Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA
* Author for correspondence (e-mail: robert.west{at}colorado.edu )
Accepted 25 November 2001
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Summary |
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Key words: Fission yeast, Kinesin, Kinetochore, Mitosis, Schizosaccharomyces pombe, Checkpoints
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Introduction |
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Two kinesin-like protein families, CENP-E
(Yen et al., 1992) and KinI
(MCAK and XKCM1) (Wordeman and Mitchison,
1995
; Walczak et al.,
1996
) have been localized to mitotic kinetochores in metazoans
(reviewed by Goldstein and Philip,
1999
). Functional studies implicate CENP-E in prometaphase
congression to the spindle equator and the maintenance of metaphase
(Wood et al., 1997
;
Schaar et al., 1997
;
Yao et al., 2000
;
Yucel et al., 2000
;
McEwen et al., 2001
), and
MCAK/XKCM1 in anaphase A chromosome movement towards the spindle poles
(Walczak et al., 1996
;
Maney et al., 1998
).
The fission yeast, Schizosaccharomyces pombe, is an excellent
model system whose suitabilities for genetic and cytological studies make it
particularly valuable for the study of kinetochore function. For example,
S. pombe contains just three chromosomes that segregate on a highly
ordered mitotic spindle, so the cytological analysis of chromosome movement
and kinetochore function is comparatively easy. Through the use of kinetochore
markers (Goshima et al., 1999)
sister chromatid separation can be monitored for each replicated chromosome
simultaneously, thus allowing distinctions between pre-anaphase motions and
the segregation of sister chromatids at anaphase. In addition, the S.
pombe kinetochores resemble those of metazoans in several ways: they are
built upon tens of kilobases of DNA, which is organized as a central core
flanked by large repeating units; they attach 2-4 microtubules; and are
visible as a region with special staining characteristics
(Ding et al., 1993
).
Here we describe the mitotic role(s) of two kinesin-like motors in fission
yeast, klp5+ and klp6+. Previous work
has shown that these two proteins have similar primary structures but that
neither of them is essential, either alone or together. Null alleles do,
however, have unusually stable microtubules; they are highly resistant to the
microtubule drug, thiabendazole, and they contain unusually long microtubules
in interphase (West et al.,
2001). Both Klp5p and Klp6p localize to cytoplasmic microtubules
in interphase, and their deletion leads to morphological defects in especially
long cells. These results led to the hypothesis that Klp5p and Klp6p foster
microtubule disassembly in vivo. In this paper, we present data indicating
that both of these kinesins are also necessary for proper sister chromatid
separation in mitosis. We propose that the defects in chromosome movement are
the result of changes in spindle dynamics and/or the interaction between the
spindle and the kinetochores.
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Materials and Methods |
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Cell cycle mutants
Cell cycle arrest and release experiments with cdc25-22 were done
as follows. Cultures were grown in YES medium to early log phase
(OD595 0.1-0.2) at permissive temperature (25°C), shifted to
restrictive temperature (36°C) for 3 hours, then returned to permissive
temperature. Samples were collected at 10 minute intervals, fixed with
methanol as described below, and stained for DNA with DAPI (4',
6-diamino-2-phenylindole) (Sigma, St Louis, MO). Mitotic cells were identified
by having >1 mass of DNA, but no septum. Abnormal mitotic cells were
defined as those containing two or more asymmetric masses of DNA.
Microscopy
For microscopy, cells were grown to early-mid log phase
(1-3x105cells/ml). Observations on strains expressing GFP
tagged genes were done at 25°C unless otherwise noted. DNA staining was
performed on cells fixed by adding one-tenth of the volume of a cell culture
to methanol at -20°C and incubating at -20°C for 2-15 minutes. Cells
were collected by centrifugation, resuspended in phosphate-buffered saline (pH
7.4) containing 1 µg/ml DAPI and mounted on glass slides.
