chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9:573-86.
Storchova, Z., and D. Pellman. 2004. From polyploidy to aneuploidy, genome instability and
cancer.Nat Rev Mol Cell Biol. 5:45-54.
Struhl, K. 2001. Gene regulation. A paradigm for precision. Science. 293:1054-5.
53
Strunnikov, A.V., J. Kingsbury, and D. Koshland. 1995. CEP3 encodes a centromere protein of
Saccharomyces cerevisiae. J Cell Biol. 128:749-60.
Sullivan, M., C. Lehane, and F. Uhlmann. 2001. Orchestrating anaphase and mitotic exit:
separase cleavage and localization of Slkl9. Nat Cell Biol. 3:771-7.
Takahashi, K., E.S. Chen, and M. Yanagida. 2000. Requirement of Mis6 centromere connector
for localizing a CENP-A-like protein in fission yeast. Science. 288:2215-9.
Tanaka, K., N. Mukae, H. Dewar, M. van Breugel, E.K. James, A.R. Prescott, C. Antony, and
T.U. Tanaka. 2005. Molecular mechanisms of kinetochore capture by spindle
microtubules. Nature. 434:987-94.
Tanaka, T., M.P. Cosma, K. Wirth, and K. Nasmyth. 1999. Identification of cohesin association
sites at centromeres and along chromosome arms. Cell. 98:847-58.
Tanaka, T., J. Fuchs, J. Loidl, and K. Nasmyth. 2000. Cohesin ensures bipolar attachment of
microtubules to sister centromeres and resists their precocious separation. Nat Cell Biol.
2:492-9.
Tanaka, T.U. 2002. Bi-orienting chromosomes on the mitotic spindle. Curr Opin Cell Biol.
14:365-71..
Tanaka, T.U., N. Rachidi, C. Janke, G. Pereira, M. Galova, E. Schiebel, M.J. Stark, and K.
Nasmyth. 2002. Evidence that the Ipl 1-Sli 15 (Aurora kinase-INCENP) complex
promotes chromosome bi-orientation by altering kinetochore-spindle pole connections.
Cell. 108:317-29.
Tempest, H.G., S.T. Homa, M. Dalakiouridou, D. Christopikou, D. Wright, X.P. Zhai, and D.K.
Griffin. 2004. The association between male infertility and sperm disomy: evidence for
variation in disomy levels among individuals and a correlation between particular semen
parameters and disomy of specific chromosome pairs. ReprodBiol Endocrinol. 2:82.
Tirnauer, J.S., .C. Canman, E.D. Salmon, and T.J. Mitchison. 2002. EB Targets to
Kinetochores with Attached, Polymerizing Microtubules. Mol Biol Cell. 13:4308-16.
Timauer, J.S., E. O'Toole, L. Berrueta, B.E. Bierer, and D. Pellman. 1999. Yeast Bimlp
promotes the Gl-specific dynamics of microtubules. J Cell Biol. 145:993-1007.
Tong, A.H., G. Lesage, G.D. Bader, H. Ding, H. Xu, X. Xin, J. Young, G.F. Berriz, R.L. Brost,
M. Chang, Y. Chen, X. Cheng, G. Chua, H. Friesen, D.S. Goldberg, J. Haynes, C.
Humphries, G. He, S. Hussein, L. Ke, N. Krogan, Z. Li, J.N. Levinson, H. Lu, P. Menard,
C. Munyana, A.B. Parsons, O. Ryan, R. Tonikian, T. Roberts, A.M. Sdicu, J. Shapiro, B.
Sheikh, B. Suter, S.L. Wong, L.V. Zhang, H. Zhu, C.G. Burd, S. Munro, C. Sander, J.
Rine, J. Greenblatt, M. Peter, A. Bretscher, G. Bell, F.P. Roth, G.W. Brown, B. Andrews,
H. Bussey, and C. Boone. 2004. Global mapping of the yeast genetic interaction network.
Science. 303:808-13.
Tournebize, R., A. Popov, K. Kinoshita, A.J. Ashford, S. Rybina, A. Pozniakovsky, T.U. Mayer,
C.E. Walczak, E. Karsenti, and A.A. Hyman. 2000. Control of microtubule dynamics by
the antagonistic activities of XMAP215 and XKCM 1 in Xenopus egg extracts. Nat Cell
Biol. 2:13-9.
Uhlmann, F. 2004. The mechanism of sister chromatid cohesion. Exp Cell Res. 296:80-5.
Vagnarelli, P., and W.C. Earnshaw. 2004. Chromosomal passengers: the four-dimensional
regulation of mitotic events. Chromosoma. 113:211-22.
Vaisberg, E.A., M.P. Koonce, and J.R. McIntosh. 1993. Cytoplasmic dynein plays a role in
mammalian mitotic spindle formation. J Cell Biol. 123:849-58.
54
Van Breugel, M., D. Drechsel, and A. Hyman. 2003. Stu2p, the budding yeast member of the
conserved Disl/XMAP215 family of microtubule-associated proteins is a plus endbinding microtubule destabilizer. J Cell Biol. 161:359-69.
Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. XMAP from Xenopus eggs promotes rapid
plus end assembly of microtubules and rapid microtubule polymer turnover. J Cell Biol.
127:985-93.
Walker, R.A., E.T. O'Brien, N.K. Pryer, M.F. Soboeiro, W.A. Voter, H.P. Erickson, and E.D.
Salmon. 1988. Dynamic instability of individual microtubules analyzed by video light
microscopy: rate constants and transition frequencies. J Cell Biol. 107:1437-48.
Wang, P.J., and T.C. Huffaker. 1997. Stu2p: A microtubule-binding protein that is an essential
component of the yeast spindle pole body. J Cell Biol. 139:1271-80.
Wei, R.R., P.K. Sorger, and S.C. Harrison. 2005. Molecular organization of the Ndc80 complex,
an essential kinetochore component. Proc Natl Acad Sci U S A. 102:5363-7.
Weiss, E., and M. Winey. 1996. The Saccharomyces cerevisiae spindle pole body duplication
gene MPS 1 is part of a mitotic checkpoint. J Cell Biol. 132:111-23.
Westermann, S., A. Avila-Sakar, H.W. Wang, H. Niederstrasser, J. Wong, D.G. Drubin, E.
Nogales, and G. Barnes. 2005. Formation of a dynamic kinetochore- microtubule
interface through assembly of the Daml ring complex. Mol Cell. 17:277-90.
Westermann, S., I.M. Cheeseman, S. Anderson, J.R. Yates, 3rd, D.G. Drubin, and G. Barnes.
2003. Architecture of the budding yeast kinetochore reveals a conserved molecular core.
J CellBiol. 163:215-22.
Wieland, G., S. Orthaus, S. Ohndorf, S. Diekmann, and P. Hemmerich. 2004. Functional
complemrnentationof human centromere protein A (CENP-A) by Cse4p from
Saccharomyces cerevisiae. Mol Cell Biol. 24:6620-30.
Wigge, P.A., and J.V. Kilmartin. 2001. The Ndc80p complex from Saccharomyces cerevisiae
contains conserved centromere components and has a function in chromosome
segregation. J Cell Biol. 152:349-60.
Winey, M., C.L. Mamay, E.T. O'Toole, D.N. Mastronarde, T.H. Giddings, Jr., K.L. McDonald,
and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the
Saccharomyces cerevisiae mitotic spindle. J Cell Biol. 129:1601-15.
Yamamoto, A., V. Guacci, and D. Koshland. 1996a. Pdslp is required for faithful execution of
anaphase in the yeast, Saccharomyces cerevisiae. J Cell Biol. 133:85-97.
Yamamoto, A., V. Guacci, and D. Koshland. 1996b. Pds lp, an inhibitor of anaphase in budding
yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Biol. 133:99110.
Yao, X., A. Abrieu, Y. Zheng, K.F. Sullivan, and D.W. Cleveland. 2000. CENP-E forms a link
between attachment of spindle microtubules to kinetochores and the mitotic checkpoint.
Nat Cell Biol. 2:484-91.
Yin, H., L. You, D. Pasqualone, K.M. Kopski, and T.C. Huffaker. 2002. Stulp is physically
associated with beta-tubulin and is required for structural integrity of the mitotic spindle.
Mol Biol Cell. 13:1881-92.
Zeng, X., J.A. Kahana, P.A. Silver, M.K. Morphew, J.R. McIntosh, I.T. Fitch, J. Carbon, and
W.S. Saunders. 1999. Slkl9p is a centromere protein that functions to stabilize mitotic
spindles. J Cell Biol. 146:415-25.
55
Zheng, L., Y. Chen, and W.H. Lee. 1999. Hec lp, an evolutionarily conserved coiled-coil protein,
modulates chromosome segregation through interaction with SMC proteins. Mol Cell
Biol. 19:5417-28.
56
CHAPTER TWO
Functional Analysis of Kinesins at
Budding Yeast Kinetochores
I have executed and designed all the experiments in this chapter with the exception of G1
imaging of Kar3 (Figure 2.9) which was performed by research technician Greg Jelson.
2.1 Abstract
Accurate chromosome segregation during mitosis requires biorientation of sister
chromatids on the microtubules of the mitotic spindle. Chromosome-microtubule binding is
mediated by kinetochores, multiprotein structures that assemble on centromeric (CEN) DNA.
The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor
proteins have yet to be determined despite evidence of their importance in higher eukaryotes.
Here we show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores
and function in three distinct processes. Kiplp and Cin8p, Kinesin-5/BimC family members,
cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a Kinesin8/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The Kinesin14 motor Kar3p, appears to function at the subset of kinetochores that become detached from
spindle microtubules. These data demonstrate roles for structurally diverse motors in the
complex processes of chromosome segregation and reveal important similarities and intriguing
differences between higher and lower eukaryotes.
2.2 Introduction
Kinetochores are multi-protein complexes that assemble on centromeric (CEN) DNA and
attach chromosomes to spindle microtubules (microtubules;Mitchison and Salmon, 2001).
Kinetochore-microtubule attachments generate the forces required for sister chromatid biorientation during metaphase and toward-the-pole movement during anaphase (Maiato et al.,
2004). Evidence from a variety of organisms suggests that regulation of microtubule dynamics
by kinetochores is critical to both of these processes, and that multiple motor and non-motor
microtubule associated proteins (MAPs) are involved (Kline-Smith et al., 2005). The
58
comparative simplicity of budding yeast centromeres makes Saccharomyces cerevisiae an
attractive organism in which to undertake a thorough study of this aspect of kinetochore biology
(McAinsh et a]., 2003).
S. cerevisiae has six kinesins and a single dynein heavy chain (for review see Hildebrandt
and Hoyt, 2000) but only the four nuclear kinesins - Cin8p, Kiplp, Kip3p, and Kar3p - are
potential kinetochore subunits. Yeast nuclear kinesins belong to different subfamilies with
distinct directionality, structure and function. Cin8p and Kip lp are members of the Kinesin-5
family of plus-end directed motors (BimC motors; Dagenbach and Endow, 2004) that form
homotetramers active in crosslinking parallel and antiparallel microtubules (Gordon and Roof,
1999; Kapitein et al., 2005). Cin8p and Kiplp function in spindle assembly and in other
microtubule-based processes (Hildebrandt and Hoyt, 2000). cin8A mutants are viable at 25°C but
have high rates of chromosome loss and undergo frequent spindle collapse (Hoyt et al., 1992); at
37°C, cin8A cells are dead. cin8A and kiplA are synthetically lethal and KIP over-expression
suppresses the spindle collapse phenotype of cin8A, though kiplA does not cause elevated
chromosome loss. cin8A (but not kiplzl) is synthetically lethal with mad2A (Geiser et al., 1997)
presumably because checkpoint-mediated cell cycle delay is required for cin8A cells to complete
mitosis successfully. Overall, these data show that Kip lp and Cin8p are functionally redundant
(Hoyt et al., 1992; Roof et al., 1992) but that Cin8p plays the larger role under normal
circumstances.
Kip3p is closely related to the Kinesin-13 family (KinI kinesins; Lawrence et al., 2004;
Moore and Wordeman, 2004; Severin et al., 2001) that includes the kinetochore motors MCAK
in mammals, XKCM1 in X. aevis, KLP1 OA, KLP59C, KLP59D in D. melanogaster, and Klp5
and Klp6 in S. pombe (Desai et al., 1999; Maney et al., 2001; West et al., 2001). Kinesin-13
59
motors destabilize microtubule protofilaments causing microtubule-depolymerization primarily
at plus-ends (Niederstrasser et al., 2002). Drosophila KLP1OA and KLP59C mediate the
disassembly of microtubules from the plus and minus ends respectively (Rogers et al., 2004). S.
cerevisiae kip3, cells are resistant to the microtubule-depolymerizing drug benomyl, consistent
with a role for Kip3p in microtubule destabilization in yeast (Cottingham and Hoyt, 1997).
Although required for chromosome movement during anaphase in all organisms examined to
date, Kinesin-13 motors are also thought to function during metaphase to correct improper
kinetochore-microtuLbuleattachment and to align chromatid pairs at the metaphase plate (for
review see Moore and Wordeman, 2004). Thus, functions of Kinesin-13 motors in vivo include
kinetochore-microtubule (kMT) attachment during metaphase and kMT depolymerization during
anaphase.
Kar3p, the fourth nuclear motor in budding yeast, is a minus-end directed Kinesin-14
family member that localizes to spindle pole bodies (SPBs) and the tips of cortical microtubules.
Kar3p destabilizes microtubule minus-ends in vitro (Endow et al., 1994; Meluh and Rose, 1990)
and has been found at low levels in biochemical preparations of the CBF3 centromere-binding
complex (Hyman et al., 1992). Like Cin8p (He et al., 2001), Kar3p associates with CEN DNA
when assayed by chromatin immunoprecipitation (ChIP; Tanaka et al., 2005). Kar3p is involved
in the sliding of minichromosomes with a GAL-regulated CEN laterally along microtubules
under circumstances in which newly induced kinetochores are captured by microtubules.
Endogenous S. cerevisiae chromosomes are bound to microtubules throughout the cell cycle
however, making it unclear whether Kar3p functions at kinetochores during normal cell division.
Functional analysis of nuclear kinesins in budding yeast is complicated by their
involvement in multiple mitotic processes either individually or in combination. This multiplicity
60
of function creates complex loss-of-function phenotypes. To begin to understand kinesin
functions specifically at kinetochores, we have applied a series of fixed and live-cell assays that
focus on kinetochore biology. We find that all four S. cerevisiae nuclear kinesins localize to
kinetochores and perform three distinct functions: Cin8p and Kip lp are required for correct
alignment and clustering of kinetochores on the metaphase spindle; Kip3p is required for
coordinated movement of chromatids to spindle poles at anaphase; and Kar3p appears to
function specifically at a subset of kinetochores on which microtubule attachments are slow to
form. Thus, while nuclear kinesins in budding yeast are best known as essential players in
spindle assembly, they also have important roles in ensuring the accurate attachment of
kinetochores to microtubules.
