REVIEWS
Role of nonhistone
mitosis
WILLIAM
C. EARNSHAW’
Department
of Cell Biology
in the chromosomal
proteins
AND
ALASTAIR
and Anatomy,
This review
is concerned
with the role of
chromosomal
nonhistone
proteins
in three
important
aspects of mitotic
events: chromosome
condensation,
sister chromatid
separation
at the metaphase:anaphase
transition,
and interactions
between
the chromosomes
and
cytoskeleton
that occur during construction
of the mitotic
spindle
and cleavage
furrow.
Emphasis
will be given to
the potential
roles of topoisomerase
II and the chromosome passenger
proteins
in these events. Other important
aspects
of mitotic
events
such as the regulation
of the
G2-’ M transition,
the structural
changes
that affect the
nuclear
envelope
and other organelles
during mitosis,
and
the mechanism
of chromosome
movement
will not be considered
here. Despite
long histories
of often elegant
experimentation,
all three of our chosen
subjects
remain
areas of lively,
ongoing
controversy.
Thus,
although
recent advances
appear to have taken us many steps closer
to an understanding
of the underlying
mechanisms,
we
suspect that final answers will be some time in coming. Earnshaw,
W. C., Mackay,
A. M. Role of nonhistone
proteins on the chromosomal
events of mitosis.
FASEB J. 8:
947-956;
1994.
Key Wordr:
topoisomerase
II’
chromosome passenger proteins . chrornosome condensation
INCENPs
#{231}ytorkeletonmetaphose:anapho.se
transition
ONE-THIRD
OF THE MASS OF MITOTIC
chromosomes
is composed
of DNA, one-third
of histones
and one-third
of
a highly
heterogeneous
and poorly
characterized
group
known as nonhistone
proteins.
The idea that members
of this
latter group of proteins
might have a role in determining
the
architecture
and biochemical
properties
of mitotic
chromosomes was developed
most fully by Laemmli
and co-workers
(1), who established
a method
for the fractionation
of chromosomes
into a relatively
soluble fraction,
containing
about
90-95%
of the chromosomal
proteins,
and an insoluble
fraction composed
of a subset of the nonhistone
proteins.
This
insoluble
fraction
was termed
the chromosome
scaffold.
It
was suggested
that the scaffold
is composed
of structural
components
that give the mitotic chromosomes
their characteristic shape and organize
the interphase
chromosome
into
loop domains
of 50-100 kb.
The methodology
used to prepare
chromosome
scaffolds
resembles
that used to prepare
nuclear
matrix fractions,
and
the biological
significance
of both fractions
remains
controversial.
Efforts to address this controversy
have principally
entailed
identification
and characterization
of specific chromosome
scaffold proteins
and the identification
of DNA sequences
(termed
SARs2 or MARs,
scaffold or matrix attachment regions)
that appear to interact
preferentially
with the
scaffold and nuclear
matrix
fractions
(2). These interactions
appear
to involve the binding
of scaffold proteins
to regions
0892-6638/94/0008-0947/$ol
.50. © FASEB
of
M. MACKAY
Johns Hopkins
ABSTRACT
ROUGHLY
events
School of Medicine,
Baltimore,
Maryland
21205, USA
of narrowed
DNA minor groove. The functional
significance
of SARs/MARs
in vivo remains
unresolved.
The most abundant
polypeptide
component
of the chromosome
scaffold is DNA topoisomerase
II (topo II). Topo II
is a widely studied enzyme
that catalyzes
the passage of both
strands
of the DNA helix through
a transient
gap in the
DNA backbone.
This activity
allows the relaxation
of supercoils, knotting
and unknotting
of DNA, and separation
of
catenated
circular
DNA molecules.
Topo II preferentially
associates with SARs in vitro (3), which suggests
a mechanism
whereby
the enzyme
could
contribute
to chromosome
higher-order
structure.
SARs typically
are spaced
approximately 50 to 200 kb apart along the chromosomal
DNA (2).
Thus,
interactions
between
SAR-binding
proteins
such as
topo II or other proteins
of the chromosome
scaffold fraction
could produce
large ioop domains,
as have been observed
by
microscopy
of swollen chromosomes
(4-6).