Live cells were analyzed by visualizing microtubules with
GFP--tubulin (Ding et al.,
1998
), Klp5p and Klp6p with their corresponding GFP fusions
(West et al., 2001
),
kinetochores with Mis 12p-GFP (Nabeshima
et al., 1998
), and DNA with Hoechst 33342 (Sigma). The
GFP-
-tubulin plasmid pDQ 105 (Ding
et al., 1998
) was transformed into cells, and the resultant
strains were grown in defined medium
(Moreno et al., 1991
)
containing 5 µg/ml thiamine (Sigma) to limit expression levels
(Maundrell, 1990
) of the
GFP-
-tubulin. Cells were collected by centrifugation (5-10 ml) at 2056
g for 2 minutes in a Beckman CS-6 centrifuge, and resuspended
in 5 ml YES, pH 7.5, containing 2 µg/ml Hoechst 33342 for
20 minutes.
Cells were then mounted on glass coverslips, and images collected with a Cooke
SensiCam slow-scan CCD camera on a Zeiss AxioplanII fluorescence microscope,
using the SlideBook software package (Intelligent Imaging Innovations, Denver,
CO) as previously described (West et al.,
2001
).
Movies
Wild-type and klp cells were followed through mitosis with
GFP-
-tubulin and Hoechst 33342 staining by deconvolution microscopy as
described above. Image stacks of eight focal planes each were collected for
every time point and processed into a 2D image as described above. The
collection of time points were compiled in Corel Photo-Paint and exported as a
QuickTime movie. The wild-type series begins towards the end of anaphase A.
The klp5
and klp6
series each start with a
single mass of DNA asymmetrically placed on a spindle. The DNA subsequently
undergoes three asymmetric divisions to produce even segregation of the DNA.
The klp5
spindle elongates and breaks (yellow arrow) at the
end of the time course. These movies are at available at
http://jcs.biologists.org/supplemental; selected images are represented in
Fig. 2C.
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Results |
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The klp cells likewise contain a single mass of DNA as they
enter mitosis, but fixed cells also frequently displayed more than one mass of
DNA distributed over an elongated spindle; the most common abnormality
observed was three unequal masses of DNA separated over 4-6 µm
(Fig. 1B;
Table 1). Other complex and
asymmetric arrangements of DNA were occasionally seen in mutant cells
(Fig. 1C), but on longer
spindles the DNA was usually present as two equal masses, as observed for
wildtype (Fig. 1D; Table 1). These data suggest
that klp
cells do not progress normally from prophase
chromosome condensation to a symmetric segregation of sister chromatids in
anaphase A, but that by late anaphase B mitosis is often like wildtype.
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The abnormal pattern of chromosome segregation was further examined by
characterizing live klp cells as they progressed from early
mitosis to the formation of daughter cells (see movies;
http://jcs.biologists.org/supplemental/). Wild-type fission yeast mitosis has
been described previously as occurring in three distinct stages
(Nabeshima et al., 1998
;
Ding et al., 1993
), and this
was confirmed by our methods (Fig.
2A). Phase 1 included spindle formation in the nucleus and the
equatorial alignment of DNA on a short spindle (
2.5 µm), indicating
the establishment of metaphase (Fig.
2A, 0 minutes). During Phase 2, the spindle elongates very slowly
(
0.1 µm/minute), but towards the end of this phase the chromosomes
segregate rapidly (
1 µm/minute) to the poles, marking the completion
of anaphase A on a spindle that is still <3.0 µm. This is apparent as an
even division of the chromatin mass (Fig.
2A, 3.1 minutes). Phase 3 is initiated by the abrupt onset of
spindle elongation from
2.5 µm to 12-15 µm
(Fig. 2A, 3.1-9.4 minutes).
Most klp cells showed Phase 1 spindles (<2.5 µm
spindle length) with chromosomes abnormally placed at or near one pole
(Fig. 2B, 0 minutes)
(Table 2, early). These cells
progressed to show several deviations from wild-type Phase 2 spindles. First,
the DNA sometimes began to separate, but then collapsed back into a single
mass before re-initiating separation (Fig.
