Download Pathways of spindle assembly

37
Pathways of spindle assembly
Jennifer
C Waters*
and ED Salmon?
Recent studies have revealed that, in some systems,
chromatin
has the ability to stabilize microtubules
organize them into bipolar spindles independently
kinetochores
and centrosomes.
and
for spindle
assembly; these include proteins that regulate microtubule
dynamics,
proteins that organize microtubule
spindle poles, and members
reside on the chromosome
on the
most
recent
of
In addition, several molecules
have been identified recently that are necessary
in animal cells, with an emphasis
literature
and unresolved
issues.
minus ends into
of the kinesin superfamily that
arms.
Addresses
*tDepartment of Biology, University of North Carolina, Chapel Hill,
NC 27599-3280, USA
*e-mail: [email protected]
te-mail: [email protected]
Current Opinion in Cell Biology 1997, 9:37-43
Electronic identifier: 0955-0674-009-00037
0 Current Biology Ltd ISSN 0955-0674
Abbreviations
NuMA
nuclear mitotic apparatus
XKCMl
Xenopus kinesin central motor 1
XKLPS
Xenopus kinesin-like protein 2
Introduction
cell division,
replicated
chromosomes
(during
mitosis or meiosis II) or homologous
chromosomes
(during
meiosis I) are segregated
on a bipolar spindle. At the onset
of each M phase, the interphase
cytoplasmic
microtubule
complex dissolves and a bipolar spindle is assembled.
The
mechanism
of spindle assembly
not only differs between
meiotic
and mitotic systems
[l]; somatic, embryonic
and
gametic cells all seem to have different ways of assembling
a bipolar spindle.
In fact, meiotic
spindle
assembly
can
even differ between
male and female gametes of the same
species [Z]. There are, however,
essential
features
of the
spindle
that are the same for all eukaryotic
cell types.
There
must be two spindle
poles from which dynamic
microtubules
of uniform polarity (with minus ends at the
poles and plus ends at the spindle equator) emanate.
The
chromosomes
must capture and stabilize microtubule
plus
ends via their kinetochores,
and, in most systems,
must
move to the metaphase
plate. This ubiquitous
design
allows the chromosomes
to be segregated
by moving
poleward
towards the minus ends of microtubules
during
anaphase.
Recent
reviews
on spindle
assembly
have focused
on
microtubule
motor proteins
[3-51, chromosomes
[Z&8],
microtubule
dynamics
[3,5], centrosome
separation
[4],
and yeast spindle
assembly
[9]. This review is a general
overview
of the various pathways
of spindle
assembly
The role of centrosomes
Somatic animal cells contain centrosomes
which nucleate
a polarized
array of microtubules,
with the minus ends
associated
with y-tubulin
complexes
[lO,l l] within
the
centrosomes,
and the faster-growing
plus ends distal to the
centrosomes.
Cells that contain
centrosomes
depend
on
them for microtubule
nucleation.
For example, the spindle
slowly dissolves
after the centrosomes
are removed
from
meiotic
grasshopper
spermatocytes
[ 12.1, and spindles
will not form at all if the centrosomes
are removed
from the cytoplasm
prior to nuclear-envelope
breakdown
[13’]. Prior to M phase, the centrosomes
replicate
and
subsequently
separate
to form the poles of the bipolar
spindle
(Fig. la) [l]. If the centrosomes
do not replicate
or separate properly, a monopolar
spindle forms (Fig. la).
Centrosomes
therefore
both nucleate
a polarized
array of
microtubules
and dictate spindle bipolarity.
Centrosomes
are thought
to separate
via pulling
forces
generated
by the astral microtubules
[14] and/or pushing
forces generated
between
the centrosomes
[15]. In the past
few years, several motor proteins
have been implicated
in the generation
of forces for centrosome
separation
[4].
These
include
the centrosomal
motor protein
XKLPZ
(Xenopus kinesin-like
protein
2) [16”] and members
of
the BimC subfamily
of kinesin-like
motor proteins
[17],
such as Xenopus Eg5 [18], all of which are necessary
for
centrosome
separation
and thought
to generate
pushing
forces [4]. Pulling forces have been hypothesized
to occur
when astral microtubules
interact with minus-end-directed
motors,
such as cytoplasmic
dynein
[19], that are anchored
in the cytoplasm
or plasma membrane.