Functional
evidence
is beginning
to accumulate
in support
of the notion that chromosome
scaffold components
such as
topo II might have a role in mitotic
chromosome
structure,
as originally
suggested
by Laemmli
et al. (1). We will discuss
a variety of evidence
suggesting
that topo II has a direct role
in mitotic
chromosome
condensation.
We will follow this
with a discussion
of the ongoing
controversy
over whether
topo II plays an additional
structural
role in chromosomes.
Next, we will discuss
the chromosomal
events required
to
render
the chromosomes
competent
to undergo
anaphase
sister chromatid
separation
(disjunction).
The assembly
of a
functional
kinetochore
is one aspect of chromosome
structure and function
that is monitored
by a cell cycle checkpoint
generally
known as the metaphase
checkpoint.
We will discuss the current
state of knowledge
concerning
the workings
of this important
cell cycle control
checkpoint.
Finally, we will close with a discussion
of the interplay
between the chromosomes
and the cytoskeleton
during mitosis.
For many years it was assumed
that chromosomes
are just so
much baggage
to be hauled
about by the cytoskeletal
proteins of the mitotic spindle.
This led to the famous expression
that “the chromosome
arms are like the corpse at the funeral:
they are the reason
for the proceedings
but take no active
part in them (7). Now this concept
has been thrown
into
question
by the discovery
of a class of molecules
with “dual
‘To whom correspondence
and requests for reprints should be addressed, at Department
of Cell Biology and Anatomy, Johns Hopkins School of Medicine,
725 N. Wolfe St., Baltimore, MD 21205,
USA.
2Abbreviations:
SARs, scaffold attachment
regions;
MARs,
matrix attachment
regions; topo II, topoisomerase
II; mad, mitotic
arrest deficient; bub, budding uninhibited
by benzimadazole;
cut,
cell untimely
torn; MKLP,
mitosis-specific
kinesinlike
protein;
MAPs, microtubule-associated
proteins; INC ENPs, inner centromere proteins.
947
.
V
citizenship”
that are chromosomal
for the bulk of the cell cycle, but become integral
cytoskeletal
components
during the
closing phases of mitosis. These proteins
are termed chromosome passengers
(8) (Table 1). It has been proposed
that the
sequestration
of these proteins
in the cell nucleus might serve
to keep them apart from the interphase
cytoskeleton.
This
could be important
if these proteins
do carry out mitosisspecific cytoskeletal
functions.
Also, the association
of the
passenger
proteins
with the chromosomes
might
serve in
part as a mechanism
for the exact positioning
of these proteins at the spindle
midplane
during
mitosis (8).
MECHANISM
CONDENSATION:
TOPOISOMERASE
OF
CHROMOSOME
THE ROLE
OF
II
DNA
Chromosome
condensation,
one of the most visually striking
of mitotic events, occurs in virtually
all eukaryotes
(although
TABLE
the condensation
is only observed
in budding
and fission
yeasts under
special
circumstances).
It is widely assumed
that condensation
of the chromosomes
into dense, discrete
bodies allows them to be moved more easily by the mitotic
spindle.
It is possible that the condensation
process serves as
a rectification
mechanism,
during
which neighboring
chromosomes
are untangled
from one another
during the transition from interphase
into mitosis;
this would allow them to
move as individual
units during
mitosis.
It has also recently
been proposed
that condensation
is needed
to prevent
the
chromosome
arms from trailing
too far behind
the centromeres as they move to the spindle poles during anaphase
(9).
Trailing
chromosome
arms could become
entrapped
in the
cytokinetic
furrow if sufficiently
extended.
The mechanism
of chromatin
condensation
is understood
only poorly. Although
it has long been known that mitotic
chromosome
condensation
is accompanied
by phosphorylation of histones
Hi and H3, the role of this modification
remains unknown.
Here we will focus on recent studies of the
1. Known chromosome passenger proteins
Molecular
weight
Possible
Antigen’
SDS/PAGE5
eDNA ORF’
MSA-36
(94)
36 kDa
-
JB antigen
(99)
TD-60
(96)
protein
Nuclear
in late 02
On prometaphase
chromosomes
At centromere
of metaphase
chromosomes
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular
bridge
38 kDa
Unknown
60 kDa
Cytokinesis?