2B) (20-30% of cells). Second, most Phase 2 spindles with more
than one mass of DNA were longer than a wild-type spindle during anaphase A,
but anaphase B did not begin until the spindle was unusually long
(Table 3). Third, DNA
segregation did not usually occur by a single, uniform separation into two
equal masses, but rather by a series of unequal divisions
(Fig. 1B,C;
Fig. 2C;
Table 2, mid). Despite this
complex pattern, proper separation was usually achieved before mitosis was
completed (Fig. 1D;
Fig. 2C, 25.5 minutes)
(Table 2, late). Phase 3
spindle elongation appeared largely normal in the klp
cells,
as no significant differences from wildtype were observed in the rate of
spindle elongation (Table 3).
However, Phase 3 frequently began before the completion of Phase 2, a
phenomenon not observed in wild-type cells
(Fig. 2C, 12.5-25.5
minutes).
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These observations showed that replicated chromosomes move back and forth
along the klp spindle, and suggested that sister chromatids
would subsequently separate at any position along the spindle. Thus, when
anaphase A started, the pairs of sister chromatids could segregate
independently of each other to reach their respective poles, at times passing
other chromatids that were moving in the opposite direction along the spindle
(Fig. 2C).
Chromosome misalignment in klp cells occurs prior to
sister chromatid separation
The abnormal chromosome movements observed in klp cells
were surprising, given that these mutants have virtually wild-type viability
(West et al., 2001
). A series
of experiments was undertaken to determine the mechanism by which
klp
cells with abnormal DNA arrangements achieved successful
chromosome segregation. Given that the most common defect in DNA segregation
was the appearance of three unequal masses of DNA (Tables
2,
3), and that fission yeast
contains three chromosomes, we inferred that each mass might be an individual
chromosome. To examine this possibility, the six kinetochores were followed
through mitosis using a tagged kinetochore protein, Mis 12p-GFP
(Fig. 3)
(Goshima et al., 1999
).
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Wild-type cells carrying this marker showed kinetochores clustered at the
spindle pole bodies (SPBs) early in mitosis, as others have previously
described (Funabiki et al.,
1993; Goshima et al.,
1999
). The kinetochores appeared briefly in early mitosis as six
dots distributed over 1.5-2 µm across the nucleus
(Fig. 3A,B), but then clustered
into two groups near the SPBs, at opposite ends of the dividing nucleus
(Fig. 3C,D).
By contrast, the kinetochores in klp cells spread out along
the spindle as it slowly elongated to
6 µm
(Fig. 3E). Each DNA mass was
associated with two Mis12p-GFP dots that moved together along the spindle,
consistent with each being a replicated but unsegregated chromosome, not an
individual chromatid. However, the distance between kinetochore pairs was
greater than that observed in wildtype (0.5-0.8 µm versus 0.2-0.3 µm),
suggesting that the klp
sister kinetochores are under tension
as a result of forces acting toward the spindle poles (compare
Fig. 3A,B to 3E). These results
demonstrated that each of the three DNA masses scattered along the
klp
spindles represents an individual chromosome. Therefore,
the abnormal chromosome movements in the klp
cells precede
anaphase A onset.