2.3 Results
2.3.1 Localization Patterns of Kinesins During the Cell Cycle
To determine whether Cin8p, Kiplp, Kip3p, and Kar3p localize to kinetochores, we
applied three criteria previously used in the analysis of other kinetochore proteins (He et al.,
2001). First, GFP-tagged kinesins were examined in fixed cells and localization patterns
compared to patterns for known kinetochore proteins; second, tagged kinesins were tested for
CEN association by ChIP; third, the role of CBF3 in CEN binding of kinesins was examined by
using a temperature sensitive mutation (ndcl]O-1) in a subunit of CBF3. CBF3 is an essential
four-protein complex required for initiating kinetochore assembly and for the recruitment of all
known kinetochore proteins to CEN DNA (Goh and Kilmartin, 1993; He et al., 2001; Lechner
and Carbon, 1991). Ndc lOp-dependent localization to kinetochores and association with CEN
DNA are diagnostic of kinetochore proteins.
61
To image motor proteins, kinesins were fused at their C-termini to GFP and integrated
into endogenous loci in a strain containing Spc42p-CFP labeled SPBs. Genetic tests established
that the tagged motors were biologically active (see materials and methods). In early mitosis,
kinetochore proteins localize to a single focus spanning the short (<1 tm) distance between the
spindle poles. Subsequently, at spindle lengths of 1.0 to 1.2 jim, kinetochores resolve into two
distinct foci lying between the SPBs. This bilobed pattern is analogous to the metaphase plate in
metazoans and is maintained until anaphase, at which time chromatids move toward the poles
and become tightly associated with SPBs (Figure 2.1A; He et al., 2000). When metaphase cells
were examined by 3D-deconvolution microscopy, Cin8p-GFP, Kiplp-GFP, and Kip3p-GFP
were found to have bilobed localization patterns similar to that of Ndc80p-GFP, a wellcharacterized kinetochore protein (Figure 2.1A; top panel). In addition, these kinesins also
decorated interpolar microtubules during metaphase (data not shown). In anaphase, Cin8p-GFP
and Kip3p-GFP were found at the spindle midzone and Kiplp-GFP localized to faint puncta
along pole to pole microtubules (pMTs) whereas Ndc80p-GFP was visible only near
kinetochores (Figure 2.1A, bottom panel; and He et al., 2001). Biochemical experiments have
shown that the majority of Kiplp is degraded at the metaphase to anaphase transition (Gordon
and Roof, 2001) implying that the Kiplp-GFP visible in anaphase cells represents a small
fraction of undegraded microtubule-bound protein. Overall, imaging data suggest that Cin8p,
Kip Ip, and Kip3p localize to kinetochores as well as to other microtubule-based structures. As
further demonstration of this point, we established that the bilobed localization of Cin8p, Kip lp,
and Kip3p was lost in ndcl 0-1 cells but that GFP fluorescence along spindle microtubules was
maintained (Figure 2.1C). Cin8p-GFP localized close to the poles in ndcl O-1cells, whereas
Kip 1p-GFP was found along the spindle (data not shown and Figure 2.1 C) and Kip3p-GFP was
62
A.
Kip3p-GFP
Q)
en
ro
..c
0-
ro
Q)
~ C
'enc
Q)
C
a..
u..
()
I
0N
1)
Q)
en
ro
0CI)
..c
0-
ro
c
<{
B.
Kar3p-GFP
Figure 2.1: Localization
of GFP-tagged
kinesins in wildtype
cells. (A) Typical 20 projections of
3D images of metaphase (top panel) and anaphase (bottom panel) cells co-expressing
the SPB
marker Spc42p-CFP (red) and Cin8p-GFP, Kip1p-GFP, Kip3p-GFP, Kar3p-GFP or Ndc80p-GFP
(green). Surface plots beneath each image depict fluorescence signal intensity distribution in arbitrary
units for CFP (red) and GFP (green). The intensity distributions were generated from 20 maximum
intensity projections of 3D image stacks. (B) Representative
20 projections of 3D images of G1 cells
co-expressing
Kip3p-GFP, Kar3p-GFP or Ndc80p-GFP (green) with Spc42p-CFP (red). (C) Images of
cells expressing Cin8p-GFP, Kip1p-GFP, and Kip3p-GFP (green) with Spc42p-CFP (red) in ndc10-1
cells. Strains were grown to mid-log phase at 25°C and shifted to 37°C for 3 hr prior to analysis.
Spindles elongate abnormally in many ndc10-1 cells, creating metaphase cells with spindles lengths
typical of anaphase (1.2- 2.0Jlm).
concentrated at the spindle midzone. We interpret these data to mean that, in the absence of
CBF3, the association of Cin8p, Kiplp, and Kip3p with kinetochores was disrupted whereas
localization to other microtubule-based structures was retained.
In contrast to Cin8p-GFP, Kipl p-GFP, and Kip3p-GFP, Kar3p-GFP was found primarily
along the nuclear face of SPBs and did not appreciably co-localize with Ndc80p-CFP (Figures
2.1A & 2.2A-B). Localization of Kar3p-GFP to SPBs in living cells confirms previous immunoEM data (Zeng et al., 1999). However, faint Kar3p-GFP foci were also visible along the spindle
in about 30% of early (<1.5 [tm spindles) and 20% of late (1.5-2.5 ptm spindles) metaphase cells
(Figures 2.2C&E). These Kar3p-GFP foci were rarely if ever seen during anaphase (Figure 2.2E)
and did not adopt the bilobed pattern typical of core kinetochore proteins. To test the idea that
Kar3p might associate specifically with detached or partially attached kinetochores (which are
more abundant early in mitosis), cells were arrested in a-factor and released into the microtubule
poison nocodazole. Most kinetochores in nocodazole-treated cells migrate to SPBs, apparently
by following shrinking microtubules (Gillett et al., 2004), but a subset becomes detached, moves
farther from the SPBs and recruits high levels of Bublp, Madlp, and Mad2p checkpoint proteins
(Gillett et al., 2004). In nocodazole-treated cells we observed that the majority of the Kar3p-GFP
signal remained associated with SPBs (visualized with Spc42p-CFP) but in 75% of cells one or
more faint foci were visible distal to the SPBs (Figure 2.2D). These fainter, more distant Kar3pGFP foci colocalized with Ndc80p-CFP (Figure 2.2D bottom). Based on our previous analysis
(Gillett et al., 2004), the Kar3p-GFP signal distant from SPBs almost certainly represents
kinetochores that have detached from microtubules (Figures 2.2D-E). Quantitation of SPB distal
Kar3p-GFP foci showed that they represented 8% + 2.5% of the total GFP signals; implying
efficient recruitment of Kar3p to detached kinetochores. We conclude that Kar3p becomes bound
64
Intensity
E.
80%
'0
.E
~
Intensity
k
Intensity
x
Q)
.;
.~
!:!l
Q)
0
0
~
0
0%
Early Late
Ana
Noc
Metaphase
Intensity
Q)
.j
"0
N
co
"0
0
0
0
z
Intensity
,J.
Figure 2.2: Kar3p-GFP recruitment to improperly attached kinetochores. (A-B) Images representative of the majority of cells that have only two foci. (A) Wildtype cells expressing Kar3p-GFP (green) and
the Spc42p-CFP (red). Surface plots as described in Figure 2.1. (B) Images of Kar3p-GFP (green)
coexpressed with kinetochore protein Ndc80p-CFP (red). (C) Typical images of the early metaphase
(spindle length <1.5Jlm; top panel) or late metaphase (spindles 1.5-2.5Jlm; bottom panel) cells in which
additional Kar3p-GFP foci are present. Spc42p-CFP labeled in red to show location of SPBs. Peak
intensity areas of additional foci are indicated by arrows. (0) Representative images of cells released
from a.-factor arrest into nocodazole for 2 hours.(top panel) Cells coexpressing Kar3p-GFP (green) with
& Spc42p-CFP(red). (bottom panel) Cells coexpressing Kar3p-GFP (green) & Ndc80p-CFP (red). (E)
Percentage of cells containing Kar3p-GFP foci in addition to those overlapping the SPBs as scored in
early metaphase (spindle length <1.5J.1m),late metaphase (spindle length 1.5 -2.5Jlm), anaphase
(spindle length <2.5J.1m)or nocodazole (treated as described in 0).
to detached or improperly attached kinetochores but not to the majority of kinetochores in an
unperturbed cell. Instead, most Kar3p is bound to SPBs in vegetatively growing cells.
2.3.2 Association of Cin8p, Kiplp, and Kip3p with Kinetochores by ChIP
Cin8p and Kar3p have previously been shown to associate with CEN DNA by ChIP (He
et al., 2001; Tanaka et al., 2005) and we therefore concentrated on the analysis of Kiplp and
Kip3p. Kip p-GFP and Kip3p-GFP were observed to crosslink efficiently by ChIP to a 200bp
region centered on CENIV but not to equal length fragments lying 400bp upstream and
downstream (Figure 2.3A). As additional specificity controls, we showed that GFP-tagged
kinesins did not crosslink appreciably with the URA3 locus, IP of untagged kinesins yielded a
negative ChIP signal at CENIV, and shifting ndclO-1 cells carrying Kiplp-GFP, Kip3p-GFP, or
Cin8p-GFP to 37°C for 3hrs lowered ChIP signals 10-fold or more (Figure 2.3B & He et al.,
2001). Overall, these data show that Cin8p, Kip lp, and Kip3p associate specifically with CEN
DNA in a CBF3-dependent manner.
2.3.3 Recruitment of Kinesins to Kinetochores
To investigate how Cin8p, Kiplp, and Kip3p are recruited to kinetochores, ChIP was
performed in ndc80-1 and spc25-7 cells. Ndc80p and Spc25p are components of the Ndc80
Complex, a multi-protein "linker" that bridges the DNA and microtubule-binding components of
kinetochores (McAinsh et al., 2003). Kinetochores partially disassemble in ndc80-1 and spc25-7
mutants and chromosomes dissociate from spindle microtubules (He et al., 2001; Janke et al.,
2002). We observed a -4-fold drop in the CEN-specific ChIP signal for Cin8p-GFP and KiplpGFP in ndc80-1 and spc25-7 cells (Figures 2.3C-D). In contrast Kip3p-GFP showed wildtype
66
A.
z 1.0
o
w
.8 0.6
a
~
~0.2
0.0_400 0 +400
bp from CENIV
C. 1.4 Cin8p-GFP
37°C
~
~
1.0
~ 1.0
.8
.8
a 0.6
~
~
0.2
0.0
~
1 4 Kip1 p-GFP
. 37°C
~
1 4 Kip3p-GFP
. 37°C
~ 1.0
.8
WT spc25-7
WT spc25-7
D. 1 4 Cin8p-GFP
. 37°C
~
~ 1.0
.8
a 0.6
0.2
~0.6
C'O
~
0.2
0.0
0.0
~
~
WT ndcBO-1
~0.6
C'O
~
WT ndcBO-1
0.2
0.0
WT
ndcBO-1
Figure 2.3: Dependence of kinesin-CEN crosslinking
by ChiP on core kinetochore components. (A-D) ChiP
of Kip1p-GFP, Kip3p-GFP and Cin8p-GFP to CENIV or
flanking DNA as assayed in (A) wildtype, (8) ndc10-1 (C)
spc25-7 or (D) ndcBO-1 cells. Asynchronous cultures were
grown to mid-log phase at 25°C and in panels 8-0, shifted
to 37°C for 3 hr prior to analysis. Results in (A) are
expressed as the ratio of the %IP from the arm regions to
the %IP of the CEN. Results in (8-0) are expressed as a
ratio of the %IP in the mutant to the %IP of the wildtype
strain.
levels of CEN-binding in ndc80-1 and spc25-7 cells (Figures 2.3C-D). Because a functional
Ndc80 Complex is required for kinetochores to bind to microtubules, we can conclude that
Kip3p is recruited to CEN DNA in an microtubule-independent fashion, implying that Kip3p is a
core kinetochore protein, as are Kinesin-13 family members in higher eukaryotes (Wordeman
and Mitchison, 1995). Data are more ambiguous for Cin8p and Kiplp: the motors could either
require microtubules for CEN association, or the Ndc80 Complex could be directly involved in
Cin8p and Kip lp recruitment to kinetochores.
2.3.4 Cin8p and Kiplp Organize Kinetochores During Metaphase
To begin to investigate the functions of kinetochore-bound motors we asked whether
mutations in kinesins would alter the localization of core kinetochore proteins such as Ndc80p
and Mtw lp. The bilobed distribution of these proteins represents the average position of
kinetochores during metaphase, and is therefore a sensitive readout of microtubule attachment
and chromosome congression (He et al., 2000; Pearson et al., 2004). We examined the
localization of Ndc80p-GFP or Mtwlp-GFP in cells carrying Spc42p-CFP tagged SPBs and
cin8A, kiplA, or cin8F467AkiplA mutations. Because cin8A kiplA double deletions are inviable
(Hoyt et al., 1992; Roof et al., 1992) we examined the effects of impairing both Kinesin-5 motors
by combining kipl]A with the temperature sensitive cin8F467A mutation (which is defective in
microtubule binding; Gheber et al., 1999). To obtain mitotic cells for imaging, cells were
synchronized in c(x-factorand released into fresh medium. In wildtype and kipl]zl cells, bilobed
metaphase configuration was visible in cells 60-75 min after release (Figure 2.4A). In cin8zAand
cin8F467AkiplNAcells, however, spindle assembly was delayed to a variable extent (Gheber et
al., 1999; Hoyt et al., 1992; Roofet al., 1992; Saunders and Hoyt, 1992) and we therefore
68
iililil~1iiii.~
B~•••
~
i•••
~
~
••• ~ iflliiil[;j
:••• ~; •••
~
~ •••
~
~
H.
Ndc80Misiocalization
~ 80
I.
Ndc80Misiocalization
~ 100 .WT DcinBIi
Q)
Q)
u
~
W
~
i•••
0 WT cin8/lkipl/lkip3/l
80
~
~
u!::
~
u
60
L~
early
late
Figure 2.4: localization of Ndc80-GFP kinetochore protein in wildtype and kinesin mutants. (A-E)
Typical 20 projections of 30 images of Ndc80p-GFP (green) and Spc42p-CFP (red) in (A) wildtype, (8)
cinB~, (C) kip1~, (0) kip3~, and (E) cinBF467Akip1~ cells. Arrow in top panel of (8) marks the location of a
kinetochore that appears to have detached from spindle microtubules. Spindles in cinBF467Akip1~ cells
rarely reach normal metaphase lengths and the top panel of (E) shows a metaphase cell with an abnormally
short spindle «1.0 11m)whereas the bottom panel shows a representative of the 10% of cells that reach
wildtype spindle length (>1.5 11m).(F-G) GFP- Tub1 p (green) and Spc42p-CFP (red) in (F) wildtype and (G)
cinB~ cells. (H) Percentage of metaphase cells with atypical Ndc80p-GFP foci as scored in wildtype, cin8~,
kip 1~, and kip3~ cells with spindle lengths 1.2-2 11m.100% of cinBF467 Akip 1~ cells had gross defects in
Ndc80p-GFP localization but the interpretation of these images is complicated by the severity of the spindle
assembly defect in these cells. (I) Percentage of atypical Ndc80p-GFP foci in metaphase cin8~ cells as a
function of time after a-factor release: 45-90 min post release is designated as "early" and 105-120 min as
"late."
focused on the 20-30% of cin8A cells at T= 60-105 min with metaphase spindles that were at
least 1.2 tm long. When these cells were examined, more than 60% contained supernumerary
Ndc80p-GFP foci along the spindle axis or had abnormally diffuse GFP lobes ("declustering";
Figures 2.4B&H). The same phenotype was observed in cells in which kinetochores were labeled
with Mtwlp-GFP (data not shown). In cin8F467Akiplz1 cells, Ndc80p-GFP and Mtwlp-GFP
localization patterns were altered to a greater extent, although only a subset of cells could be
scored due to spindle collapse. However, in the 5-10% of cells that formed metaphase-length
bipolar spindles, kinetochores were distributed all over the spindle and 10-15 partially resolved
foci were visible (Figure 2.4E). In contrast, kinetochore distribution was only slightly altered in
kipl]A and kip31 single mutants (Figures 2.4C-D&H). Taken together, these data suggest that
Cin8p is involved in establishing or maintaining the normal metaphase configuration of yeast
chromosomes, perhaps by bundling kMTs. kiplA alone does not disrupt kinetochore localization
to a significant extent but the severity of the cin8F467AkiplA phenotype suggests that Cin8p and
Kip I p work together to cluster kinetochores, as they do during spindle assembly.