Recent
experiments
suggest
that pulling
forces could also be
generated
by interactions
of astral microtubules
with other
cytoskeleton
components.
The product
of the Drosophila
gene &nstnr
is an actin-severing
protein that is necessary
for centrosome
separation
in meiotic spermatocytes
[ZO*].
Time-lapsed
microscopy
of fluorescently
labeled
nuclear
lamins has been used to demonstrate
that centrosome
movements
and nuclear lamin rearrangements
are tightly
coupled,
suggesting
an interaction
between
the two [al].
Recent
data also suggest
that phosphorylation
may be
necessary
for the regulation
of centrosome
separation.
Mutations
that cause the loss of function
of the protein
kinase aurora prevent centrosome
separation
in Drosophila
[Z?], while phosphorylation
of Eg5 has been shown to be
necessary
for its localization
to spindle poles [23’].
Although
assembly
centrosomes
during the
are clearly necessary
early stages of mitosis,
for spindle
they do not
38
Figure
Cytoskeleton
1
.
Kinetochore
0
X
Antiparallel microtubule cross-linker
0
Plus-end chromokinesin
Minus-end motor
Centrosome
0 1997 Current Opinion in Cell Biology
A model for the pathways of spindle assembly in cells with centrosomes (a) and cells without centrosomes (b). Chromosomes are represented
by gray shapes. (a) In cells with centrosomes, such as somatic animal cells, spindle bipolarity is dependent on centrosome replication and
separation. If centrosomes do not separate, a monopolar spindle forms (left). In this case, centrosomes nucleate a polarized radial array of
microtubules
(black lines), with microtubule minus ends at the pole (i.e. at the centrosome)
and plus ends distal. The forces for centrosome
separation are generated by minus-end motors that are anchored in the cytoplasm, and plus-end motors that cross-link antiparallel microtubules
(antiparallel microtubule cross-linkers)
between spindle poles (centre right diagram). Microtubules are dynamic, allowing them to search for
kinetochores on chromosomes. Kinetochores (black dots) capture and stabilize microtubules, resulting in a proper bipolar spindle (bottom
diagram). (b) In cells without centrosomes,
such as meiotic frog eggs, microtubules
(black lines) nucleated at randomly dispersed
in the cytoplasm bind to, and are stabilized by, the chromosomes. Kinetochores and kinetochore
Plus-end-directed chromokinesins and minus-end-directed motors work together to organize the
minus (-) ends are found at the spindle poles and plus (+) ends are located distal to the spindle
microtubule motors that cross-link antiparallel microtubules (antiparallel microtubule cross-linkers)
places
microtubules are not shown for clarity.
microtubules according to polarity. Microtubule
poles (i.e. at the spindle equator). Plus-end
are found in the spindle midzone.
Pathways
appear to be necessary
for spindle maintenance
during late
metaphase
and anaphase.
In newt lung epithelial
cells [24]
and Xenopus egg M-phase
extracts [25-l, the centrosomes
can wander
away from the spindle
in late metaphase
or anaphase,
while the microtubule
minus ends remain
focused
into spindle
poles.
Chromosome
segregation
occurs normally
under
these
conditions.
The
same is
true when centrosomes
are removed
from the spindle
by micromanipulation
during
anaphase
in grasshopper
spermatocytes
[26] and sand dollar eggs [27]. Spindle
microtubule
dynamics
decrease
in anaphase
in PtK cells
[28], so it may be that the spindle
does not require the
nucleating
capacity of the centrosomes
during anaphase.
Evidence
is also accumulating
that shows that the minus
ends of the microtubules
are organized
by components
that are separate
from the centrosomes
(see below). The
centrosomes
could, therefore,
leave a mature spindle and
the microtubule
minus ends would remain focused into a
pole.