klp5 and klp6
mutants are delayed in
mitotic progression
The observations of individual live klp cells suggested
that klp
cells are slow to complete mitosis. To test this
possibility on populations of cells, we followed synchronous cultures of
klp
and control strains through mitosis. Cells were
synchronized at G2/M by constructing klp
, cdc25-22
double mutants and placing these cells at restrictive temperature for this
cell cycle regulating phosphatase. After cells had accumulated at the
G2/M boundary, they were released into mitosis by returning the
cultures to permissive temperature. Previous work showed no temperature
sensitivity in the klp
mutants alone
(West et al., 2001
). Cells
with two or more masses of DNA in the absence of a septum were scored as
`mitotic', and these were further distinguished as `normal' (two equal masses
of DNA) or `abnormal' (two or more unequal DNA masses). The frequency of
abnormal DNA distributions in the cdc25-22 cells after block/release
was less than 1%, indicating that the cdc25-22 mutation does not, in
itself, disorganize chromosome segregation (data not shown). The
klp
, cdc25-22 mitotic cells showed a high frequency of an
abnormal DNA distribution as the culture entered mitosis, but abnormalities
decreased rapidly as the culture completed mitosis
(Fig. 4A). These data indicate
that abnormal chromosome movements occur early in mitosis but are corrected
later, a conclusion that is consistent with the observations made on
individual cells (Figs 1,
3;
Table 3).
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These experiments also revealed a delay in the progression through mitosis
in klp strains. Progress of a synchronized cell culture
through M phase is revealed by the fraction of mitotic cells as a function of
time after release from cell cycle arest
(Fig. 4B). The klp
,
cdc25-22 mutants showed a significant increase in the total time spent in
mitosis, compared with cdc25-22 alone. This was caused by a delay in
the completion of mitosis, but not in its initiation, as indicated by the
positions of the ascending and descending lines of the two mitotic
distributions (Fig. 4B). The
klp
, cdc25-22 cultures were mitotic 25-50% longer than
cdc25-22 alone.
klp5 and klp6
are synthetically
lethal with bub1
and mutants of the anaphase-promoting
complex
The delay in mitotic progression, together with abnormal spindle elongation
prior to anaphase onset, suggested that klp cells might be
activating their spindle assembly checkpoint, thus allowing time to rectify
their mitotic abnormalities. This checkpoint is a regulatory pathway that
monitors the attachment of kinetochores to the spindle and/or the tension on
these kinetochores, delaying sister chromatid separation until all the
chromosomes are properly attached to the spindle (reviewed in
Amon, 1999
;
Burke, 2000
). Disruption of the
checkpoint should then lead to a loss of viability in a klp
background. To test this prediction, double and triple mutants of the
klp
alleles and components of the spindle assembly checkpoint
were constructed and assayed for viability. A significant loss of viability
was observed in the klp
, bub1
mutants
(Bernard et al., 1998
) but not
in klp
strains with backgrounds in mph1
[MPS1 homologue (He et al.,
1998
)], mad2
(He et al., 1997
), or
cdc16-116ts [BUB2 homologue
(Minet et al., 1979
)]. The
klp
bub1
cells produced small colonies at
25°C which then failed to growth when replica plated to 36°C (44
tetrads analyzed) (data not shown).
One of the regulatory targets of the spindle assembly checkpoint is the
cyclosome, or anaphase-promoting complex (APC), which catalyzes the loss of
sister chromatid cohesion at the onset of anaphase A (reviewed by
Page and Hieter, 1999;
Nasmyth et al., 2000
). Double
and triple mutants with the klp
strains and mutants of the APC
were constructed and assayed for viability. Double and triple mutants of
klp
and nuc2-663ts
(Hirano et al., 1988
;
Yamada et al., 1997
) were not
isolated by tetrad analysis (36 tetrads) or random spores (
35,000).
Double and triple mutants with klp
and
cut4-533ts (Yamashita
et al., 1996
) were not found from either tetrads (12 tetrads) or
random spores (5300) indicating that these mutant combinations were
synthetically lethal. Mutants with klp
and
cut9-665ts (Samejima
et al., 1993
) produced small colonies, but failed to grow upon
re-streaking to new plates (6 tetrads/3800 random spores) data not shown).