Because cin8A mutants are known to have unstable mitotic spindles, one concern with the
localization data described above is that declustering might be a simple consequence of failing to
form a spindle. To explore this possibility, spindle morphology was compared in wildtype and
mutant cells using GFP-Tublp (-tubulin;
Straight et al., 1997). In wildtype cells, microtubules
were visible as a thick bar with a slight increase in intensity near the SPBs, reflecting the
termination of many kMTs near the spindle poles (Figure 2.4F; Maddox et al., 2000). kMTs are
particularly prominent in budding yeast because they outnumber pMTs (Winey et al., 1995).
GFP-Tub Ip morphology was similar in cin8A cells, indicating that bipolar spindles had formed,
although the GFP-Tublp signal was less highly concentrated near SPBs (Figure 2.4G). This is
70
precisely what one would expect if pMTs were correctly assembled but kMTs mislocalized due
to defects in congression. We conclude from these data that gross defects in spindle morphology
are not responsible for the disruption of kinetochore clustering in cin8A cells.
We were also concerned that kinetochore declustering might be a consequence of spindle
collapse and regrowth. Were this the case, we would expect the mutant phenotype to be more
severe as mitosis progressed and spindles had time to undergo multiple cycles of collapse and
regrowth. However, when metaphase declustering was measured in cin8A cells as a function of
time after a-factor release, the fraction of cells with Ndc80p-GFP declustering early (T=60-75)
and late (T=90---105)was similar (Figure 2.4I). We therefore conclude that declustering was not a
consequence of altered mitotic timing in cin8A cells or of rounds of spindle collapse and
regrowth.
A third possible explanation for kinetochore declustering was the presence of large
numbers of unattached or improperly attached chromosomes. To investigate this possibility, we
assayed the degree of transient separation in a-factor synchronized cells carrying a CENIVproximal TetO/TetR-GFP tag, and Spc42p-GFP labeled SPBs (Ciosk et al., 1998; He et al.,
20(00;Straight et al., 1996). Transient separation arises when kMTs pull sister kinetochores in
opposite directions (Goshima and Yanagida, 2000; He et al., 2000; Tanaka et al., 2000) and
represents an in vivo measure of force generation. No significant decrease in transient separation
was observed in cin8A and kiplA single mutants relative to wildtype cells (Figure 2.5A).
Quantitation was difficult in cin8F467AkiplA cells due to their very short spindles, but live-cell
imaging revealed transient separations qualitatively similar to those seen in wildtype (Figures
2.5B-C). While kip3A cells and kipl]Akip3A cells both exhibited wildtype levels of transient
separation, cin84Akip3Acells had a statistically significant 30% decrease (Figure 2.5A). Overall
71
A.
c 80
.2
10
(ij
c..
60
CD
en
C 40
CD
'en
~'- 20
~
?ft. 0
B.
C' .
16
_
~
CD
g
1.2
cinBF467Akip16
-SPBa-SPBb
(d3)
--SPBa-ChromA
(d1)
-- SPBa-ChromB (d2)
0.8
ro
en
o
0.4
a
co
a
N
.....
a
co
.....
a
a
N
time (sec)
Figure 2.5: Measurements of transient separation In klnesln mutants.
(A) Percentage of metaphase cells undergoing transient sister separation in synchronized cultures of
wildtype, cinB/1, kip1/1, kip3/1, cinBD.kip3/1, and kip1D.kip3/1. Transient separation was determined in
cells tagged near CENIV with TetOlTetR and coexpressing Spc42p-CFP as a reference for the SPBs
(spindle length 1.5-2.5J,1mat T=75 and T=90 after release from a-factor). (B) Geometry of 3D live-cell
tracking experiments. Chromatids are labeled using the TetOfTetR-GFP system and compared to
GFP-tagged SPBs. Distances are measured between each sister chromatid and the same reference
SPB (d1 - black and d2 - green) allowing for visualization of chromatid separation. The spindle length
(d3 - red) is also plotted to show cell cycle progression. (C) Chromatid trajectories in a representative
cinBF467Akip16 cell showing occurrences of transient separations; yellow fill denotes separations.
Transient separations highlighted in yellow
these data show that chromosome-microtubule attachment is not severely disrupted by the
deletion of individual kinesins but that CIN8 and KIP3 might work together in force generation
during metaphase. Moreover, with respect to declustering in cin8zl cells, we conclude that it is
not simply a consequence of defective kinetochore-microtubule attachment. Instead, Kiplp and
Cin8p appear to have a specific role in maintaining the metaphase configuration of budding yeast
kinetochores, and thus to function in the process of sister chromatid "congression."
2.3.5 Kip3p Regulates Anaphase A Movement
Since many Kinesin- 13 motors play a role in the movement of chromatids poleward we
asked whether KIP3 deletion would alter the anaphase movement of budding yeast kinetochores.
We observed sister chromatid disjunction to be complete in wildtype cells with 2.0-4.0 pm
spindles spanning the bud neck (a morphological marker of anaphase) as evidenced by the
appearance of two bright puncta of Ndc80p-GFP or Mtwlp-GFP immediately adjacent to the
SPB. The puncta represent clusters of disjoined kinetochores (Figure 2.6A). In -20-25% of these
cells, particularly those very early in anaphase, an extra focus of Ndc80p-GFP or Mtwlp-GFP
was visible away from the SPBs (Figure 2.6A and data not shown). In contrast, kip3A cells with
2.0-4.0 im spindles had multiple supernumerary kinetochore foci, typically two to five (Figures
2.6 B&C), and they persisted for longer. The number of supernumerary GFP foci in kip3A cells
fell as anaphase progressed and none were visible when spindles had reached their maximum
anaphase length of 7-10 tm, indicating that all chromatids had eventually moved to the poles
(Figure 2.6C-E). We propose that supernumerary Ndc80p-GFP and Mtwl p-GFP foci represent
lagging chromosomes.
73
Wildtype
A.
kip3~
B.
Il..
LL
<.9
I
0-
o
CO
()
"0
Z
Il..
LL
U
I
0N
'3000
Q)
....
Q)
C>
~
Mid
D.
C.
Late
>7 m
E.
47
- 'urn
~ 100
~
Q)
()
'0
100
~
6
~o
2
n
~
6
~o
2n
o
o
o
1 2 345
# foci
6 7
100
Q)
Q)
~6
o
~o
2
••
1234567
# foci
III
o
1 234
567
# foci
kip3~mad2~
Figure 2.6: Localization of kinetochore foci during anaphase.
(A-B) Representative images of Ndc80p-GFP (green) and Spc42pCFP (red) in (A) wildtype, and (B) kip3~ cells during anaphase. Arrow
along bottom denotes time progression during anaphase (C-E)
Quantitation of additional anaphase kinetochore foci throughout
anaphase by spindle length. (F) Anaphase localization of Ndc80pGFP and Spc42p-CFP in kip3tlmad26 anaphase cells.
An alternative explanation for supernumerary kinetochore foci is that kip3A cells with
abnormal metaphase spindle morphology are transiently delayed in metaphase by the spindle
checkpoint. However when checkpoint-dependent cell cycle arrest was abolished by deleting
M4D2 (Li and Murray, 1991), the number of lagging chromosomes was similar to that in kip3A
cells (Figure 2.6F). Thus supernumerary Ndc80p-GFP or Mtwlp-GFP foci in kip3A cells are
unlikely to arise from malorientation of chromatid pairs during an extended metaphase.
Moreover, the observation that anaphase movement is not so delayed as to prevent chromatid
disjunction prior to cytokinesis supports previous data showing that chromosome loss rates are
normal in kip3A cells (DeZwaan et al., 1997). Taken together our findings argue that in the
absence of Kip3p, synchronous movement of chromatids toward the spindle poles is disrupted.
To obtain further evidence for lagging chromatids in kip3A cells, we filmed anaphase in live cells
carrying TetO/TetR-GFP-tagged CENIV and Spc42p-GFP-tagged SPBs. In wildtype cells, CENproximal GFP tags were briefly stretched into a line along the spindle axis. Such
"hyperstretching" presumably reflects increased pulling forces on CEN DNA prior to the
dissolution of sister chromatid cohesion leading to unraveling of chromatin ultrastructure. After
15-45 sec (mean 25 sec) of hyperstreching, individual TetO/TetR tags resolved in two compact
dots (Figures 2.7A & C-E) and moved rapidly to within 0.4 tm of the SPBs, where they
remained for the duration of anaphase (Figure 2.7E). In -15% of kip3A5cells (n=13) one
chromatid made a swift movement toward the pole but then paused for several minutes before
finally moving all the way to the pole (Figures 2.8A&D with pause highlighted in yellow). In
other kip3A cells, chromosomes remained hyperstretched for significantly longer than in
wildtype (30-100 sec, mean 70 sec; Figures 2.7B&D), implying imposition of pulling forces
prior to the dissolution of sister cohesion. The accumulation of lagging chromatids demonstrates
75
A. WT
B. kip3/1
-110
c
•
.
.
•
'
-55
OJ
o
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CJ)
co
a.
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co
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.c.
~o
-70
-20
Amount of
-40
.
.
•
.e
40
Q)
o
kip3/1
E.
Post-Anaphase
0.5 kMT length
0.4
I
I
.m 0.1 I
O. .1
C)
o
WT
WT
kip3/1
•
•
0
-5
~0.3
:6 0.2
~E 30
20
.
.
-
~ 60
60
30
Hyperstretching
80
.
.
•
.
Duration of
90
100 Hyperstretching
.
.•
-60
-35
-10
-15
D.
;
•
•
.
.
-65
.
-45
-90
.
•
.
.. •
25
.!!l
.
-50
.
.
•
-95
.
-75
-80
C.
.
.
•
-85
-100
.
.
•
•
-105
•
.
•
•
•
-115
.
.
.
..
WT
I
I
I
I
I
I
I
I
kip3/1
Figure 2.7: Live cell analysis of kip3/1 defects in anaphase. (A-B) Consecutive frames
from representative movies of (A) wildtype and (B) kip3/1 cells demonstrating preanaphase
"hyperstretching." Numbers on bottom left of each image indicate seconds prior to anaphase
A completion. (C) Percent of anaphase cells where "hyperstretching" occurs prior to
anaphase A completion. (D) Average time in seconds of "super-stretching". Error bars are
standard error of the mean (SEM). (E) Average difference in chromosome to pole distance
after anaphase A completion .
A. kip36.
B.
C.
WT
5.0
2.5
J
--------*-----
2.5
-
[ +
E
r-
+
1.25 ~
'0
06
::1..
r
E
3 2.5
1.25 ~
'0
~
06
+
+
...--
...--
'0
0.0
~ g ~
-SPBa-SPBb
(d3)
-SPBa-ChromA
(d1)
--SPBb-ChromB
(d4)
~
I
-r-'
a
I
~
g
~
0.0
0.0
a
a
a
~
~
u;>
I
a
a
a
ll)
~
I
time (sec)
time (sec)
Figure 2.8: Live cell analysis of a lagging chromatid in kip36. cells. (A) Consecutive frames from representative movies of kip36. cells containing a lagging chromosome. White arrows denote locations of
TetOlTetR-GFP tags and yellow arrows denote SPBs (B) Geometry of 3D live-cell tracking showing
distances between labeled chromatids and the nearest SPB (d1and d4) and spindle length (d3). Chromatid
trajectories in (C) wildtype and (0) kip36. cells; yellow fill denotes a pause in which one chromatid remained
ca. 1.5 ~m from the nearest SPB for 100 sec. Note the difference in scales for d1 and d4 v. d3. Times for
A,C and 0 displayed relative to anaphase onset which is set to t=O.
'0
failure of kip3l kinetochores to complete poleward movement in a timely fashion; the existence
of hyperstretching suggests abnormally early imposition of anaphase A forces. Thus, Kip3p may
function both to mediate kMT depolymerization and to coordinate pulling forces with the
metaphase-anaphase transition.
2.3.6 Kip3p Regulates microtubule length and dynamics in G1 and a-factor arrested cells
The involvement of Kip3p in chromosome movement during anaphase suggested that it
might regulate microtubule dynamics. To measure directly the effects of kinesin mutations on
chromosome movement we used fast-acquisition live-cell imaging coupled with machine vision
tools. Our software makes it possible to track TetO/TetR-tagged CENs with great precision and
to extract rates of microtubule growth, shrinkage, rescue, and catastrophe (Dom, 2005).
Currently, accurate tracking is possible only within the simple monopolar spindle geometry of
G1 cells. Cin8p and Kiplp are not present in G1 and we therefore focused on analyzing Kip3p
and Kar3p (Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001). Kip3p deletion led to a
statistically significant decrease in mean microtubule growth (p <10 - 3 ) and shrinkage speeds (p
<10-3) implying a role for Kip3p in the regulation of G1 microtubule dynamics (Figure 2.9A). In
contrast, kar3A did not affect either of these key parameters to a significant degree. As a second
means to show that Kip3p alters kMT behavior, we examined the distribution of Ndc80p-GFP or
Mtwlp-GFP in a-factor-arrested cells. After 2 hr in a-factor, Ndc80p-GFP or Mtwlp-GFP foci
in wildtype cells averaged 0.8 ,um from SPBs and were rarely > 1.5 tm away (Figures 2.9B-D);
in kip3A cells under identical conditions, kinetochore foci averaged 1.2 pim from SPBs and were
often as far as 2.5 tm away (Figures 2.9B-D). Taken together, these data show that Kip3p is
involved in regulating the dynamics and mean lengths of kMTs in GI1and u1-factorarrested cells.
78
A.
5.0
Growth
5.0 Shrinkage
OJ
24.0
~ 4.0
:J
C
c
E
E
E
E
.........
.........
:1.
3.0
2.0
:1.
WT
3.0
2.0
k'Ip 3 ~ k ar3~
WT kip3~ kar3~
0.7 Catastrophe
0.8 Rescue
\J
C
\J
C
0
0
u 0.6
QJ
~ 0.7
Ul
Ul
1-
1-
QJ
QJ
0-
0-
Ul
......
c 0.5
Ul
QJ
C
w
>
0.6
QJ
>
w
0.4
0.5
WT kip3~ kar3~
WT
k'Ip 3 ~ k ar3~
B.