Absent or masked in interphase
and prophase
On prometaphase
chromosomes
of some cells
At centromere
of metaphase
chromosomes
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular
bridge
Absent or masked in interphase
At centromeres
from prometaphase-early
anaphase
At spindle midzone, midanaphase-telophase
At cleavage furrow, midanaphase-telophase
133 kDa
145 kDa
37A5 antigens
140 kDa
155 kDa
96,000
101,000
-330
330d
(87)
Structural
protein?
Anaphase onset
Cytokinesis?
Unknown
300 kDa
CENP-E
(86, 92)
Description
Unknown
INCENP,
INCENP,,
(55, 85, 93)
(97)
functions
312,000
motor
Unknown
(doublet)
-400
kDa
948
Vol. 8
numbers
appear
Molecular
Unknown
September
in parentheses.
weight
1994
calculated
from
bApparent
deduced
until
metaphase
midbody
in intercellular
bridge
Probably identical to CENP-F
Absent or masked in 01, S, nuclear in G2
At kinetochores
from prometaphase-early
anaphase
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular
bridge
Probably identical to p330”
(88)
‘Reference
(SDS-PAGE).
bridge
At centromere
of metaphase
chromosomes
At spindle midzone, anaphase-telophase
On both sides of midbody in intercellular
bridge
Absent in Gl, S, accumulates
in cytoplasm in G2
At kinetochores
from prometaphase-early
anaphase
At spindle midzone, mid-anaphase-telophase
Flanking midbody in intercellular
bridge
Absent in Gl, accumulates
in nucleus in S, 02
At centromeres
from prometaphase-early
anaphase
At spindle midzone, anaphase-telophase
Flanking
CENP-F
midbody
in intercellular
in interphase
On prometaphase
chromosomes
At centromere
of metaphase
chromosomes
At spindle midzone, late metaphase-telophase
At cleavage furrow, anaphase-telophase
Flanking midbody in intercellular
bridge
Masked
Kinesin-like
Microtubule
kDa
Flanking
Nuclear
molecular
weight
derived
from mobility
open reading
frames
(ORFs)
of full-length
The FASEB Journal
on sodium
cDNAs.
dodecyl
sulfate-polyacrylamide
EARNSHAW
AND
gels
MACKAY
REVIEWS
role of topo II in chromosome
condensation.
These studies
are important
because
they provide
the first evidence
for a
role of any chromosome
scaffold
protein
in chromosome
structure.
In vivo
studies
Genetic
studies of fission yeast first showed that topo II is required
for chromosome
condensation
during
prophase.
Individual
Schizosaccharomyces
pombe chromosomes
normally
cannot
be resolved
in mitosis
by light microscopy
(10).
However,
if spindle
assembly
is blocked
by incubating
cells
harboring
a cold-sensitive
f3-tubulin
mutation
at the nonpermissive
temperature,
the chromosomes
become
hypercondensed
and can be observed
as separate
entities
(11). When
such a mitotic
block is performed
under
conditions
where
topo II function
is absent, the chromosomes
appear less condensed
than
when
the mitotic
block
is imposed
in the
presence
of functional
enzyme
(12).
Several
initial attempts
to examine
the role of topo II in
chromosome
condensation
in higher eukaryotes
yielded
inconclusive
results (reviewed
in ref 13). However,
it has recently been shown that injection
of anti-topo
II antibodies
into Drosophila embryos
results
in local regions
where
the
condensation
of the chromosomes
is grossly aberrant
(14). So
far there is no Drosophila topo II mutant,
but the existence
of
many late-larval
lethal mutations
affecting
chromosome
condensation
suggests
that other,
as yet unidentified,
chromosomal
proteins
also participate
in this process (15).
In vitro
studies
Mitotic
extracts
from Xenopns eggs cause added
interphase
nuclei to undergo
mitotic chromosome
condensation
(16, 17),
thereby
providing
a system where the condensing
chromosomes
are accessible
to direct
biochemical
manipulation.
These extracts
have also yielded evidence
supporting
a role
of topo II in mitotic
chromosome
condensation.