klp mutants are synthetically lethal with
dis1-1cs
The aberrant chromosome behavior described here in the klp
mutants, together with the effects of these mutants on cytoplasmic
microtubules and cell morphology described previously
(West et al., 2001
), suggested
that klp5+ and klp6+ affect both
microtubule and kinetochore functions. Similar functions have been described
for Dis1p, a member of the TOG1/XMAP215 family of microtubule-associated
proteins; Dis1p binds to both microtubules and centromeric DNA, and mutants
display abnormalities in both microtubule organization and chromosome
segregation (Nabeshima et al.,
1995
; Nakaseko et al.,
1996
; Nakaseko et al.,
2001
). We therefore looked for genetic interactions between the
klp
mutants and the cold-sensitive mutant
dis1-1cs (Ohkura et
al., 1988
), by constructing double and triple mutants using tetrad
analysis. The dis1-1cs strain failed to grow at 20°C,
as previously reported (Ohkura et al.,
1988
), whereas both klp
and
dis1-1cs parental strains grew virtually at wild-type
levels at 32°C. By contrast, the klp
,
dis1-1cs double and triple mutants failed to grow at
32°C, although the viability of the sibling spores was normal in these
crosses (20 recombinant tetrads examined for each klp
dis1-1cs cross). Spore formation in these crosses
resembled wildtype, suggesting normal karyogamy and meiosis, but the
klp
dis1-1cs spores often failed to
germinate. When colonies formed, they never progressed past two or three
divisions. These results indicate a strong interaction between
klp5+ and klp6+ and the
dis1+ gene.
Klp5p and Klp6p localize to early mitotic kinetochores and the late
spindle mid-zone
Previous work showed that Klp5p and Klp6p localize to microtubules in both
interphase and mitotic cells (West et al.,
2001). Here, we examined the mitotic localization with finer
temporal resolution as cells progressed through mitosis. As the cells entered
mitosis, each Klp-GFP localized as two or three spots within the nucleus
(Fig. 5A, t=0
minutes). These spots briefly separated into 4-6 resolvable spots
(mean=4.8±0.5, n=18) lying along a
2 µm line
(mean=1.8±0.4 µm, n=18)
(Fig. 5B, and
t=3.7-13.7 minutes). This pattern resembles that of wild-type
kinetochores (Fig. 3B)
(Saitoh et al., 1997
;
Goshima et al., 1999
;
Wigge and Kilmartin, 2001
). As
the DNA mass became oblong, indicating the completion of anaphase A
(Fig. 3C), the Klp-GFP signal
re-localized to the entire spindle (mean length of spindle
staining=3.1±0.4 µm, n=10), rather than to the
pole-associated dots characteristic of a kinetochore marker
(Fig. 5D; 16 minutes). The
Klp-GFP remained in this arrangement as the spindle elongated, thus becoming
restricted to a decreasing fraction of the spindle midzone
(Fig. 5D; 27.4 minutes). The
Klp-GFP re-appears on interphase cytoplasmic microtubules coincident with the
formation of the post-anaphase array, which arises from the point of septation
as the cells enter G1 (West et
al., 2001
).
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To better discern the localization of Klp-GFP proteins during pre-anaphase,
we attempted to take advantage of the clarity of kinetochore positions seen in
klp cells, as revealed by expressing Mis12-GFP
(Fig. 3). Double mutant strains
were constructed in which Klp5p-GFP was expressed in the klp6
background, and vice versa. In these cells, there was no concentration of GFP
signal on kinetochores when the chromosomes were distributed across a long
spindle (4-6 µm); these cells usually showed spindle staining (data not
shown). These results imply a co-dependence for kinetochore localization
between Klp5p and Klp6p at this stage, or that each Klp requires the other to
be restricted to the kinetochores before anaphase A. In any case, these
results suggest that proper localization of both Klp5p and Klp6p in
pre-anaphase A cells requires the expression of both of these kinesins.