WT
kip3~
D.50
C''E1.5
d
co
ckip3~
40
~ 1.0
~l1> 30
E
o
'~0.5
U
'020
I--
~~
~
u
c
o
co
(;)
:a 0.0
-WT
WT
kip3~
10
I--
~~f- f-
o
'--
I~
-
OI.()OI.()OI.()I.()
OO""':""':NNN
1\
distance from SPB (~m)
Figure 2.9: Analysis of chromosome
dynamics and microtubule length in
G1 and a-factor-arrested cells. (A)
Comparison of microtubule growth,
shrinkage, catastrophe, and rescue in
wildtype, kar36., and kip36. cells as
determined using automated tracking
methods (Dorn, 2005). (B) Representative images in a-factor arrested cellsof
Ndc80p-GFP
localization (green) in
wildtype or kip3D. cells relative to
Spc42p-CFP
reference (red). (C)
Quantitation of data in B showing
average distance of kinetochore foci to
SPBs (in 11m). Error bars are SEM from
biological repeats. (D) Distribution of
data in C.
2.4 Discussion
2.4.1 Cin8p, Kiplp, and Kip3p Localize to Kinetochores
In this paper we determine which of the six kinesin motor proteins in S. cerevisiae
localize to kinetochores and analyze the functions of kinetochore kinesins using fixed and livecell microscopy. Because S. cerevisiae has a closed mitosis, only the four nuclear kinesins Cin8p
and Kip p (Kinesin-5/Bim-C family members), Kip3p (a Kinesin-13/KinI motor) and Kar3p (a
minus-end directed Kinesin-14) have the potential to bind to kinetochores. ChIP has previously
established that: Cin8p and Kar3p associate with CENand we show that this is also true of Kiplp
and Kip3p, the two remaining nuclear motors. Live and fixed-cell imaging shows that
kinetochores are one of the primary structures to which Kiplp-, Cin8p-, and Kip3p-GFP are
localized during mitosis in normally growing cells. However, association with other structures is
observed in cells lacking active kinetochores (as a consequence of a mutation in the core CENbinding complex CBF3) consistent with previous data showing that kinesins play important roles
in spindle assembly.
How are kinesins recruited to kinetochores? In the case of Kip3p, it appears that the
motor binds directly to core kinetochore components: Kip3p remains CEN-bound in ndc80-1 and
spc25-7 mutants despite the dissociation of chromosomes from microtubules. In this respect,
Kip3p is similar to the human Kinesin- 13/KinI motor MCAK, which is a component of the inner
kinetochore (Wordeman and Mitchison, 1995). The finding that CEN-binding by Cin8p and
Kip lp is partially but not entirely dependent on NDC80 and SPC25 is ambiguous with respect to
the role of microtubule attachment, but it seems likely that both motors require microtubules to
associate with kinetochores. Other yeast kinetochore proteins, including members of the
microtubule-binding Dam 1/DASH complex require microtubules for kinetochore association
80
(McAinsh et al., 2003; Miranda et al., 2005; Westermann et al., 2005) as do plus-end MAPs such
as CLIP- 170 in higher eukaryotes (Maiato et al., 2004). It therefore seems that microtubulebinding kinetochore proteins in S. cerevisiae fall into two classes: those that are recruited directly
by core kinetochore proteins and those that bind to, or are transported to, microtubule plus ends
and then associate with kinetochores. These two classes of k-MAPs must then dock together to
form a fully functional kinetochore-microtubule attachment site.
In contrast to Kip lp, Kip3p and Cin8p, which mainly localize to kinetochores, Kar3pGFP is found primarily on the nuclear face of SPBs. It has been suggested that Kar3p might be a
kinetochore motor, based on its co-purification with CBF3 (Hyman et al., 1992) and genetic
interaction with other motors (Hildebrandt and Hoyt, 2000). However, previous immuno-EM
data (Zeng et al., 1999) are consistent with our live-cell imaging in showing Kar3p to be
primarily SPB bound. Low levels of Kar3p can be detected at a subset of kinetochores early in
mitosis and higher levels on kinetochores that are detached from microtubules by nocodazoletreatment. Kar3p has recently been implicated in lateral microtubule sliding of newly captured
kinetochores formed de novo on GAL-regulated CENs (Tanaka et al., 2005). However,
kinetochores normally remain microtubule-bound throughout the cell cycle (Dom, 2005), and
microtubule capture is probably important only during a brief period in S phase. This may
explain both the low levels of Kar3p on kinetochores under normal conditions and the absence of
elevated chromosome loss in kar3A cells. Moreover, while Kar3p may function in de novo
microtubule-kinetochore attachment, we have not been able to detect a kinetochore function for
Kar3p in cells under normal growth conditions. Deletion of KAR3 has been shown to partially
suppress the lethality of cin8Akipla double mutants (Saunders and Hoyt, 1992) and kinetochore
81
localization does not explain this phenotypic suppression. Instead, it must reflect functions for
Kar3p, Cin8p and Kip lp in processes other than kinetochore-microtubule interaction.
2.4.2 Organization of metaphase chromosomes by Cin8p and Kiplp
Our data show that Cin8p, and to a lesser extent Kipl p, are involved in the generation or
stablization of the distinctive bibobed kinetochore clusters found in budding yeast from mid to
late metaphase. The disruption of biblobedclustering in cin8Azmutants does not appear to reflect
gross disorganization of the spindle, dramatic increases in the number of detached chromosomes
or changes in the fraction of transiently separated sister CENs. Instead, we speculate that Cin8p
and Kip p, like other Kinesin-5 motors that can crosslink parallel and anti-parallel microtubules
(Gordon and Roof, 1999), are involved in crosslinking kMTs. Because S. cerevisiae CENs
associate with a single microtubule, kinetochore-bound Cin8p and Kiplp must crosslink
microtubules from different kinetochores. Metaphase sister kinetochores can transiently separate
by 0.5 [tm or more and therefore it seems unlikely that Cin8p and Kiplp are able to crosslink
sisters; instead, we propose that crosslinking involves kMTs from different chromatids, though
not necessarily kMTs emanating from the same pole (see figure 2.10B). If Cin8p and Kiplp, like
human Eg5, can remain attached to microtubule plus ends (Kapitein et al., 2005), the motors may
link kinetochores together directly. The net effect of microtubule and kinetochore crosslinking
would be to create kMT bundles in which the polymerization of multiple microtubules was
coupled, perhaps explaining the requirement for Cin8p/Kiplp in forming biblobedkinetochore
clusters. The significance of clustering is suggested by the appearance of detached kinetochores
in cin8A mutants and an elevated rate of chromosome loss. It is interesting in this regard that
higher eukaryotes contain kinetochore fibers made up of 20 or more microtubules. Bundles of
82
A.
(II) both kinetochores
unattached (nocodazole)
(I) single unattached
kinetochore (S phase)
(III) both kinetochores
properly attached
cp Cin8p&
cb
Kip1p
CPKiP3p
<p
Kar3p
0
Kinetochore
•
SPB
B.
(I) With Cin8p - Kinetochore bundling
(II) Without Cin8p - Kinetochores mislocalized
(I) With Kip3p - synchronous anaphase movement
(II) Without Kip3p - asynchronous anaphase movement
Figure 2.10
Figure 2.10. Models of kinesin functions at budding yeast kinetochores. (A) Kar3p is
recruited to (I) improperly attached or (II) unattached kinetochores but not (III) bioriented
kinetochores as well as to SPBs. In contrast, Cin8p, Kiplp and Kip3p are present on bioriented
kinetochores and on spindle microtubules. (B) Model for Cin8p/Kip lp-mediated bundling of
kinetochores. (C) Model depicting the role of Kip3p at microtubule plus ends in synchronizing
the movement of chromatids toward spindle poles during anaphase A.
84
yeast kMTs created by Cin8p and Kip lp may therefore resemble the multi-stranded kinetochore
fibers found in higher cells, except that, multiple chromatids would be involved in the yeast
microtubule bundles. Further analysis of Cin8p and Kiplp function during metaphase will
require deeper understanding of the forces that generate the bilobed configuration of yeast
kinetochores, an effort that is currently underway in several labs.
2.4.3 Kip3p and Anaphase Chromosome Movement
Live and fixed cell imaging of kip3i cells reveals abnormally asynchronous sister
chromatid separation during anaphase. A subset of chromatids in kip3A cells lags behind the
majority and is found arrayed along spindle microtubules when the bulk of disjoined sisters have
already arrived at the spindle poles. Surprisingly, a second subset of kip3A chromatids exhibits
the opposite behavior: prolonged centromere hyperstretching. Transient sister separation and
chromosome stretching are observed in wildtype cells, but coordinated dissolution of sister
cohesion and poleward movement generate only a brief period of CEN hyperstretching at
anaphase A onset. In kip3i cells stretching is greater in magnitude and duration. Hyperstretching
presumably reflects the initiation of poleward movement prior to the complete degradation of
cohesin. However, despite these problems early in anaphase, all chromatids in kip3A cells are
disjoined correctly by the end of anaphase B, consistent with a normal rate of chromosome loss
in kip3A mutants (DeZwaan et al., 1997). The simultaneous generation of lagging and
hyperstretched chromatids in kip3A cells can be understood if Kip3p plays a role in ensuring the
synchronicity of poleward movement, presumably by coupling plus-end microtubule
depolymerization to the release of tension on sisters following cohesion degradation. In
Drosophila, a similar role has been proposed for Kinesin- 13 motors in triggering timely
85
destabilization of microtubules (Rogers et al., 2004). Kip3p function does not appear to be
restricted to anaphase however, because kinetochore dynamics during G 1 and microtubule length
in c-factor are altered in kip3A cells. Moreover, Cin8p and Kip3p function together during
metaphase to generate pulling forces on kinetochores, as evidenced by the 30% decrease in
transient sister separation observed in cin8Akip3A double mutants. Overall we conclude that
budding yeast Kip3p, like Kinesin-13/KinI motors in higher eukaryotes, plays an important role
in the timely and efficient depolymerization of kMTs during anaphase, and probably also during
other phases of the cell cycle.
2.4.4 Conclusions
We have established that all four nuclear kinesins localize to mitotic kinetochores in S.
cerevisiae, implying a surprising degree of complexity in kinetochore-microtubule interaction,
During normal cell division Cin8p, Kiplp, and Kip3p are found at high levels on most, if not all,
kinetochores whereas Kar3p is found transiently only on a subset of maloriented or unattached
kinetochores. The absence of Kar3p from the majority of metaphase chromatids suggests that
kinetochores do not normally move poleward along the sides of microtubules, though such
motion may by observed during microtubule capture by newly assembled ectopic
minichromosomes (Tanaka et al., 2005). Instead, it appears that in yeast, as in other organisms,
the primary way in which kinetochores move is by binding to microtubule plus-ends and then
altering their dynamics. Our data suggest that Kip3p is the motor involved in this regulation.
Among our most striking observations is that Cin8p and Kip lp, are important in organizing the
bilobed metaphase configuration of yeast kinetochores. No precedent exists for this in higher
cells, but we speculate that kinetochores with a single bound microtubule, such as those in S.
86
cerevisiae, present mechanical problems not found in complex kinetochores that bind multiple
microtubules. Perhaps by bundling 16 or so kMTs (the number bound to one pole in a haploid) in
S. cerevisiae cells create a structure similar to a kinetochore fiber in higher cells, thereby
strengthening microtubule attachment.
87
Materials and Methods
YeastStrains and Manipulations
Strains in this study were derived from W303 or S288C. GFP-tagged proteins were constructed
as described (Gillett et al., 2004) and integrated into the genome so as to replace the endogenous
wild type copy. Because loss-of-function phenotypes for individual motor deletions are subtle,
GFP-tagged kinesins were tested for function in strains carrying deletions in other motors
exhibiting synthetic lethality and the resulting compound mutants tested for viability and growth:
Kiplp-GFP was examined in cin8A cells; Cin8p-GFP in kip]lA cells; Kar3p-GFP in kip3A cells
and Kip3p-GFP in kar3A cells. In all cases, compound mutants were viable with growth rates
indistinguishable from wild-type. KanMX deletion strains were constructed by amplifying the
deleted gene of interest from ATCC deletion strains by PCR using primers 500bp upstream and
downstream of the deleted gene. PCR products were then transformed into fresh cells and correct
integrants confirmed by PCR. pFA6a-HisMX6 deletion strains were made as described in
(Longtine et al., 1998) using primers with at least 50bp of homologous sequence
MicroscopyAnalysis
Image acquisition and processing were performed as described previously (Gillett et al., 2004)
on an Applied Precision Deltavision RT microscope with 1OOX1.4NA optics and CoolSnap
camera. Fixed cells were prepared by treatment with 2% formaldehyde for 2 to 5 min followed
by 0.1M phosphate buffer (pH6.6) for at least 10 minutes prior to microscopy analysis. Live cells
were grown in SD media for several hrs and then resuspended in fresh media prior to imaging
either at room temperature or at 30°C.
ChIP
88
Temperature-sensitive strains and wildtype controls were grown at 37°C for 3 hrs before
crosslinking; protocol described in (Megee et al., 1999) with minor modifications as described
(Gillett et al., 2004). Untagged strains served as a control. To establish the linearity of the ChIP
assay, serial dilutions of immunoprecipitated DNA or total DNA were used as substrates for
PCR amplification of amplify 200bp CENIV or fragments 200bp fragments lying 400bp
upstream or downstream. The ChIP signal was determined as a ratio of CENIV DNA recovered
by IP to CENIV in the total DNA preparation. ChIP data is presented as a ratio of signals for
mutant vs. wildtype strains or CENIV vs. flanking DNA.
89
Acknowledgements
We thank J. Dom for aid with statistical analyses and image processing, G. Jelson for
microscopy and image analysis and members of the Sorger lab for critical readings of the
manuscript. J.D.T. was supported by a fellowship from the HHMI. This work was funded by
NIH grants GM51464 and GM64524 to PKS.
Abbreviations list
CEN- centromere
MAP - microtubule associated protein
ChIP - chromatin immunoprecipitation
SPB - spindle pole body
pMTs - pole-to-pole microtubules
kMTs - kinetochore microtubules
90
References
Ciosk, R., W. Zachariae, C. Michaelis, A. Shevchenko, M. Mann, and K. Nasmyth. 1998. An
ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to
anaphase transition in yeast. Cell. 93:1067-76.
Cottingham, F.R., and M.A. Hoyt. 1997. Mitotic spindle positioning in Saccharomyces
cerevisiae is accomplished by antagonistically acting microtubule motor proteins. J Cell
Biol. 138:1041-53.
Dagenbach, E.M., and S.A. Endow. 2004. A new kinesin tree. J Cell Sci. 117:3-7.
Desai, A., S. Verma, T.J. Mitchison, and C.E. Walczak. 1999. Kin I kinesins are microtubuledestabilizing enzymes. Cell. 96:69-78.
DeZwaan, T.M., E. Ellingson, D. Pellman, and D.M. Roof. 1997. Kinesin-related KIP3 of
Saccharomyces cerevisiae is required for a distinct step in nuclear migration. J Cell Biol.
138:1023-40.
Dom, J.F., K. Jaqaman, D. R. Rines, G. S. Jelson, P. K. Sorger, and G. Danuser. 2005.
Interphase kinetochore microtubule dynamics in yeast analyzed by high-resolution
microscopy. Biophys. J. accepted.