The role of kinetochores
In the ‘search-and-capture’
model,
which
applies
to
most mitotic
animal cells, the kinetochores
capture
and
stabilize microtubules
that are nucleated
from the centrosomes, thereby
stabilizing
the bipolar spindle morphology
(Fig. la) [‘29]. In CHO cells, kinetochores
and centrosomes
can assemble
a bipolar
spindle
on their
own after
kinetochores
are experimentally
detached
from the bulk
of the chromosome
arms [30]. Echinoderm
egg [31] and
newt lung cell [32] kinetochores
appear to stabilize
the
spindle;
in the absence
of chromosomes,
centrosomes
nucleate
two separate
asters instead of a bipolar spindle.
One interpretation
of these data is that the kinetochores
are necessary
for bipolar spindle formation.
Those
kinetochore
proteins
whose
function
has been
determined
do not appear to be essential
for microtubule
stabilization
[33]. Overexpression
of the p50 subunit
of
the dynactin
complex in mammalian
tissue cells does not
inhibit
bipolar
spindle
formation,
although
the bipolar
spindles
that form are aberrant
in size and symmetry
[34’]. In addition,
microinjection
of antibodies
to the
kinesin-related
protein
CENP-E
(centromere
protein-E)
disrupts
chromosome
attachment
to the spindle,
but
does
not completely
inhibit
chromosome
attachment
and bipolar
spindle
formation
(BT Schaar, P Maddox,
ED Salmon, TJ Yen, unpublished
data). Functions
have
not been determined
for all of the known
kinetochore
proteins,
and further
analysis
may reveal that one of
these proteins
is necessary
for kinetochore
microtubule
stabilization
and spindle assembly.
In addition,
it is likely
that there are many more kinetochore
to be identified.
proteins
that remain
The role of chromosomes
Plants and many meiotic systems do not contain microtubuleorganizing
centers.
They
must, therefore,
organize
microtubules
and establish
spindle
bipolarity
in a different
of spindle
assembly
Waters
and Salmon
39
way to that of cells with centrosomes.
Over ten years
ago, Karsenti
and coworkers
[35,36] showed
that phage
DNA induces
spindle formation
when microinjected
into
meiotic
Xenopus eggs. This led to the hypothesis
that
chromosomes
induce the organization
of microtubules
into
a spindle. In the past few years, much of the research into
spindle
assembly
has once again focused
on the role of
chromosomes
in spindle
formation.
Recent
data support
the hypothesis
that chromosomes
can have at least three
roles in spindle
assembly:
stabilization
of microtubules;
organization
of microtubules
by polarity; and chromosome
congression
to the metaphase
plate. The contribution
of
chromosomes
to spindle
assembly
differs
between
cell
types, however.
Microtubules
in meiotic
grasshopper
spermatocytes
are
nucleated
at the centrosomes.
Spindles
in these
cells
maintain
their morphology
in the absence
of chromosomes,
as long as centrosomes
are present
[la*]. The
chromosomes
do affect
spindle
microtubule
assembly,
however. When early prophase
chromosomes
are exposed
to the centrosomes
by mechanical
disruption
of the nuclear
envelope
with a microneedle,
a bipolar
spindle
forms
prematurely
[13’]. This suggests that there is a dominant
component
of the chromosomes
that is required
to induce spindle assembly.
In addition,
micromanipulation
of
metaphase
chromosomes
has shown that the microtubule
mass increases
when a chromosome
is present
in the
spindle
[la*]. The effect on microtubule
dynamics
was
correlated
with an increase
in chromosome
mass, and
not with the number
of kinetochores
present.
Murray
and coworkers
[25*] found
that chromosome
position
relative to the spindle poles also influences
the extent of
microtubule
assembly
in Xenopus egg M-phase
extracts.
Dogterom
and colleagues
[37”] directly
measured
the
effect of chromatin
that does not contain
kinetochores
on individual
microtubule
dynamics
in Xenopus
egg
M-phase
extracts.
They
found evidence
for both longrange ‘guidance’
of microtubules
towards chromatin
and
short-range
stabilization
of microtubules
in the vicinity of
the chromatin.