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Discussion |
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The localization of Klp5p and Klp6p to the kinetochores from prophase to
the onset of anaphase A suggests that these motors play a role in chromosome
alignment prior to chromatid separation. The strong genetic interactions
between klp and one component of the spindle assembly
checkpoint, bub1+, as well as three components of the APC,
are consistent with a function for these kinesins before or at anaphase A
onset. However, once chromosome separation has occurred in klp
cells, the chromatids migrate to the poles normally (i.e. there is no apparent
`lagging chromosome' phenotype, as has been described for other mitotic
mutants) (Pidoux et al.,
2000
).
Our results suggest that anaphase A can be delayed when proper metaphase
chromatid alignment is not achieved, but that chromatid separation can
eventually be initiated from any point on the spindle. A strikingly similar
phenotype has been observed in other S. pombe mutants, including a
dominant allele of the cdc2+ kinase
(Labib et al., 1995) and a
disruption of dis1+ or its related protein
mtc1+/alp14+, proteins that bind to
both microtubules and kinetochores
(Nabeshima et al., 1998
;
Garcia et al., 2001
;
Nakaseko et al., 2001
). In
each of these mutants, three masses of DNA are observed on an unusually long
spindle prior to sister chromatid separation, indicating a block to sister
chromatid separation without inhibition of spindle elongation. The
dis1+ disruption mutants are, however, distinct from the
klp
cells with respect to spindle length in that they skip
mitotic Phase 2 (metaphase/anaphase A) and appear to proceed directly into
Phase 3 (anaphase B); klp
cells have a longer Phase 2 with a
distinct transition to anaphase B (Table
3). In this way, the klp
phenotype more closely
resembles mutants of two kinetochore components, mis6+ and
mis12+ (Goshima et
al., 1999
).
Spindle forces
The extensive, albeit unusual, chromosome movements observed in
klp mutants demonstrate that the spindle in S. pombe
contains mechanisms other than Klp5/6p that are capable of moving chromosomes.
Several candidate motors have been identified, including two KAR3
homologues (Pidoux et al.,
1996
; Troxell et al.,
2001
), dynein (Yamamoto et
al., 1999
), and a potential chromokinesin (pombe genome project).
A role in chromosome segregation for one of the KAR3 homologues,
pkl1+, is implied from the chromosome missegregation
phenotype of a cold-sensitive allele of
-tubulin, which is
synthetically lethal with a pkl1
mutation
(Paluh et al., 2000
). However,
the quadruple pkl1
, klp2
,
klp5
, klp6
mutant is viable, indicating that
fission yeast possesses some other mechanism to segregate its chromosomes
(West et al., 2001
).
Alternatively, microtubule dynamics, in the absence of motor activity per se,
have been shown to move chromosomes in vitro
(Lombillo et al., 1995
).
Sorting out the roles of each of these different sources of motive force will
required a systematic analysis of all the genes involved.
klp5+, klp6+ and the spindle assembly
checkpoint
Our observations of both fixed and live cells may be summarized by the
statement that the deletion of either klp5+ or
klp6+ disrupts the balance of forces exerted on the
kinetochores, which in turn leads to the misalignment of chromosomes at
metaphase and a delay in the onset of anaphase. This interpretation motivated
our search for interactions between the klp mutations and
spindle assembly checkpoint genes, but only bub1
showed an
effect. This implies that either bub1+ functions in a
spindle checkpoint independently of the other checkpoint genes, or that
bub1+ has functions outside the checkpoint. At present, we
cannot distinguish between these possibilities and, indeed, they are not
mutually exclusive. Recent work in HeLa cells supports a model in which a
BUB1-dependent checkpoint detects tension on the kinetochores, while a
MAD2-dependent checkpoint detects kinetochore attachment to the spindle
(Skoufias, et al., 2001
). In
vitro assays have also demonstrated that BubR1 can interact with Cdc20
independently of Mad2 to inhibit the APC complex
(Tang et al., 2001
). By
analogy, the synthetic effects of klp
with
bub1
, but not mad2
or mph1
,
can be interpreted as a requirement only for Bub1p to keep klp
cells from a self destructive anaphase onset. Since the kinetochores in
klp
cells appear to be under greater tension than in wild-type
cells, this interpretation leads to a disagreement with Skoufias et al.