Endow, S.A., S.J. Kang, L.L. Satterwhite, M.D. Rose, V.P. Skeen, and E.D. Salmon. 1994. Yeast
Kar3 is a minus-end microtubule motor protein that destabilizes microtubules
preferentially at the minus ends. Embo J. 13:2708-13.
Geiser, J.R., E..J. Schott, T.J. Kingsbury, N.B. Cole, L.J. Totis, G. Bhattacharyya, L. He, and
M.A. Hoyt. 1997. Saccharomyces cerevisiae genes required in the absence of the CIN8encoded spindle motor act in functionally diverse mitotic pathways. Mol Biol Cell.
8:1035-50.
Gheber, L., S.C. Kuo, and M.A. Hoyt. 1999. Motile properties of the kinesin-related Cin8p
spindle motor extracted from Saccharomyces cerevisiae cells. JBiol Chem. 274:9564-72.
Gillett, E.S., C.W. Espelin, and P.K. Sorger. 2004. Spindle checkpoint proteins and
chromosome-microtubule attachment in budding yeast. J Cell Biol. 164:535-46.
Goh, P.Y., and J.V. Kilmartin. 1993. NDC10: a gene involved in chromosome segregation in
Saccharomyces cerevisiae. J Cell Biol. 121:503-12.
Gordon, D).M.,and D.M. Roof. 1999. The kinesin-related protein Kip p of Saccharomyces
cerevisiae is bipolar. JBiol Chem. 274:28779-86.
Gordon, D.M., and D.M. Roof. 2001. Degradation of the kinesin Kiplp at anaphase onset is
mediated by the anaphase-promoting complex and Cdc20p. Proc Natl Acad Sci U SA.
98:12515-20.
Goshima, G., and M. Yanagida. 2000. Establishing biorientation occurs with precocious
separation of the sister kinetochores, but not the arms, in the early spindle of budding
yeast. Cell. 100:619-33.
He, X., S. Asthana, and P.K. Sorger. 2000. Transient sister chromatid separation and elastic
deformation of chromosomes during mitosis in budding yeast. Cell. 101:763-75.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochoremicrotubule attachment in budding yeast. Cell. 106:195-206.
Hildebrandt, E. R., and M.A. Hoyt. 2000. Mitotic motors in Saccharomyces cerevisiae. Biochim
Biophys Acta. 1496:99-116.
91
Hildebrandt, E.R., and M.A. Hoyt. 2001. Cell cycle-dependent degradation of the
Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdhl) and a bipartite
destruction sequence. Mol Biol Cell. 12:3402-16.
Hoyt, M.A., L. He, K.K. Loo, and W.S. Saunders. 1992. Two Saccharomyces cerevisiae kinesinrelated gene products required for mitotic spindle assembly. J Cell Biol. 118:109-20.
Hyman, A.A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubulemotor activity of a yeast centromere-binding protein complex. Nature. 359:533-6.
Janke, C., J. Ortiz, T.U. Tanaka, J. Lechner, and E. Schiebel. 2002. Four new subunits of the
Dam 1--D)uo
1 complex reveal novel functions in sister kinetochore biorientation. Embo J.
21:181-93.
Kapitein, L.C., E.J.G. Peterman, B.H. Kwok, J.H. Kim, T.M. Kapoor, and C.F. Schmidt. 2005.
The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature.
435:114-118.
Kline-Smith, S.L., S. Sandall, and A. Desai. 2005. Kinetochore-spindle microtubule interactions
during rmnitosis.Curr Opin Cell Biol. 17:35-46.
Lawrence, C.J.. R.K. Dawe, K.R. Christie, D.W. Cleveland, S.C. Dawson, S.A. Endow, L.S.
Goldstein, H.V. Goodson, N. Hirokawa, J. Howard, R.L. Malmberg, J.R. McIntosh, H.
Miki, T.J. Mitchison, Y. Okada, A.S. Reddy, W.M. Saxton, M. Schliwa, J.M. Scholey,
R.D. Vale, C.E. Walczak, and L. Wordeman. 2004. A standardized kinesin nomenclature.
J CellBiol. 167:19-22.
Lechner, J., and J. Carbon. 1991. A 240 kd multisubunit protein complex, CBF3, is a major
component of the budding yeast centromere. Cell. 64:717-25.
Li, R., and A.W. Murray. 1991. Feedback control of mitosis in budding yeast. Cell. 66:519-31.
Longtine, M.S.. A. McKenzie, 3rd, D.J. Demarini, N.G. Shah, A. Wach, A. Brachat, P.
Philippsen, and J.R. Pringle. 1998. Additional modules for versatile and economical
PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14:95361.
Maddox, P.S., K.S. Bloom, and E.D. Salmon. 2000. The polarity and dynamics of microtubule
assembly in the budding yeast Saccharomyces cerevisiae. Nat Cell Biol. 2:36-41.
Maiato, H., J. DeLuca, E.D. Salmon, and W.C. Earnshaw. 2004. The dynamic kinetochoremicrotubule interface. J Cell Sci. 117:5461-77.
Maney, T., M. Wagenbach, and L. Wordeman. 2001. Molecular dissection of the microtubule
depolymerizing activity of mitotic centromere-associated kinesin. J Biol Chem.
276:34753-8.
McAinsh, A.D., J.D. Tytell, and P.K. Sorger. 2003. Structure, function, and regulation of
budding yeast kinetochores. Annu Rev Cell Dev Biol. 19:519-39.
Megee, P.C., C. Mistrot, V. Guacci, and D. Koshland. 1999. The centromeric sister chromatid
cohesion site directs Mcdlp binding to adjacent sequences. Mol Cell. 4:445-50.
Meluh, P.B., and M.D. Rose. 1990. KAR3, a kinesin-related gene required for yeast nuclear
fusion. Cell. 60:1029-41.
Miranda, J.J., P. De Wulf, P.K. Sorger, and S.C. Harrison. 2005. The yeast DASH complex
forms closed rings on microtubules. Nat Struct MolBiol. 12:138-43.
Mitchison, T.J., and E.D. Salmon. 2001. Mitosis: a history of division. Nat Cell Biol. 3:E17-21.
Moore, A., and L. Wordeman. 2004. The mechanism, function and regulation of depolymerizing
kinesins during mitosis. Trends Cell Biol. 14:537-46.
92
Niederstrasser, H., H. Salehi-Had, E.C. Gan, C. Walczak, and E. Nogales. 2002. XKCM1 acts on
a single protofilament and requires the C terminus of tubulin. J Mol Biol. 316:817-28.
Pearson, C.G., E. Yeh, M. Gardner, D. Odde, E.D. Salmon, and K. Bloom. 2004. Stable
kinetochore-microtubule attachment constrains centromere positioning in metaphase.
Curr Biol. 14:1962-7.
Rogers, G.C., S.L. Rogers, T.A. Schwimmer, S.C. Ems-McClung, C.E. Walczak, R.D. Vale,
J.M. Scholey, and D.J. Sharp. 2004. Two mitotic kinesins cooperate to drive sister
chromatid separation during anaphase. Nature. 427:364-70.
Roof, D.M., P.B. Meluh, and M.D. Rose. 1992. Kinesin-related proteins required for assembly of
the mitotic spindle. J Cell Biol. 118:95-108.
Saunders, W.S., and M.A. Hoyt. 1992. Kinesin-related proteins required for structural integrity
of the mitotic spindle. Cell. 70:451-8.
Severin, F., B. Habermann, T. Huffaker, and T. Hyman. 2001. Stu2 promotes mitotic spindle
elongation in anaphase. J Cell Biol. 153:435-42.
Straight, A.F., A.S. Belmont, C.C. Robinett, and A.W. Murray. 1996. GFP tagging of budding
yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid
cohesion. CurrBiol. 6:1599-608.
Straight, A.F., W.F. Marshall, J.W. Sedat, and A.W. Murray. 1997. Mitosis in living budding
yeast: anaphase A but no metaphase plate. Science. 277:574-8.
Tanaka, K., N. Mukae, H. Dewar, M. van Breugel, E.K. James, A.R. Prescott, C. Antony, and
T.U. Tanaka. 2005. Molecular mechanisms of kinetochore capture by spindle
microtubules. Nature. 434:987-94.
Tanaka, T., J. Fuchs, J. Loidl, and K. Nasmyth. 2000. Cohesin ensures bipolar attachment of
microtubules to sister centromeres and resists their precocious separation. Nat Cell Biol.
2:492-9.
West, R.R., T. Malmstrom, C.L. Troxell, and J.R. McIntosh. 2001. Two related kinesins, klp5+
and klp6+, foster microtubule disassembly and are required for meiosis in fission yeast.
Mol Biol Cell. 12:3919-32.
Westermann, S., A. Avila-Sakar, H.W. Wang, H. Niederstrasser, J. Wong, D.G. Drubin, E.
Nogales, and G. Barnes. 2005. Formation of a dynamic kinetochore- microtubule
interface through assembly of the Daml ring complex. Mol Cell. 17:277-90.
Winey, M., C.L. Mamay, E.T. O'Toole, D.N. Mastronarde, T.H. Giddings, Jr., K.L. McDonald,
and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the
Saccharomyces cerevisiae mitotic spindle. J Cell Biol. 129:1601-15.
Wordeman, L., and T.J. Mitchison. 1995. Identification and partial characterization of mitotic
centromere- associated kinesin, a kinesin-related protein that associates with centromeres
during mitosis. J Cell Biol. 128:95-104.
Zeng, X., J.A. Kahana, P.A. Silver, M.K. Morphew, J.R. McIntosh, I.T. Fitch, J. Carbon, and
W.S. Saunders. 1999. Slkl9p is a centromere protein that functions to stabilize mitotic
spindles. J Cell Biol. 146:415-25.
93
CHAPTER THREE
Cell Cycle Regulation of Kinetochore-Microtubule
Attachment
I planned and executed all experiments in this chapter.
3.1 Abstract
Attachment of centromeric DNA to microtubules is vital to the fidelity of chromosome
segregation. This connection is mediated by the kinetochore, a large, multi-protein complex
bridging chromosomes to microtubules. Although the identity of proteins mediating attachment
is becoming clearer, the dynamics and regulation of kinetochore-microtubule attachment during
the cell cycle are still poorly understood. In this work, I demonstrate the existence of a period
early in the cell cycle during which treatment with the microtubule depolymerizing drug
nocodazole results in an increase in chromosome detachment. Interestingly, nocodazole addition
after this period causes only minor detachment, indicating that kinetochore-microtubule
attachments are not uniform throughout mitosis. In addition, I show that in populations of cells
exhibiting little detectable chromosome detachment, metaphase arrest is still robust. This
indicates that loss of attachment may not be required to activate the spindle checkpoint.
3.2 Introduction
Proper chromosome segregation requires that sister chromatids become attached to
opposite poles of the mitotic spindle and maintain this attachment during complex changes in
force and microtubule dynamics. Attachment occurs via kinetochores, large protein structures
that assemble onto centromeres and tether them to the plus-ends of microtubules. During G1,
each kinetochore is attached to a single kinetochore-microtubule (k-MT) emanating from the
spindle pole body (SPB) (Winey et al., 1995). As the cell progresses into S-phase, the
chromosomes become replicated and the new sister chromatid must become attached to a
microtubule emanating from a newly replicated SPB. These attachments are not rigid. Instead the
kinetochores must remain attached to the dynamic k-MTs as they undergo frequent catastrophe
and rescue events (Maiato et al., 2004). The cohesin complex, which holds together newly
95
formed sister chromatids, opposes the forces generated by the k-MTs pulling toward the pole,
resulting in the generation of tension across the kinetochores (Tanaka et al., 2000). In higher
eukaryotes, an increase in tension leads to larger inter-kinetochore distances and a higher number
of microtubules per attachment (King and Nicklas, 2000; Shelby et al., 1996). In budding yeast,
tension across kinetochores results in the transient separation of the sisters and stretching of the
centromeric chromatin (Goshima and Yanagida, 2000; He et al., 2000; Tanaka et al., 2000).
Additional force across sister kinetochores is generated by the elongation and antiparallel sliding
of the interpolar microtubules of the mitotic spindle (p-MTs). Subsequently, at anaphase onset,
cohesin is cleaved and the kinetochores remain attached to the rapidly depolymerizing k-MTs.
Despite the fact that budding yeast kinetochores are attached to only a single, dynamic
microtubule, kinetochore-microtubule attachments are extremely stable. Many kinetochore
mutants weaken these chromosome-microtubule attachments resulting in chromosome
missegregation or arrest by a surveillance mechanism known as the spindle checkpoint (for
review see McAinsh et al., 2003; Taylor et al., 2004). However, with the exception of mutations
in the CBF3 Complex, which completely abrogate kinetochore assembly, only mutations in the
Ndc80 Complex have been shown to cause detachment of all kinetochores from the spindle
microtubules (e
et al., 2001). This suggests that microtubule attachment is accomplished
through the interaction of many proteins with a high degree of partial redundancy.
The spindle checkpoint is a surveillance mechanism that monitors kinetochoremicrotubule attachment to protect cells from chromosome loss. Lesions in attachment trigger the
spindle checkpoint to generate a "wait anaphase" signal. This checkpoint signal results in
sequestration of Cdc20p, the activator for the ubiquitin ligase Anaphase Promoting Complex
(APC) (Visintin et al., 1997). Madl -3p, Bublp, and Bub3p respond to spindle defects and
96
sequester Cdc20p, and all these proteins are essential for checkpoint function (for review see
Gillett and Sorger, 2001).
Treatment of higher eukaryotic cells with the microtubule depolymerizing drug
nocodazole causes spindle collapse and kinetochore detachment from microtubules resulting in
sister chromatid pairs floating freely throughout the cell. Activation of the spindle checkpoint
prevents further mitotic progress. Although nocodazole is routinely used in budding yeast to
detach chromosomes and arrest cells in mitosis, nocodazole addition results in limited
detachment of chromosomes from microtubules (Gillett et al., 2004). Instead, treatment of
asynchronous cells with nocodazole leads to p-MT and k-MT collapse, with the majority of
kinetochores colocalizing with SPBs by microscopy. Only a subset of cells contain chromosomes
distant from those clustered at the SPBs, and only these foci recruit the spindle checkpoint
proteins Madlp, Mad2p, and Bublp (Gillett et al., 2004). These data indicate that not all
chromosomes have equal attachment strengths.
I have utilized this observation to probe kinetochore-microtubule attachments during the
cell cycle by differential timing of nocodazole treatment. I observed a window early in the cell
cycle during which chromosomes were significantly more sensitive to nocodazole. When cells
are exposed to nocodazole after this period, the overall amount of detachment was extremely
low. Surprisingly, even in populations with low chromosome detachment, cells established an
extremely robust metaphase arrest, indicating that arrest did not depend on detached
chromosomes. These results indicate that chromosome-microtubule attachments vary with cell
cycle progression and provide a powerful new assay to detect subtle defects in chromosomemicrotubule attachment.
97
3.3 Results
3.3.1 Nocodazole Treatment Causes Limited Chromosome Detachment
To evaluate the strengths of chromosome-microtubule attachments, I observed the effect
of nocodazole on asynchronous cultures of S. cerevisiae coexpressing the kinetochore protein
Ndc80p-GFP and the SPB protein Spc42p-CFP. Prior to the addition of nocodazole, Ndc80pGFP exhibited the prototypical bilobed kinetochore distribution between the SPBs, indicating
that all chromosomes were properly attached. After treatment with nocodazole, many cells were
found to contain faint Ndc80p-GFP foci distant from the chromosomes (Figure 3.1A).