Microtubules
near the chromatin
showed a
decrease in the number of catastrophes
(i.e. switches from
growing to shortening
states), an increase in the number of
rescues (i.e. switches
from shortening
to growing states),
and a slower
growth
velocity.
This
stabilizing
effect
resulted
in an increased
number
of microtubules
near the
chromatin.
Meiotic
Xenopus
eggs, and consequently
Xenopus
egg
M-phase
extracts,
do not contain
focused
nucleating
centers;
instead,
microtubules
are nucleated
from randomly dispersed
sites [38”]. Recently,
magnetic
beads
coated with plasmid
DNA were shown to induce
bipolar spindle
assembly
in Xenopus egg M-phase
extracts
[38”].
As plasmid
DNA
does not contain
any centromeric
sequences,
the chromatin
that forms from it
does not contain
kinetochores.
Time-lapsed
recordings
40
Cytoskeleton
of chromatin-induced
spindle
assembly
[38”]
showed
that microtubules
first aggregated
around the chromatin
and then
coalesced
into bundles.
In the final stages
of spindle
assembly,
the distal ends of the microtubule
bundles
were ‘pinched’
together
to form focused spindle
poles.
Short microtubules,
asymmetrically
labeled
with
rhodamine,
were shown to move toward the spindle poles.
This movement
was dependent
on the microtubule
motor
protein
cytoplasmic
dynein.
This demonstrated
that the
microtubule
minus ends were found at the poles in these
in vitro spindles.
This centrosomeand kinetochore-free
system is the clearest
example
that chromatin
alone can
organize microtubules
into a bipolar spindle.
Like Xenopus eggs, Drosophila oocytes
do not contain
nucleating
centers.
This
system
is different,
however,
in that kinetochores
seem
to be responsible
for the
generation
of a bipolar spindle
[a]. In the early stages of
spindle
assembly,
microtubules
gather around the chromatin. Single meiotic
chromosomes
which are expelled
from the karyosome
in various
mutants
can assemble
minispindles.
Bivalent
chromosomes
(which
have two
kinetochore
regions)
organize
bipolar spindles,
whereas
univalent
chromosomes
(which
have one kinetochore
region) organize monopolar
spindles.
What’s on the chromosomes?
The evidence
above suggests that chromosomes
associate
with molecules
that can stabilize microtubules,
in addition
to molecules
that can organize
microtubules.
Of course,
these functions
are not necessarily
mutually
exclusive.
The observation
that exogenous
DNA added to Xenopus
eggs or egg extracts can assemble
into chromatin
that is
able to induce spindle formation
[35,36,38”]
suggests that
these unidentified
molecules
are present in the cytoplasm.
A class of chromosomal
proteins
that may function
in
spindle
assembly,
as well as in chromosome
congression,
is the chromokinesins
[6]. The chromokinesins
constitute
a class of kinesin-like
motor proteins
that bind DNA
and localize
to the chromosome
arms. To date,
the
chromokinesin
family includes
Nod (in Drosophila) [39],
XKLPl
(in Xenopus) [40”], chromokinesin
(in chicken)
[41], and Kid (in human)
[42]. The chromokinesins
have
been proposed
to be responsible
both for ‘polar ejection
forces’ [43] and for the organization
of microtubules
into
spindles
(Fig. 1) [3,4]. Polar ejection
forces push the
chromosomes
away from the spindle poles and are thought
to contribute
to chromosome
congression
to the metaphase
plate [6,43]. Although both Kid and chromokinesin
localize
to chromosomes,
however,
their function
has not yet
been
determined.
Nod
appears
to be necessary
for
achiasmatic
chromosome
alignment
to the metaphase
plate during
meiosis,
suggesting
that it interacts
with
spindle microtubules
to produce polar ejection forces [39].
XKLPl
is necessary
for spindle assembly
in Xenopus egg
M-phase
extracts
[40”]. Asters assemble
after XKLPl
is immunodepleted
from extracts,
but they are smaller
and more symmetrical
than control
spindles.