(Skoufias et al., 2001
),
concerning the tension versus microtubule attachment aspects of the spindle
assembly checkpoint. Alternatively, it may be that the delay in anaphase onset
is not required to maintain viability in klp
cells, but that
the synthetic lethality of klp
with bub1
arises from a bub1+ function distinct from checkpoints,
such as a role in the integrity of the kinetochore.
The specificity for bub1+ among the checkpoint
components for the viability of klp strains was surprising and
suggests that klp5+ and klp6+ interact
more directly with bub1+ than with other checkpoint genes.
Previous work indicated that BUB1 and MPS1 genes function at
the top of a regulatory hierarchy, with MAD2 downstream of both of
these genes (reviewed by Amon,
1999
; Abrieu et al.,
2001
). However, in fission yeast the interactions among the
klp
and checkpoint mutants indicate that
bub1+ has functions independent of
mad2+. The fission yeast mad2+ and
mph1+ genes do share some features of checkpoint
regulation with the budding yeast homologues, including a co-dependence for
metaphase arrest (He et al.,
1997
; He et al.,
1998
). However, differences between these yeast genes in the
regulation of SPB duplication have been previously described
(He et al., 1998
).
A distinction between BUB1/BUBR1 and MAD2 has also been described recently
(Skoufias et al., 2001;
Tang et al., 2001
). In
mammals, MAD2 is thought to respond to the attachment of kinetochores to the
spindle microtubules, while BUB1/BUBR1 respond to tension on kinetochores
(Skoufias et al., 2001
;
Li and Nicklas, 1995
;
Waters, et al., 1998
). If an
analogous system functions in S. pombe, the interaction between the
klp
mutants and bub1
would imply that the
chromosomes are attached but not under tension in klp
cells.
The lack of obviously unattached chromosomes, together with active movement of
the chromosomes in klp
cells are consistent with the
chromosomes remaining attached to the spindle. However, increased distance
between sister kinetochores implies that the chromosomes are under increased
tension (Fig. 3).
The synthetic interactions between the klp mutants and the
APC components are also consistent with a kinetochore function for these
kinesins, but the relation between these results and the checkpoint genes
remains to be determined. Mad2p is thought to be the principle regulator of
the APC through its interactions with CDC20 (reviewed by
Amon, 1999
), but BubR1 also
interacts with CDC20 independently of Mad2p
(Tang et al., 2001
). Although
our results show no interactions between the klp
and
mad2
mutants, both of these mutants show synthetic lethal
interactions with the same APC components (cut4+,
cut9+ and nuc2+)
(He et al., 1997
). By
contrast, in mammalian cells, BUBR1 co-immunoprecipitates with Cdc16p
(cut9+ homologue) and Cdc27p (nuc2+
homologue) (Chan et al., 1999
).
The fission yeast bub1+ gene has checkpoint functions
(Bernard et al., 1998
) and
synthetic interactions with the klp
mutants, but it remains to
be determined whether it also interacts with APC components. Clearly,
additional work will be required to sort out the complex roles of both motor
proteins and checkpoint regulators at the kinetochore.
Kinetochore kinesins
Prometaphase congression in metazoans may be dependent on the kinesin-like
protein CENP-E (Wood et al.,
1997; Schaar et al.,
1997
; Yucel et al.,
2000
). Although the sequences of Klp5p and Klp6p are not closely
related to those of known CENP-E proteins, there are striking similarities in
mutant phenotypes. Disruption of CENP-E function, either by depletion with
antibodies (Yen et al., 1991
;
Wood et al., 1997
;
Schaar et al., 1997
) or
mutation (Yucel et al., 2000
)
causes a failure in metaphase chromosome alignment and a delay in the onset of
anaphase. Microinjection of CENP-E antibodies into CF-PAC cells did not show
the same degree of chromosome misalignment, but a decrease in the number of
kinetochore microtubules and a loss of tension at the kinetochores
(McEwen et al., 2001
).