Chromosomes distant from SPBs recruit high concentrations of the spindle checkpoint proteins
Bublp, Madlp and Mad2p, and are believed to be detached from the mitotic spindle (Gillett et
al., 2004). The percentage of cells with additional Ndc80p-GFP foci increased from 0% before
the addition of nocodazole (n=451) to 31% after 1 hour in nocodazole (n=179) and 49% after 2
hours (n= 151)(Figure 1B). Of the cells with extra foci, 80% had only a single focus far from the
SPBs (n=74). In all cells the majority of Ndc80p-GFP colocalized with the SPBs. The low
percentage of kinetochores distal to the SPBs is in stark contrast to nocodazole treatment in
ndc80 mutants. In these cells, numerous kinetochore foci (visualized with Mtwlp-GFP) are
visible throughout the nucleus, indicating that most of the kinetochores have lost their
attachments (Figure 3. 1C). Because chromosomes are held together by cohesin until anaphase
onset, we believe that each Ndc80p focus is a single pair of sister chromatids. Therefore, we
conclude that in the vast majority of nocodazole-treated cells, only a single chromosome pair
became detached. These data suggest that k-MTs depolymerize in nocodazole, and though
chromosome-microtubule attachments in budding yeast are very strong and the majority
remained attached to chromosomes, a small percentage become detached.
98
A.
Nocodazole
B.
Kinetochore
Attachment
C.
Nocodazole
100%
J!1
Qi
CJ
I
75%
r
50%
r
I
0
~
D
Wildtype
I
25%
f
I
0%
0
I
60
time in nocodazole
• attached
Figure 3.1: Chromosome
detachment
120
ndcBO-7
(min)
c detached
in asynchronous
populations.
(A) Representative
image from asynchronous cells treated for two hours with nocodazole coexpressing the
kinetochore marker Ndc80p-GFP (green) and the SPB marker Spc42p-CFP (red). White arrows
indicate collapsed SPBs.Yellow arrow indicates detached chromosome. (B) Percentage of
asynchronous cells with at least one detached kinetochore as a function of time. Nocodazole
was added just after the 0 minute time point. (C) Representative image of Mtw1 p localization
(green) in ndcBO-7 cells incubated at 37° for 2 hours in nocodazole.
3.3.2 Response to Nocodazole Varies During the Cell Cycle
It remained unclear why some, but not all, cells exhibited detached chromosomes. I
postulated that there might be a window of sensitivity during which the attachments were less
mature. During S-phase, kinetochores are presumed to be displaced from the DNA in order to
allow replication of centromeric DNA. I speculated that this displacement, albeit transient, might
increase the sensitivity to nocodazole. To test this hypothesis I arrested cells coexpressing
Ndc80p-GFP and Spc42p-CFP with a-factor and then released half of the culture directly into
nocodazole (early addition) and released the other half into fresh medium for one hour before
nocodazole addition (late addition) to allow progression through S phase. Time points for
microscopy were taken every hour after c-factor arrest and scored by microscopy for the
proximity of the Ndc80p-GFP foci to the SPBs. Before release from c-factor, all chromosomes
were in close proximity to the SPBs (Figure 3.2A). In cells released directly into nocodazole,
53% of cells had at least one detached chromosome after one hour (n=299), and this rose to 78%
of cells after 2 hours (n=232)(Figure 3.2A). The cells with late addition of nocodazole showed
significantly different results. One hour after nocodazole addition, only 3.5% of cells exhibited
any detached chromosomes (n=172). After 2 hours in nocodazole only 8% of cells had detached
chromosomes (n=120)(Figure 3.2B). These data indicate that there is a period early in the cell
cycle - perhaps S-phase - during which kinetochore-microtubule attachments are sensitive to
nocodazole, and that exposure to nocodazole after this period leads to collapse of kinetochoremicrotubules but not the detachment of chromosomes from the spindle.
100
B.
Late Nocodazole Addition
100%
.!!!.
Q)
-
• attached
[J detached
u
o
?f!.
0%
o
60
120
180
time after a-factor release (min)
o
o
60
120
time in nocodazole (min)
Figure 3.2: Cell-cycle-dependence
of nocodazole sensitivity. (A) Percentage of cells with
attached and detached chromosomes after a-factor release into nocodzole. x-axis indicates
time after a-factor on top and time after nocodazole addition on the bottom. (B) Percentage
of cells with attached and detached chromosomes before and after nocodazole treatment.
Nocodazole added 60 minutes after a-factor release. x-axis indicates time after a-factor on
top and time after nocodazole addition on the bottom. In both graphs the dotted line
indicates time of nocodazole addition.
3.3.3 Checkpoint arrest does not depend on timing of nocodazole addition
It has been suggested that the spindle checkpoint might require detached chromosomes to
generate the "wait anaphase" signal (Gillett et al., 2004). Therefore I tested whether the
checkpoint mediated arrest was as robust in cells with late nocodazole treatment as in
asynchronous cells. The extremely low levels of chromosome detachment observed in cells with
late nocodazole treatment suggested that checkpoint proteins might be recruited in just this small
subset of cells. If the checkpoint only senses detached kinetochores, then the checkpoint should
only be activated in the >10% of these cells with detached chromosomes, and just those few cells
should arrest. The remaining 90% of cells should proceed into anaphase unencumbered.
However, FACS analysis demonstrated that both "early" and "late" addition of nocodazole leads
to robust arrest with 2N DNA content lasting for at least 21/2hours after addition of nocodazole
(Figures 3.3A&B). Therefore, based on our observations, activation of the checkpoint and
metaphase arrest are not solely dependent on the presence of unattached kinetochores. However,
it remains a distinct possibility that the attached, but collapsed, kinetochores might represent
incomplete attachments that might activate the spindle checkpoint without recruiting levels of
checkpoint proteins visible by microscopy.
3.4 Discussion
3.4.1 Kinetochores are Sensitive to Nocodazole Early in the Cell Cycle
It is commonly presumed that nocodazole eliminates microtubule attachments through
depolymerizatio)n in yeast, as in humans cells. Here I show evidence that, contrary to these
general assumptions, the majority of kinetochore attachments survive nocodazole treatment.
Only half of asynchronous cells treated with nocodazole have detectable detached chromosomes.
Because chromosome loss in nocodazole was non-uniform, I suspected that there might be cell
102
Early Nocodazole Addition
A.
t190
t150
t135
t120
t105
t90
t75
t60
t45
t30 noc
to ~
B.
Late Nocodazole Addition
1210
t190
t150
t135
t120
t105
t90
t75 noc
t60~
t45
t30
to
Figure 3.3: FACS analysis of early and late nocodazole addition. (A) FACS plots of
cultures to which nocodazole was added immediately upon release from a-factor. (B)
FACS plots of cultures to which nocodazole was added immediately after the GO-minute
time point.
cycle dependent weaknesses in chromosome attachment. Further investigation uncovered such a
cell cycle dependent weakness of kinetochore-microtubule attachment; nearly 80% of cells
released directly into nocodazole exhibit detached chromosomes after 2 hours, whereas those
cells allowed to enter the cell cycle for one hour in the absence of nocodazole had less than 10%
detachment after two hours in nocodazole. Notably, even in cells with visibly detached
chromosomes, the majority of chromosomes were closely associated with the SPBs, suggesting
that they maintained chromosome-microtubule attachment.
As DNA replication proceeds, all bound proteins are presumed to be removed from the
DNA for the replication machinery to pass through. Therefore, kinetochore complexes and their
attached microtubules become dissociated from centromeric DNA, at least briefly, during Sphase. It is possible that under wildtype conditions, microtubule plus-ends might retain partial
kinetochore binding, stabilizing the microtubules and keeping them near the centromeres until
the DNA-recruited kinetochore proteins reassemble following replication. Addition of
nocodazole might depolymerize these microtubules, thereby removing the prebound
microtubules from the vicinity of the centromeres and making reattachment by the sisters much
more difficult (Figure 3.4A). This model would explain the sensitivity to early but not late
nocodazole addition.
However, if all kinetochores are released from the centromere during S-phase, why are
most chromosomes still attached after early release into nocodazole? When treated with
nocodazole after a-factor release, the chromosomes collapse to the poles and the mitotic spindle
never elongates. Therefore, chromosomes are likely in close proximity to the duplicated SPBs
during S-phase and when kinetochore dissociate, the microtubules cannot depolymerize far away
104
A.
/
Without Nocodazole
With Nocodazole
Before Replication
S Phase
After Replication
B.
II. Bipolar Attachment
I. Syntelic Attachment
Figure 3.4: Models for nocodazole response. (A) Model for S-phase progression. Before replication
kinetochores are attached to a microtubule. (top panel) During S-phase, the CEN binding kinetochore
components transiently dissociate from the DNA but the microtubule remains nearby, stabilized by the
microtubule associated proteins (MAPs). Once the replication fork has passed, the proximity of the
microtubule allows it to be recaptured by the remainder of the kinetochore. (bottom panel) In the
presence of nocodazole, the CEN binding kinetochore components still dissociate from DNA but the
microtubule depolymerizes away from the DNA, making recapture difficult. After replication the
CEN-binding kinetochore components bind the centromere, but the microtubule cannot be captured.
(B) Model for kinetochore recapture in nocodazole. Upon addition of nocodazole both kinetochoremicrotubules and interpolar microtubules collapse. Therefore all chromosomes are nearby the SPBs.
If short microtubules CCWl be nucleated, these can capture chromosomes that lose attachments
forming either (I) syntelic or (II) bipolar attachments. Either attachment is sufficient to sequester the
chromosomes near the SPBs.
from the centromeres. In addition, the two SPBs are in close proximity to the sister chromatids.
Therefore, the nucleation of short microtubules from either SPB might capture the nearby
centromeres by raising the effective concentration of microtubules and centromeres (Figure
4.4B).
The intermediate level of detachment in nocodazole suggests that nocodazole treatment
might be an excellent assay for detecting proteins that make small contributions to attachment or
for factors that are loaded during S-phase. It is possible that many kinetochore proteins play
small redundant roles in chromosome attachment, and because of their redundancy, cause no
obvious defects in normal cell cycles. Nocodazole treatment of these mutant cells might then
increase the number of detached chromosomes. Similarly, this assay could be used to assess the
ability of various kinetochore mutants to form novel attachments after chromosome detachment.
To this end, one could release cells from a-factor into nocodazole and quantitate the detached
chromosomes. Subsequently, nocodazole could be washed out and one could quantitate the
ability of the cells to reattach the detached chromosomes.
3.4.2 The Spindle Checkpoint Signals in the Absence of Detached Chromosomes
Although a great deal is known about the spindle checkpoint, there are still many open
questions. Notably, debate exists regarding the types of lesions sensed by the checkpoint;
specifically, whether the checkpoint can directly sense tension defects or whether detachments
are required for checkpoint activation (Biggins and Walczak, 2003). Studies from cdc6 mutant
cells indicate that unreplicated chromosomes that have made monopolar attachments signal the
checkpoint indicating that cells can respond to tension (Stem and Murray, 2001). However, these
106
data are not conclusive and there is also significant evidence that the spindle checkpoint cannot
recognize tension alone (Draviam, 2005; Gillett et al., 2004).
I have found that only a low percentage of cells treated with nocodazole after S-phase
have kinetochores distant from the SPBs. Previous studies found that only these chromosomes
recruited checkpoint proteins (Gillett et al., 2004). Taken together, these data suggest that the
majority of chromosomes in such cultures are attached to microtubules. However, these cells
robustly arrest, indicating that detached chromosomes are not required to signal the checkpoint.
It is tempting to consider the possibility that loss of tension triggers the checkpoint in these cells.
However, there are several caveats to this conclusion. First, the Ipl lp kinase is functional in these
cells and could be causing the transient detachment of chromosomes in response to the loss of
tension. Also, it is possible that some of the kinetochores have only monopolar attachments,
sequestering them to the SPBs, and that the unattached sisters are signaling the checkpoint
without recruiting high concentrations of the checkpoint proteins. Low levels of checkpoint
proteins could go undetected by microscopy. Further investigation is required to distinguish
between checkpoint response to tension and attachment.
107
References:
Biggins, S., and C.E. Walczak. 2003. Captivating capture: how microtubules attach to
kinetochores. Curr Biol. 13:R449-60.
Draviam, V.M. 2005. Misorientation and reduced centromeric stretching promote chromosome
missegregation.manuscript in preparation.
Gillett, E.S., C.W. Espelin, and P.K. Sorger. 2004. Spindle checkpoint proteins and
chromosome-microtubule attachment in budding yeast. J Cell Biol. 164:535-46.
Gillett, E.S., and P.K. Sorger. 2001. Tracing the pathway of spindle assembly checkpoint
signaling. Dev Cell. 1:162-4.
Goshima, G., and M. Yanagida. 2000. Establishing biorientation occurs with precocious
separation of the sister kinetochores, but not the arms, in the early spindle of budding
yeast. Cell. 100:619-33.
He, X., S. Asthana, and P.K. Sorger. 2000. Transient sister chromatid separation and elastic
deformation of chromosomes during mitosis in budding yeast. Cell. 101:763-75.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochoremicrotubule attachment in budding yeast. Cell. 106:195-206.
King, J.M., and R.B. Nicklas. 2000. Tension on chromosomes increases the number of
kinetochore microtubules but only within limits. J Cell Sci. 113 Pt 21:3815-23.
Maiato, H., J. D)eluca, E.D. Salmon, and W.C. Earnshaw. 2004. The dynamic kinetochoremicrotubule interface. J Cell Sci. 117:5461-77.
McAinsh, A.D., J.D. Tytell, and P.K. Sorger. 2003. Structure, function, and regulation of
budding yeast kinetochores. Annu Rev Cell Dev Biol. 19:519-39.
Shelby, R.D., K.M. Hahn, and K.F. Sullivan. 1996. Dynamic elastic behavior of alpha-satellite
DNA domains visualized in situ in living human cells. J Cell Biol. 135:545-57.
Stern, B.M., and A.W. Murray. 2001. Lack of tension at kinetochores activates the spindle
checkpoint in budding yeast. Curr Biol. 11:1462-7.
Tanaka, T., J. Fuchs, J. Loidl, and K. Nasmyth. 2000. Cohesin ensures bipolar attachment of
microtubules to sister centromeres and resists their precocious separation. Nat Cell Biol.
2:492-9.
Taylor, S.S., M.I. Scott, and A.J. Holland. 2004. The spindle checkpoint: a quality control
mechanism which ensures accurate chromosome segregation. Chromosome Res. 12:599616.
Visintin, R., S. Prinz, and A. Amon. 1997. CDC20 and CDH1: a family of substrate-specific
activators of APC-dependent proteolysis. Science. 278:460-3.
Winey, M., C.L. Mamay, E.T. O'Toole, D.N. Mastronarde, T.H. Giddings, Jr., K.L. McDonald,
and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the
Saccharomyces cerevisiae mitotic spindle. JCellBiol. 129:1601-15.