If chicken erythrocyte
nuclei (which lack endogenous
topo
II) are added to Xenopus extracts,
mitotic chromosome
condensation
is observed
(18). However,
if topo II is removed
from the extract by immunoadsorption
before addition
of the
erythrocyte
nuclei,
then condensed
chromosomes
are not
seen. Chromosome
condensation
can be restored
by supplementing
the depleted
extract
with purified
S. pombe topo
II. These results indicate
that topo II is required
for chromosome condensation
in this in vitro system.
Because
of the
stoichiometry
of purified topo II needed to restore condensation in the immunodepleted
extracts,
it was suggested
that
the protein
has both structural
and enzymatic
roles (18).
Subsequent
studies that used either naked DNA or nuclei
from Xenopus sperm as substrates
for the in vitro condensation reaction
confirmed
that topo II is involved
in chromosome condensation
in these extracts
(19, 20). Treatment
of
the Xenopvs mitotic
extracts
with teniposide
(VM26,
which
inhibits
the enzymatic
function
of topo II) or immunodepletion with anti-topo
II antibody
severely
inhibited
chromosome condensation.
IS TOPOISOMERASE
II A STRUCTURAL
IN MITOTIC
CHROMOSOMES?
PROTEIN
These foregoing
studies provide
concrete
evidence
for a role
of topo II in mitotic
chromosome
condensation.
Does this
bolster the argument
that chromosome
scaffold components
play an essential
structural
role in mitotic
chromosomes?
NONHISTONE
PROTEINS IN MITOSIS
This had been suggested
by the observation
that topo II is
the most abundant
component
of the chromosome
scaffold
fraction
(6, 21). However,
two recent studies have challenged
the notion that this protein plays a structural
role in chromosomes.
One study examined
the association
of topo II with chromosomes
assembled
in vitro in the Xenopu.c extract
system
(20) and found that the enzyme
can be extracted
from these
chromosomes
rather
easily by increasing
the concentration
of monovalent
cations.
Furthermore,
the enzyme
apparently
is distributed
throughout
the entire
chromosome,
whereas
chromosomal
nonhistone
proteins
recognized
by
the
MPM-2
antibody
are concentrated
along
the chromatid
axes.
(The
MPM-2
monoclonal
antibody
recognizes
a
mitosis-specific
phosphoepitope
present
on a number
of cellular proteins.)
The MPM-2
antigens
on these in vitro condensed chromosomes
are much more resistant
to extraction
with salt than is topo II. Thus,
it was concluded
that topo
II is not a scaffold protein
in mitotic chromosomes,
whereas
one or more MPM-2
antigens
might be (20).
Two difficulties
arise with this interpretation.
First, topo II
is definitely
a major component
of mitotic
chromosomes
in
living cells. This has been shown by indirect
immunofluorescence, biochemical
fractionation
(6, 21), and direct observation of labeled protein
in vivo (22 see below). Thus, the relative solubility
of the enzyme
at nearly
physiological
ionic
strengths
in the Xenopus extracts
raises the possibility
that
binding
of the enzyme to chromosomes
may be altered in the
unusual
buffer used in this study. Second,
others have shown
that topo II is itself the major chromosomal
MPM-2
antigen
when
appropriate
protocols
are
used
to
minimize
dephosphorylation
(23). As the phosphorylation
state of topo
II is an important
determinant
of its enzymatic
activity (24)
and its ability to exhibit high-affinity
binding
to SAR DNA
(25),
phosphatase
activity
in the extracts
could
have
significant
consequences
for both the distribution
and solubility of the enzyme.
Finally,
the modification
status of the
enzyme
in embryonic
extracts
may differ from that in somatic cells, and this could affect the interaction
of the enzyme with chromosomes.
Technical
considerations
could at
least partly
explain
the differences
between
this and the
previous
studies of topo II in mitotic
chromosomes.
A second recent study has suggested
that only a subpopulation
of topo II could serve a structural
role in chromosomes. This study used elegant light microscope
image analysis
techniques
to
follow
the
cellular
location
of
rhodamine-labeled
topo II in living Drosophila embryos
(22).
The authors found that topo II is clearly detectable
in nuclei
and on chromosomes
as they condense.
However,
the levels
of detectable
topo II on the chromosomes
unexpectedly
dropped
during mitosis,
concomitant
with an increase
in the
levels of topo II detected
in the cytoplasm.