It will be
interesting
to see if any of the chromokinesins
affect
microtubule
dynamics
in vitro. Although
there is no hard
evidence
that chromokinesins
organize microtubules
into
spindles,
it is easy to imagine
how a plus-end-directed
motor (such as chromokinesin)
that is anchored
to the
chromosomes
could work with minus-end-directed
motors
to organize microtubules
into spindle poles with uniform
polarity (Fig. lb).
Organization
of the microtubule
minus ends
Cells that do not contain microtubule-nucleating
centers
must organize
the microtubules,
which bind to and are
stabilized
by the chromosomes,
into two focused spindle
poles.
In Drosophila oocytes,
the minus-end-directed
microtubule
motor protein
Ned is necessary
for spindle
integrity
and the formation
of ‘focused’
spindle
poles
[44,45-l. When cytoplasmic
dynein,
another
minus-enddirected
microtubule
motor
protein,
is disrupted
in
Xenopus egg M-phase
extracts
the minus
ends of the
microtubules
are less focused
than in control
extracts
[38”].
These
data have led to a model
for spindle
assembly
in which
minus-end-directed
motor proteins
cross-link
and pull microtubule
minus ends together
to
form spindle poles (Fig. lb) [4].
The protein NuMA (nuclear mitotic apparatus)
has been
shown to be necessary
for organizing
microtubules
into
asters in HeLa
mitotic
extracts
[46’] and into bipolar
spindles
in Xenopus egg M-phase
extracts
[47”]. Neither
of these systems contains centrosomes.
In addition,
microinjection
of antibodies
against NuMA into mammalian
tissue culture cells (which do contain centrosomes)
results in
aberrant spindle morphology
[46’]. Recent immunodepletion experiments
suggest that NuMA functions
in HeLa
cell extracts
by associating
with a minus-end-directed
microtubule
motor that opposes
the plus-end-directed
motility
of the
motor
Eg5
[48”].
The
minus-enddirected
motor cytoplasmic
dynein also opposes Eg5, but
immunodepletion
of Eg5, NuMA and cytoplasmic
dynein
from the same extract suggests
that cytoplasmic
dynein
is not the minus-end-directed
protein that associates
with
NuMA
[48”].
However,
NuMA,
dynein
and dynactin
coimmunoprecipitate
in a complex
from Xenopus
egg
M-phase
extracts [47”]. The inconsistency
of these results
could represent
a difference
between
somatic and early
embryonic
systems.
Alternatively,
NuMA could associate
with one of the different
isoforms of dynein [49], whereas
a second isoform of dynein functions
without NuMA.
The role of microtubule
and poleward flux
dynamic instability
Nonkinetochore
spindle
microtubules
turn over quickly
in metaphase
(t1/2560
seconds)
relative
to interphase
microtubules
(t1/2 210 minutes)
[28,50]. This appears to be
the result of both an increase in the number
of transitions
from microtubule
growth to shortening
(i.e. an increase
in the number
of catastrophes)
and an increase
in the
Pathways
rate
of microtubule
growth in metaphase
relative
to in
interphase
[51,52]. In 1996, the first endogenous
regulators
of catastrophe
were discovered
[5,53”,54”,55].
XKCMl
(Xenopus kinesin
central
motor 1) is a Xenopus kinesinrelated
protein
that is homologous
to the human
kinetochore
protein
MCAK
(mitotic
centromere-associated
kinesin)
[53”]. Op18/stathmin
is a phosphoprotein
that
is present
at elevated
levels in some cancer cells [54”].
Immunodepletion
of either
XKCMl
or Op 18/stathmin
from Xenopus egg M-phase
extracts
results
in aberrant
spindle
assembly;
spindles
in these extracts
have centrally localized
chromatin
from which an array of long
microtubules
emanates
[53”,54”].
Analysis of the effect
of immunodepletion
of XKCMl
[53”] or Opl@tathmin
[54”] on individual
microtubule
dynamics
showed
that
these proteins
increase the number
of catastrophes,
without affecting
other properties
of microtubule
dynamics.
These
results show that microtubule
dynamics
must be
regulated
in order to ensure proper spindle formation.