108
CHAPTER FOUR
Conclusions and Future Directions
4.1 Conclusions
Accurate chromosome segregation is vital to the genetic stability of all organisms. Proper
segregation requires that every sister chromatid pair establishes a bipolar attachment to the
mitotic spindle, whereby each sister binds a microtubule extending from an opposing pole. This
attachment is mediated by the kinetochore, a multi-protein complex that bridges centromeric
DNA to the microtubules.
When I began my thesis research (2000), the complexity of the kinetochore was not yet
evident. Many kinetochore proteins required for assembling the kinetochore structure were still
being discovered. It was also just becoming clear that kinetochores do not simply form
attachments -they
also regulate the microtubules to which they are attached (He et al., 2001).
In addition, the dynamic nature of chromosome movement and its interrelation with the mitotic
spindle were not yet fully appreciated. The main cause of chromosome movement was thought to
be the transport of chromosomes as cargo along microtubules by motor proteins. However, it is
now apparent that kinetochores are not simply static attachment factors, but instead are active
regulators of microtubule dynamics and chromosome movement. It is also clear that the coupling
of kinetochores to the microtubule plus-ends, and the concurrent regulation of microtubule
dynamics, generate a significant fraction of chromosome movement.
This thesis presents data that advances our understanding of the role of the kinesin motors
in the regulation of budding yeast chromosome movement and organization, as well as
chromosome-microtubule attachment. Using fluorescence microscopy and chromatin
immunoprecipitation, I show that three of the four nuclear kinesins in budding yeast are present
at kinetochores with bipolar attachments: Cin8p, Kiplp, and Kip3p. The Kinesin-5 motors Cin8p
and Kip p function redundantly to align chromosomes during metaphase into a biblobed
110
distribution - the typical metaphase kinetochore localization pattern that is analogous to the
metaphase plate of higher eukaryotes. I propose that these motors crosslink k-MTs and bundle
centromeres. The plus-end binding microtubule-destabilizing kinesin, Kip3p, regulates
microtubule dynamics throughout the cell cycle and is required for synchronous chromosome
movement during anaphase A. The minus-end directed motor, Kar3p, localizes specifically to
improperly or unattached kinetochores, but not to bioriented kinetochores during the cell cycle.
Through careful study of asynchronous and synchronous cultures treated with nocodazole, I have
discovered that kinetochore-microtubule attachments are not uniform throughout the cell cycle
and that nocodazole does not detach the majority of chromosomes. I also have preliminary data
suggesting that cells can sustain a robust spindle checkpoint arrest in the absence of detached
chromosomes. Together these findings represent a significant advancement in kinetochore
biology.
4.1.1 Kinesins Localize to Budding Yeast Kinetochores
It was originally hypothesized that motors were responsible for the majority of
chromosome movement in yeast (McIntosh et al., 2002). Motors were thought to bind
chromosomes as cargo and walk them along microtubules. According to this model, plus-end
motors carried chromosomes to the metaphase plate while minus-end directed motors transported
chromosomes poleward during anaphase. Further examination of both yeast and higher
eukaryotic chromosome movement has shown that microtubule dynamics are regulated by MAPs
such as the +TIPs, and are responsible for a considerable fraction of force regulation in the
mitotic spindle (for review see Akhmanova and Hoogenraad, 2005). Further, once captured,
kinetochores maintain an end-on attachment, calling into question the model of chromosomes as
cargo. When I began my research, the role of the motor proteins in budding yeast had yet to be
111
determined and their roles in higher eukaryotes were still debated. Therefore, I set out to
determine the complement of motors at budding yeast kinetochores. I found that all four nuclear
kinesins function at budding yeast kinetochores. The plus-end directed motors Cin8p, Kip lp, and
Kip3p localize to mature kinetochores, whereas the minus-end directed motor Kar3p localizes to
kinetochores with attachment defects, but not to kinetochores that have established bipolar
attachments. The presence of only plus-end directed motors during anaphase rules out the model
of motors walking chromosomes as cargo toward the poles. Instead their presence supports the
model whereby kinetochores remain attached to the depolymerizing plus-ends of microtubules.
Deletio)n of KAR3 slows transport of newly captured kinetochores to the poles (Tanaka
et al., 2005). It has been proposed that Kar3p might function at kinetochores throughout the cell
cycle. However, I have found that Kar3p rarely colocalizes with kinetochores during unperturbed
cell cycles. When treated with nocodazole, Kar3p colocalizes with kinetochores - indicating that
it is only recruited to detached kinetochores. Furthermore, GI1chromosome dynamics in kar3
mutants demonstrate that Kar3p plays little or no role in regulating chromosome motion in G1.
Therefore, I hypothesize that Kar3p is recruited only to immature or detached kinetochores and
that it functions to return detached chromosomes to the SPBs. It is important to note that, unlike
with mature attachments, captured chromosomes move laterally along microtubules. Therefore
Kar3p may transport chromosomes as cargo to the poles before they establish bipolar
attachments.
This role for Kar3p is analogous to the role of dynein in higher eukaryotes. Dynein aids
in the capture of kinetochores and transports them laterally along microtubules toward the pole.
Dynein dissociates from kinetochores and moves to the spindle as chromosomes establish mature
112
attachments. Therefore, despite the substitution of a different motor type, I believe that the
mechanism of chromosome capture has been conserved through evolution.
One important focus for future research is the process of establishing end-on attachments
after recruitment to microtubule minus-ends. The microtubules along which newly captured
kinetochores are transported, are preferentially stabilized at the plus-ends by the +TIP proteins:
Stu2p and Bik lp (Tanaka et al., 2005). This suggests that the microtubule to which a captured
chromosome forms its lateral attachment might not be the one to which it makes its mature
attachment. There are several models to explain how microtubules could transition from a lateral
to a mature attachment. Kar3p could walk captured kinetochores toward the SPB where
chromosomes could be efficiently captured by the high concentration of searching microtubules.
This idea is appealing in that it does not require any additional protein function. A second
attractive model proposes that the Ipll p kinase responds to tensionless kinetochores once they
are returned to the poles by detaching them from the microtubule as it does for syntelic
attachments (Tanaka et al., 2005). Another possibility is that the minus-end depolymerizing
activity of Kar3p preferentially depolymerizes the microtubule from the minus-end after
transporting the chromosome to the SPB. This third model is in keeping with the known
biochemical activity of Kar3p, which depolymerizes stabilized microtubules preferentially from
the minus-ends (Endow et al., 1994). However, no evidence of treadmilling has been observed in
budding yeast and, therefore, the minus-ends of budding yeast microtubules are thought to be
nondynamic (Maddox et al., 2000). Also, kinetochores and chromosomes are bulky and may be
stericly inhibited from reaching the extreme minus-end of the microtubule. Current data suggest
that one of the first two models are correct -Kar3p
walks chromosomes to the SPB embedded
minus-ends of microtubules, where they are captured either with or without the help of
113
AuroraB/Ipl lIp.However, further experiments will be required to distinguish between these
models.
4.1.2 Differential Recruitment of Kinesins to Kinetochores
Kinesins are recruited to kinetochores by different subcomplexes. While all three
metaphase motors require the CBF3 Complex in order to associate with centromeric DNA, only
Cin8p and Kip Ilp also require the Ndc80 Complex. Because kinetochores dissociate from
microtubules in the absence of the Ndc80 Complex, dependence on this complex implies that
microtubule binding is also required for association with centromeres. Along with other studies
of kinetochore architecture, these data indicate that not all kinetochore proteins assemble directly
onto DNA. Instead, I hypothesize that only a subset of proteins - consisting mainly of DNA
binding proteins and linkers - assemble directly onto DNA. The majority of the plus-end
microtubule associated proteins and the ring structure formed by the Damlp Complex, combine
to form a plus-end substructure. Mature kinetochore attachments are established by docking of
the centromere-assembled subcomplex with the plus-end subcomplex (Figure 4.1A).
Unlike the other MAPs and motors, Kip3p does not require the Ndc80 Complex for
centromere association. This indicates that Kip3p may be recruited to the centromere-associated
subcomplex of kinetochores rather than the microtubule plus-ends, mimicking the localization of
the Kip3p homolog MCAK to the inner kinetochore region in higher eukaryotes (Wordeman and
Mitchison, 1995). hypothesize that Kip3p may colocalize with the ends of microtubules
embedded in kinetochores, allowing the protein to directly regulate the microtubule plus-ends
(Figure 4. 1B).
114
A.
Microtubule-Associated
comp~
Kinetochore Mega-Complex
Centromere Binding Complexes
Figure 4.1: Model for kinetochore architecture. (A) Most microtubule associated proteins (MAPsyellow), including the Dam1 Complex are recruited to microtubules, while the centromere binding
proteins (pink) and linker complexes (blue) and a few kinetochore recruited MAPs (including Kip3p)
assemble onto centromeric DNA. The centromere binding component then captures the microtubule
via the associated complexes forming the mature funtional kinetochore. (B) Model for differential
recruitment of kinesins to kinetochores
It remains unclear, however, how Kar3p is specifically recruited to detached or
improperly attached kinetochores. One hypothesis is that recruitment of Kar3p to kinetochores
may involve the spindle checkpoint protein Bublp. Bub lp localization mirrors that of Kar3p,
with the exception that a much lower percentage of prometaphase cells show Kar3p localization
than show Bub p localization (Gillett et al., 2004). However, Kar3p localization to unattached
kinetochores disappears in ndc80-1 mutants as assayed by chromatin immunoprecipitation and
microscopy, whereas Bub lp does not require Ndc80p for kinetochore localization (though it
does require the Ndc80 Complex members Spc24p and Spc25p) (Gillett et al., 2004 and P. De
Wulf personal communication). The mammalian checkpoint protein, BubR1, recruits the kinesin
CENP-E to kinetochores and CENP-E, in turn, modulates BubR1 activity (Mao et al., 2003; Yao
et al., 2000). Thus, it is tempting to speculate that Kar3p and Bub may interact in a similar
manner to CENP-E and BubRI1.Experiments are currently underway to clarify the mechanism of
Kar3p recruitment to detached kinetochores in yeast.
4.1.3 Cin8p and Kiplp Regulate the Establishment of the Budding Yeast Metaphase Plate
Cin8p and Kip lp are closely related kinesins that form homotetramers (Gordon and Roof,
1999). The higher eukaryotic homolog Eg5 can crosslink and walk along parallel and antiparallel
microtubules in vitro (Kapitein et al., 2005). While budding yeast do not form the stereotypical
metaphase plates of higher eukaryotes, they do have an analogous localization pattern - the
bilobed kinetochore distribution (He et al., 2000). I have found that mutation of Cin8p and Kip lp
in budding yeast leads to defects in chromosome alignment during metaphase. Based on
biochemical data, hypothesize that Cin8p and Kiplp crosslink adjacent kinetochores during
metaphase. This crosslinking of multiple centromeres could coordinate chromosome movement
116
and stabilize the spindle (Figure 4.2). Bundling kinetochores and microtubules might also
strengthen chromosome-microtubule attachments, and could explain the high rate of
chromosome loss in cin8 mutants.
While budding yeast kinetochores are bound to a single microtubule via an end-on
attachment, higher eukaryotes recruit an average of 20-25 microtubules per kinetochore
(Brinkley and Cartwright, 1971; Rieder, 1982; Winey et al., 1995). However, some evidence
suggests that the regional centromeres could be composed of multiple individual centromere
units. These units are specified by the specialized histone CENP-A and interspersed with regions
of non-centromeric histone H3 (Vafa and Sullivan, 1997; Zinkowski et al., 1991). Each
individual centromere could recruit a single microtubule. In this case, mammalian kinetochores
could be composed of dozens of interconnected point-centromere-like modules. I speculate that
the crosslinking of adjacent centromeres in budding yeast is analogous to the bundling of
centromere modules in higher eukaryotes. These data suggest that centromere organization and
microtubule binding may be more evolutionarily conserved than previously believed.
4.1.4 Kinesins function in regulating chromosome dynamics
1 have discovered that Kip3p regulates microtubule dynamics throughout the cell cycle.
Loss of Kip3p causes alterations in GI1microtubule dynamics. In a-factor, k-MTs are longer in
kip3A cells than in wildtype cells, indicating a loss of depolymerizing ability. In metaphase,
kip3Acin8A double mutants have decreased transient separation, indicating that Cin8p and Kip3p
regulate microtubule dynamics during metaphase. In the absence of Kip3p, cells initiate
anaphase asynchronously, suggesting that Kip3p is a switch-like signal regulating microtubule
dynamics. In wildtype cells, anaphase A is extremely fast, and microtubules depolymerize
117
Figure 4.2: Models for kinetochore architecture. Cin8p and Kip1 p bundle separate centromeres
together to form linked units from individual kinetochores (green).
without pausing until they reach the SPBs. In contrast, in some kip3A mutant cells chromosomes
move toward the SPBs but then pause before completing anaphase A. Other kip3A mutant cells
destabilize their chromosomes too early (before cohesin is cleaved) as evidenced by the duration
of preanaphase stretching. Therefore, Kip3p does not simply act as a microtubule-destabilizing
factor, but instead it acts as a complex modulator of microtubule dynamics. The premature
anaphase A movement in kip3A mutant cells suggests that Kip3p may actually stabilize k-MTs
during metaphase. The change in Kip3p activity from stabilization to destabilization of
microtubules suggests that Kip3p itself is regulated in a cell cycle dependent manner.
This leads to the important question of the mechanism of Kip3p regulation. The most
likely mechanism is phosphoregulation. Kip3p is a phophoprotein that can be phosphorylated by
the Cdc28p kinase in vitro (Sullivan et al., 2001; Ubersax et al., 2003). Dephosphorylation by the
Cdc 14p phophatase regulates spindle dynamics at the metaphase-to-anaphase transition and is a
good candidate for Kip3p regulation. It has been suggested, however, that Kip3p is not a
substrate of Cdc 14p (Sullivan et al., 2001). Evidence from higher eukaryotes demonstrates that
the Kip3p homolog, MCAK, is phosphorylated by the Aurora B/Ipllp kinase in vitro and that
phosphorylation of MCAK reduces its ability to destabilize microtubules and alters its
localization (Andrews et al., 2004; Gorbsky, 2004; Lan et al., 2004). Therefore, Kip3p may also
be a substrate of Ipllp in S. cerevisiae.
Phosphorylation of Kip3p by Ipl lp should occur early in the cell cycle, perhaps early in
metaphase after kinetochore capture. Since Ipllp moves from kinetochores to the spindle
midzone at the metaphase-to-anaphase transition, this could allow for the dephosphorylation of
Kip3p. Dephosphorylation may then act to increase MT depolymerization. In higher eukaryotes,
Aurora B activity is opposed by type 1 protein phosphotase (Gorbsky, 2004). In budding yeast,
119
the type 1 phosphatase Glc7p, localizes to kinetochores and opposes Ipl p function in vivo,
though its substrates are unknown (Francisco et al., 1994). Therefore, Glc7p might
dephosphorylate Kip3p, as well as other Ipl p targets, to regulate kinetochore-microtubule
attachment microtubule dynamics during metaphase and anaphase and perhaps in repairing
syntelic attachments early in metaphase as well. The regulation of Kip3p function is an important
question that remains to be addressed.