This decrease
occurred in two stages, with about 30-60%
of the enzyme
leaving between
prophase
and metaphase,
and a further
10%
leaving
by late telophase.
Thus, it was concluded
that 30%
of the topo II at most could serve a structural
role. These
results
are in agreement
with earlier
studies
that found
significant
levels of topo II immunostaining
in the cytoplasm
of mitotic
Drosophila cells.
This finding
does not, of course,
rule out the possibility
that a portion of the chromosomal
topo II does serve a structural role in these cells. As will be described
later in this article, topo II must remain
on chromosomes
at least until the
metaphase:anaphase
transition,
when it fulfills an essential
function
in sister chromatid
disjunction;
exactly what percentage of the topo II is required
for this purpose is not known.
949
I5V
IL.WT
There
may be differences
in the role of topo II between
Drosophila and vertebrates
during
mitosis.
First, it must be
borne in mind that these experiments
used Drosophila syncitial early embryos,
which are known to contain
a maternal
pool of topo II in addition
to the injected
labeled molecules.
Studies have shown topo II to be highly concentrated
in mitotic chromosomes
of vertebrates
(6, 21, 23), and quantitative
studies with avian cultured
cells showed that about 70% of
the chromosomal
topo II in prometaphase
cells is associated
with the insoluble
chromosome
scaffold fraction.
These contrasting
findings
may be explained
by the fact that vertebrates
have two isoforms
of topo II, whereas
only a single
form of this protein
has been detected
in Drosophila. The
170-kDa
a-form
is apparently
bound
to chromosomes
throughout
mitosis,
whereas
the 180-kDa fl-form is released
(26); thus, in Drosophila, that portion of topo II that fulfills the
role of topo II may be released from chromosomes
in mitosis.
Considering
both the new and old experiments,
the original
suggestion
that topo II plays a role in one or more aspects
of mitotic chromosome
condensation
and structure
appears
to best explain the bulk of current
data, although
the biological significance
of the chromosome
scaffold remains
an area
where further
experiments
are needed.
CHROMOSOMAL
PREPARATIONS
FOR
ANAPHASE
The classical
literature
lists several requirements
that must
be accomplished
by the chromosomes
to prepare
them for
the orderly
segregation
that occurs during
anaphase.
Essential preparations
that occur during
interphase
include replication of the DNA molecules,
their assembly
into chromatin,
and repair of any DNA damage
incurred
since the previous
mitosis. Upon entry into mitosis, the chromosomes
must first
condense
and then subsequently
form correct bipolar attachments to the mitotic spindle (the latter is not discussed
in this
review).
Recent
experiments
have added at least two more
requirements
to this list.
First, chromosomes
must assemble
a functional
kinetochore
that is capable
of interacting
with microtubules
and directing
chromosome
movements.
Microinjection
of IgG from autoimmune
patients
with antibodies
to the CENP
antigens
(27) during
the G, and S phases of the cell cyde blocks assembly of a normal
trilaminar
kinetochore
(28). Under
these
circumstances,
chromosomes
can still bind to microtubules,
but cannot move along them. Upon entry into mitosis, these
cells delay for many hours in prometaphase
or metaphase
(29). Some injected
cells eventually
do enter anaphase
and
undergo
a highly abnormal
completion
of mitosis.
More recently, it has been shown that microinjection
of specific antibodies recognizing
the CENP-C
protein
alters kinetochore
structure,
reducing
both size and stability
(30). These
injected cells also show an extensive
delay at metaphase
before
embarking
on an abnormal
completion
of mitotic
events.
It is not surprising
that chromosomes
must assemble
a
functional
kinetochore
in order to move normally
in mitosis.
However,
a second, much more subtle, structural
change occurs in chromosomes
during
metaphase.
This change
has
been detected
only with antibodies
recognizing
the INCENP
proteins,
a doublet
of 135- and 155-kDa
polypeptides
that
were the first chromosome
passengers
to be identified
(see
below). At the onset of chromosome
condensation,
these proteins are distributed
along the entire chromosome.
However,
during
metaphase
they gradually
disappear
from the chromosome
arms and become
concentrated
at the centromere
region. The mechanism
underlying
this redistribution
is not
understood.
It could be driven by specific motor proteins
or
950
Vol. 8
September
1994
it could reflect the ongoing
process of chromosome
sation that continues
throughout
metaphase.