A
key question
is whether
XKCMl
and Op18/stathmin
are
targets for the chromosomal
factors that modulate
spindle
microtubule
assembly.
of spindle
assembly
Waters
and Salmon
41
forms solely because
minus-end-directed
motors zipper
up the microtubule
minus
ends into poles,
the most
favorable
conformation
would be a monopolar
spindle.
The answer probably
lies in interactions
of microtubules
from opposing
spindle poles. If antiparallel
microtubules
become cross-linked
early on, then a bipolar conformation
would be favorable (Fig. lb).
An interesting
unresolved
issue is why cells need centrosomes
at all if some cells types can make perfectly
good spindles without them. Why can’t the chromosomes
in grasshopper
spermatocytes,
for example,
assemble
a
spindle in the absence
of centrosomes?
The answer may
be that cells that require
centrosomes
to assemble
a
bipolar spindle may simply need them because
they are
their only source of microtubule
nucleation.
Nucleation
is
probably
necessary
until late metaphase
to keep up with
microtubule
dynamic instability.
In addition,
centrosomes
may be important
in cells that rely on astral microtubules
for positioning
the division plane relative to the spindle
and to segregated
chromosomes
during cytokinesis.
Acknowledgements
In addition
to displaying
an increase
in growth rate and
catastrophe
frequency,
M-phase
microtubules
also exhibit
poleward
microtubule
flux; net kinetochore
microtubule
polymerization
at the plus ends is balanced
by constant
depolymerization
at the minus ends, resulting
in a slow
flux of the tubulin
subunits
within
the kinetochore
microtubule
lattice
poleward
[56-581. When
plus-end
dynamics
at the kinetochore
are inhibited,
microtubule
poleward
flux continues
[59’]. The motor for microtubule
poleward
flux is, therefore,
likely to be found at microtubule minus ends or associated
with the spindle matrix
[58,59-l. This motor has not been identified,
however.
Indeed,
no-one
has found a way to specifically
inhibit
microtubule
poleward
flux. When
the motor
for flux
is finally discovered,
or when a reliable
pharmaceutical
inhibitor
is identified,
it will be interesting
to see if flux
is essential
for spindle
assembly,
as catastrophe
appears
to be. Microtubule
poleward
flux has been shown to be
capable
of producing
tension
across the centromeres
of
newt lung epithelial
cells [59’]. It may be that microtubule
poleward
flux produces
tension at the kinetochore
that is
necessary
to stabilize
kinetochore
microtubules
[59’,60],
and, therefore,
bipolar spindle morphology.
Conclusions
There
are still plenty
of unanswered
questions
about
spindle assembly
that will undoubtedly
keep those of us
who are enamored
with the subject busy for many more
years. One particularly
perplexing
question is: how do cells
that do not contain
centrosomes
ensure
that there are
two, and only two, spindle
poles? Animal cells regulate
centrosome
replication,
allowing for only one replication
per cell cycle. Cells without centrosomes
assemble
bipolar
spindles
with remarkable
fidelity, however.
If a spindle
We thank Kerry Bloom, Mike Caplow, and R Scott Hawley for stimulating
discussions
and helpful suggestions.
We are also grateful to Don Cleveland,
Duane Compton,
and Andres Merdes for sending us manuscripts
prior to
publication.
References
and recommended
reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
.
l
*
of special interest
of outstanding interest
1.
Rieder CL, Ault JG, Eichenlaub-Ritter U, Sluder G:
Morphogenesis of the mitotic and meiotic spindle: conclusions
from one svstem are not necessarilv aDDliCable to the other.
In Chromosbme Segregation and Aneupioidy
Edited by Vig BK.
Berlin, Heidelberg: Springer-Verlag; 1993:183-l
97. [NATO series,
vol H72.1
2.
McKim KS, Hawley RS: Chromosomal control of meiotic cell
division. Science 1995, 270:1595-l
601.
3.
Hyman AA, Karsenti E: Morphogenetic properties of
microtubules and mitotic spindle assembly. Cell 1996,
84:401-410.
4.
Karsenti E, Haralabia B, Vernos I: The role of microtubule
dependent motors in centrosome movements and spindle
pole organization during mitosis. Semin Cell Dev Biol 1996,
7~367-378.
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