4.1.5 Cell Cycle Regulation of the Kinetochore
Unlike higher eukaryotes where chromosomes condense and must become attached to the
mitotic spindle following nuclear envelope break down, budding yeast kinetochores remain
attached to microtubules throughout the cell cycle (Dom, 2005). Although kinetochores remain
attached to microtubules, it is unknown if this attachment is uniform or if kinetochoremicrotubule attachments change or mature as the cell cycle progresses. I found that when k-MTs
proceed into the cell cycle in the presence of nocodazole, a large percentage of cells lose some
kinetochore attachment. However, when cells are allowed to proceed past S-phase in the absence
of nocodazole, they lose a significantly smaller percentage of chromosomes. These results
suggest that chromosomes proceeding through S-phase in the presence of nocodazole are unable
to establish mature attachments. It is initially tempting to assume that the detached chromosomes
we observe dissociate during S-phase. One model is that kinetochores dissociate briefly from the
centromeres during replication. In the absence of nocodazole, the microtubules may remain near
the DNA, stabilized by the microtubule-associated proteins of the kinetochore. The addition of
nocodazole at this point would depolymerize the microtubules, resulting in spatial separation
between centromeres and the microtubules. However, this explanation may be too simplistic or
120
may address only part of the process since chromosome detachment continues 2 hours after
nocodazole addition whereas DNA replication is complete after the first hour.
Another possible explanation for differences in kinetochore-microtubule attachment
strengths is cell-cycle-dependent changes in kinetochore architecture and regulation. Kinetochore
composition is not uniform throughout the cell cycle. Instead, there is mounting evidence that the
kinetochore is highly regulated during the cell cycle both by differential recruitment of subunits
and by posttranslational modifications - especially phosphorylation. At least three separate
phosphorylation pathways function at kinetochores during the cell cycle. The cyclin dependent
kinases phosphorylate many kinetochore proteins, including Ipl lp and Kip3p (Ubersax et al.,
2003). Ipl p in turn, has been shown to phosphorylate many kinetochore proteins, presumably in
response to inappropriate microtubule attachments (Cheeseman et al., 2002; Hsu et al., 2000;
Shang et al., 2003). Finally the spindle checkpoint kinases, Bub p and Mpslp, are likely to
phosphorylate kinetochore proteins and/or kinetochore regulatory proteins (Gillett and Sorger,
2001). Dephosphorylation of kinetochore components has also been shown to regulate their
function and localization (Higuchi and Uhlmann, 2005). Investigation of subunit composition
demonstrates that the make-up of the linker COMA Complex changes during the cell cycle (De
Wulf et al., 2003). Cell cycle regulation of protein stability also regulates kinetochore function.
Cin8p and Kip p are degraded at mitotic exit and anaphase onset respectively, and do not
associate with kinetochores until S-phase (Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001).
Therefore, it is evident that cell cycle regulation plays a significant role in kinetochore
regulation, and that a complete understanding of cell-cycle-dependent regulation will be required
for understanding kinetochore function.
121
4.1.6 Triggers for the Spindle Checkpoint
Although the data are preliminary, it appears that populations of cells can establish and
maintain a robust spindle checkpoint arrest in response to nocodazole even when less than 10%
of these cells have a single detached kinetochore. Since spindle checkpoint proteins are only
detectable by microscopy at unattached kinetochores, these data suggest that the checkpoint is
responding to a signal other than detachment. Several issues could account for this observation.
Most simply, the chromosomes that have collapsed back to the SPBs could be recruiting low
levels of checkpoint proteins to an unattached sister in a monopolarly attached chromosome pair.
Simniliarly,the checkpoint could be responding to syntelic or bipolar attachments that are lacking
tension. In these cases, spindle checkpoint proteins would be directly recruited to chromosomes
with subtle defects. It is also possible that transient detachments caused by Ipllp-mediated
detachments from microtubules signal the checkpoint. Another option is that nocodazole
treatment might alter kinetochore structure itself (distinct from mutation in kinetochore
components), and that displaced kinetochore proteins are engaging the spindle checkpoint in the
absence of checkpoint recruitment to kinetochores. Circumstantial evidence supporting this last
idea comes from the observation that that some kinetochore proteins lose CEN association in
nocodazole treatment - but not in mutants of the Ndc80 complex. This indicates that nocodazole
treatment alters kinetochores differently than detachment by kinetochore mutants (J. Tytell
unpublished observations). Further experiments will be required to distinguish between these
mechanisms.
122
4.1.7 Modeling of the Budding Yeast Mitotic Spindle
Many motor and non-motor MAPs function in higher eukaryotes to regulate spindle and
chromosome dynamics at both microtubule plus- and minus-ends. The extreme complexity of
spindle regulation in higher eukaryotes has made dissecting kinetochore-specific functions of
microtubule binding proteins extremely difficult. Although complicated, budding yeast
kinetochores are much easier to study because the minus-ends of microtubules appear to be
embedded in the SPBs and nondynamic, suggesting that all changes in chromosome movement
are regulated from the kinetochore-attached plus-ends (Maddox et al., 2000). Therefore, in
collaboration with the Danuser lab at Scripps Research Institute, we have undertaken
experiments to study chromosome dynamics with high levels of precision. Tandem arrays of Tet
operator regions are integrated into a single chromosome near the centromere in cells expressing
the Tet repressor (TetR) and the SPB component Spc42p tagged to GFP.
We then acquire live-cell movies with images taken every second. The SPBs and
centromeres are tracked using sophisticated algorithms to measurements chromosome and SPB
position in three dimensions over time. From these data we acquire rates of microtubule
dynamics (Dorn, 2005; Thomann et al., 2002). Computational tracking allows us to acquire a
much larger number of accurate data points than would ever be possible to measure by hand.
This allows for statistically significant changes to be calculated from live-cell movies. Our
technique has proven successful in measuring GI1dynamics in which distinctions could be made
between different mutant phenotypes (Dom, 2005).
Measurement of microtubule dynamics using this technique suggest that the standard
descriptors of microtubule dynamics do not adequately describe true microtubule motion.
Therefore, we are evaluating more sophisticated methods for modeling microtubule dynamics to
123
find more a more comprehensive set of descriptors (Jaqaman, 2005). These show that
chromosome motion in attached kinetochores at a given time point is linked to the previous time
point, whereas motion of unattached kinetochores is not. We believe that these improved
descriptors will aid in the analysis of kinetochore mutants. We plan to combine these modeling
approaches with more traditional techniques - including localization studies and biochemistry
-
to fully explore the mechanisms of the budding yeast kinetochore.
4.2 Summary
In this thesis I have evaluated the mechanisms of chromosome movement and dynamics.
The goal of my research has been to increase the understanding of basic cellular processes as it
relates to chromosome segregation with the hope of providing insight into the treatment and
prevention of human disease. I have discovered that all four S. cerevisiae nuclear kinesins
function at kinetochores. Two, Cin8p and Kip lp, are involved in organizing centromeres into the
budding yeast equivalent of a metaphase plate. Another, Kip3p, is involved in regulating the
coordinated onset of anaphase at the metaphase-to-anaphase transition, and in regulating
microtubule dynamics during the cell cycle. I have found that Kar3p does not localize to mature
kinetochores, but that it is found at detached kinetochores and in a small subset of immature
spindles. This suggests that Kar3p is recruited only to improperly attached kinetochores. I have
also discovered that the commonly used drug nocodazole, does not detach the majority of
kinetochores as previously believed. However, it does increase chromosome detachment when
administered at a discrete period early in the cell cycle. Moreover, regardless of when
nocodazole is added, or whether chromosomes appear to be detached, the spindle checkpoint is
activated. This indicates that the spindle checkpoint may be sensitive to lesions other than
124
detachment. These findings represent a significant advance in the field of kinetochore biology
and will hopefillly serve as a building block for further research.
125
References
Akhmanova, A., and C.C. Hoogenraad. 2005. Microtubule plus-end-tracking proteins:
mechanisms and functions. Curr Opin Cell Biol. 17:47-54.
Andrews, P.D., Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Wordeman, and J.R.
Swedlow. 2004. Aurora B regulates MCAK at the mitotic centromere. Dev Cell. 6:25368.
Brinkley, B.R., and J. Cartwright, Jr. 1971. Ultrastructural analysis of mitotic spindle elongation
in mammalian cells in vitro. Direct microtubule counts. J Cell Biol. 50:416-31.
Cheeseman, I.M., S. Anderson, M. Jwa, E.M. Green, J. Kang, J.R. Yates, 3rd, C.S. Chan, D.G.
Drubin, and G. Barnes. 2002. Phospho-regulation of kinetochore-microtubule
attachments by the Aurora kinase Ipllp. Cell. 111:163-72.
De Wulf, P., A.D. McAinsh, and P.K. Sorger. 2003. Hierarchical assembly of the budding yeast
kinetochore from multiple subcomplexes. Genes Dev. 17:2902-21.
Dom, J.F., K. Jaqaman, D. R. Rines, G. S. Jelson, P. K. Sorger, and G. Danuser. 2005.
Interphase kinetochore microtubule dynamics in yeast analyzed by high-resolution
microscopy. Biophys. J. accepted.
Endow, S.A., S.J. Kang, L.L. Satterwhite, M.D. Rose, V.P. Skeen, and E.D. Salmon. 1994. Yeast
Kar3 is a minus-end microtubule motor protein that destabilizes microtubules
preferentially at the minus ends. Embo J. 13:2708-13.
Francisco, L., W. Wang, and C.S. Chan. 1994. Type 1 protein phosphatase acts in opposition to
IpL 1 protein kinase in regulating yeast chromosome segregation. Mol Cell Biol. 14:473140.
Gillett, E.S., C.W. Espelin, and P.K. Sorger. 2004. Spindle checkpoint proteins and
chromosome-microtubule attachment in budding yeast. J Cell Biol. 164:535-46.
Gillett, E.S., and P.K. Sorger. 2001. Tracing the pathway of spindle assembly checkpoint
signaling. Dev Cell. 1:162-4.
Gorbsky, G.J. 2004. Mitosis: MCAK under the aura of Aurora B. Curr Biol. 14:R346-8.
Gordon, D.M., and D.M. Roof 1999. The kinesin-related protein Kip p of Saccharomyces
cerevisiae is bipolar. JBiol Chem. 274:28779-86.
Gordon, I).M., and D.M. Roof. 2001. Degradation of the kinesin Kiplp at anaphase onset is
mediated by the anaphase-promoting complex and Cdc20p. Proc Natl Acad Sci U S A.
98:12515-20.
He, X., S. Asthana, and P.K. Sorger. 2000. Transient sister chromatid separation and elastic
deformation of chromosomes during mitosis in budding yeast. Cell. 101:763-75.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochoremicrotubule attachment in budding yeast. Cell. 106:195-206.
Higuchi, T., and F. Uhlmann. 2005. Stabilization of microtubule dynamics at anaphase onset
promotes chromosome segregation. Nature. 433:171-6.
Hildebrandt, E.R., and M.A. Hoyt. 2001. Cell cycle-dependent degradation of the
Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdhl) and a bipartite
destruction sequence. Mol Biol Cell. 12:3402-16.
Hsu, J.Y., Z.W. Sun, X. Li, M. Reuben, K. Tatchell, D.K. Bishop, J.M. Grushcow, C.J. Brame,
J.A. Caldwell, D.F. Hunt, R. Lin, M.M. Smith, and C.D. Allis. 2000. Mitotic
phosphorylation of histone H3 is governed by Ipl /aurora kinase and Glc7/PP1
phosphatase in budding yeast and nematodes. Cell. 102:279-91.
126
Jaqaman, K., Jelson, G.S, Dorn,J.F ,Sorger, P.K, and Danuser, G. 2005. Comparative Time
Series Analysis of Stochastic Molecular Processes: Application to KIlnetochore
Microtubule Dynamics in Yeast. PNAS. submitted.
Kapitein, L.C., E.J. Peterman, B.H. Kwok, J.H. Kim, T.M. Kapoor, and C.F. Schmidt. 2005. The
bipolar mitotic kinesin EgS moves on both microtubules that it crosslinks. Nature.
435:114-8.
Lan, W., X. Zhang, S.L. Kline-Smith, S.E. Rosasco, G.A. Barrett-Wilt, J. Shabanowitz, D.F.
Hunt, C.E. Walczak, and P.T. Stukenberg. 2004. Aurora B phosphorylates centromeric
MCAK and regulates its localization and microtubule depolymerization activity. Curr
Biol. 14:273-86.
Maddox, P.S., K.S. Bloom, and E.D. Salmon. 2000. The polarity and dynamics of microtubule
assembly in the budding yeast Saccharomyces cerevisiae. Nat Cell Biol. 2:36-41.
Mao, Y., A. Abrieu, and D.W. Cleveland. 2003. Activating and silencing the mitotic checkpoint
through CENP-E-dependent activation/inactivation of BubR . Cell. 114:87-98.
McIntosh, J.R.., E.L. Grishchuk, and R.R. West. 2002. Chromosome-microtubule interactions
during mitosis. Annu Rev Cell Dev Biol. 18:193-219.
Rieder, C.L. 1982. The formation, structure, and composition of the mammalian kinetochore and
kinetochore fiber. Int Rev Cytol. 79:1-58.
Shang, C., T.R. Hazbun, I.M. Cheeseman, J. Aranda, S. Fields, D.G. Drubin, and G. Barnes.
2003. Kinetochore protein interactions and their regulation by the Aurora kinase Ipllp.
Mol Biol Cell. 14:3342-55.
Sullivan, M., C. Lehane, and F. Uhlmann. 2001. Orchestrating anaphase and mitotic exit:
separase cleavage and localization of Slkl 9. Nat Cell Biol. 3:771-7.
Tanaka, K., N. Mukae, H. Dewar, M. van Breugel, E.K. James, A.R. Prescott, C. Antony, and
T.U. Tanaka. 2005. Molecular mechanisms of kinetochore capture by spindle
microtubules. Nature. 434:987-94.
Thomann, D., D.R. Rines, P.K. Sorger, and G. Danuser. 2002. Automatic fluorescent tag
detection in 3D with super-resolution: application to the analysis of chromosome
movement. J Microsc. 208:49-64.
Ubersax, J.A., E.L. Woodbury, P.N. Quang, M. Paraz, J.D. Blethrow, K. Shah, K.M. Shokat, and
D.O. Morgan. 2003. Targets of the cyclin-dependent kinase Cdkl. Nature. 425:859-64.
Vafa, O., and K.F. Sullivan. 1997. Chromatin containing CENP-A and alpha-satellite DNA is a
major component of the inner kinetochore plate. Curr Biol. 7:897-900.
Winey, M., C.L. Mamay, E.T. O'Toole, D.N. Mastronarde, T.H. Giddings, Jr., K.L. McDonald,
and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the
Saccharomyces cerevisiae mitotic spindle. J Cell Biol. 129:1601-15.
Wordeman, L., and T.J. Mitchison. 1995. Identification and partial characterization of mitotic
centromere- associated kinesin, a kinesin-related protein that associates with centromeres
during mitosis. J Cell Biol. 128:95-104.
Yao, X., A. Abrieu, Y. Zheng, K.F. Sullivan, and D.W. Cleveland. 2000. CENP-E forms a link
between attachment of spindle microtubules to kinetochores and the mitotic checkpoint.
Nat Cell Biol. 2:484-91.
Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. The centromere-kinetochore complex: a
repeat subunit model. J Cell Biol. 113:1091-110.
127
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