Further, the genetic interactions between klp5,
klp6
and bub1
in fission yeast are reminiscent
of the physical association reported between CENP-E and the
kinetochore-associated spindle assembly checkpoint kinases BUB1 and BUBR1 in
metazoans (Taylor and McKeon,
1997
; Chan et al.,
1998
; Yao et al.,
2000
). A functional role for this interaction is indicated by the
BUBR1-dependent mitotic arrest in systems deficient in CENP-E
(Chan et al., 1999
;
Yao et al., 2000
), and the
requirement for CENP-E in the activation of the checkpoint upon spindle damage
(Abrieu et al., 2000
). The
synthetic lethal interactions reported here between klp
and
bub1
strains indicate a similar functional relation in fission
yeast.
In vertebrates, BUBR1 is required for the localization of CENP-E to the
kinetochores (Chan et al.,
1999; Sharp-Baker and Chen,
2001
). Bub1p in fission yeast also localizes to kinetochores, but
it is not known whether this is dependent on other proteins
(Bernard et al., 1998
).
CENP-E in vertebrates localizes to kinetochores from prophase to the end of
anaphase A, when it re-localizes to the spindle midzone
(Brown et al., 1996). Klp5p and
Klp6p also localize to kinetochores prior to anaphase A, and then they
relocalize to the spindle midzone during anaphase B.
Previous work has shown that Klp5p and Klp6p may foster microtubule
disassembly (West et al.,
2001), reminiscent of the KinI (MCAK, XKCM1) subfamily of
kinesins, which also localize to kinetochores
(Walczak et al., 1996
;
Maney et al., 1998
).
Furthermore, sequence similarity has been described among the KIP3 subfamily
members and the metazoan KinI subfamily
(Severin et al., 2001
). The
activity of the KinI motors may be balanced by the activity of
microtubule-associated proteins of the TOG/XMAP215 family (reviewed by
Andersen, 2000
). For example,
in budding yeast, deletion of the KIP3 gene partially rescues
anaphase B defects in a mutant of the TOG/XMAP215 homologue,
stu2-10ts (Severin et
al., 2001
). Here, we show that the klp
mutants are
synthetically lethal with a cold sensitive allele of the fission yeast
TOG/XMAP215 homologue, dis1+. Given that the
klp
, dis1-1cs mutants failed to grow
beyond one or two divisions, we cannot assess the nature of the defects in
these mutants. However, we have not detected any abnormalities in anaphase B
in our klp
strains (Table
3). It remains to be determined whether klp5+
and klp6+ also interact with the second TOG/XMAP215
homologue, mtc1+/alp14+
(Garcia et al., 2001
;
Nakaseko et al., 2001
).
With the completion of the genome sequencing projects for both yeasts, it
is evident that they both lack obvious sequence homologues to CENP-E, and the
kinesins they do express have only limited similarities to members of the KinI
family (Severi, et al., 2001). We suggest that the KIP3 subfamily, represented
by klp5+ and klp6+ in S.
pombe and KIP3 in S. cerevisiae
(West et al., 2001), is the
functional analogue of one or both of these metazoan kinesin subfamilies.
Severin et al. have previously suggested that Kip3p is the orthologue of the
KinI subfamily (Severin et al.,
2001
). We emphasize functional similarities among
klp5+, klp6+ and both CENP-E and KinI
based on similarities in loss-of-function phenotypes. We conclude that mitotic
functions can be subsumed by a variety of molecular entities and that a broad
view of mitosis will require an emphasis upon mitotic functions, rather than
on the identity of specific molecular players.
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Acknowledgments |
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Footnotes |
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References |
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