THE
METAPHASE
conden-
CHECKPOINT
It is conceptually
useful to consider
that the five phases of
mitosis form two distinct
groups.
The first group consists
of
the preparations
for chromatid
segregation,
including
chromosome
condensation,
assembly
of a bipolar mitotic spindle,
and attachment
of the chromosomes
to this spindle via their
kinetochores
(prophase,
prometaphase,
and metaphase
in
the conventional
terminology).
It is now widely accepted
that
except for certain
early embryos,
eukaryotic
cells have a cell
cycle checkpoint
mechanism
to monitor
completion
of the
preparation
stages, particularly
the correct
attachment
and
alignment
of the chromosomes
on a bipolar
spindle.
The
metaphase
checkpoint
has been proposed
to hold the cells in
prometaphase/metaphase
until the last chromosome
has attained a bipolar orientation
or until an underlying
cell cycle
clock overrides
the checkpoint
delay. The metaphase
checkpoint appears
to be sensitive
to both the quantity
of assembled microtubules
and to their organization
into a bipolar
spindle (31). Although
the metaphase
checkpoint
is regarded
as an integral part of mitotic regulation,
little is known about
its biochemical
basis.
The second
overall
stage of mitosis
includes
the active
events of chromatid
separation
and movement,
spindle movements, and cytokinesis
(anaphase,
telophase,
and cytokinesis
in the conventional
terminology).
This “execution”
stage appears to lack quality control circuitry,
and thus is entirely
dependent
on preparations
being completed
correctly
before it
begins.
Studies of yeast mutants
and of microinjected
mammalian
cells reveal that when cells commence
mitotic execution without properly
completing
the necessary
preparations,
the resulting
division
is aberrant.
If cytokinesis
occurs, chromosome
damage or gross aneuploidy
may ensue, often resulting in cell death. If cytokinesis
fails, polyploid
progeny result.
The monitoring
of spindle architecture
by the metaphase
checkpoint
has been subjected
to genetic analysis in the budding yeast. Although
yeast chromosomes
have not been observed to form a metaphase
plate, the fact that antimicrotubule drugs or mutant
centromeres
(32) produce
a mitotic
delay suggests
that a metaphase
checkpoint
is an integral
aspect of mitosis in these organisms.
Furthermore,
centromere alignment
at a metaphase
plate has recently
been observed by in situ hybridization
in the fission yeast (33). Two
groups have used genetic screens to identify putative
components of the regulatory
circuitry
of this checkpoint
by looking
for mutants
that fail to arrest in mitosis when microtubule
assembly
is inhibited.
These
mutants
have been termed
either
mad (mitotic
arrest deficient)
or bub (budding
uninhibited
by benzimadazole).
Initial
screening
yielded
three
mad and seven bub mutants
(34, 35).
Whether
the mad and bub mutants
affect the metaphase
checkpoint
directly, and if so, how they do it, is unknown.
Of
the 10 mad and bub mutants,
two have been partially
characterized on a molecular
level. Although
MAD2 was at first believed to encode an isoprenyl
transferase
(36), it was recently
shown to correspond
to a separate,
adjacent
open reading
frame (A. Murray,
personal
communication).
The product
of the BUBJ gene appears
to encode an unusual
118-kDa protein kinase. The protein contains
a carboxyl-terminal
kinase
domain
and a large amino-terminal
domain
of unknown
function.
Its physiological
substrates
are unknown,
although
it is known to form a complex
with Bub3 protein
(A. Hoyt,
personal
communication).
The FASEB Journal
EARNSHAW
AND
MACKAY
REVIEWS
It has been suggested
that as well as sensing the assembly
of a bipolar
spindle,
the metaphase
checkpoint
is also sensitive to the presence
of maloriented
chromosomes.
Newt lung
cells with all chromosomes
but one properly
congressed
into
a metaphase
plate on a bipolar
spindle apparently
delay their
entry into anaphase
for a significant
time (37-39).
Zirkle
(38), in a widely cited abstract,
argued
that this delay was
likely to be due to a signal transmitted
by the kinetochores
of the maloriented
chromosome,
as irradiation
of the centromere region of such a chromosome
with a UV microbeam
appeared
to abolish
its inhibitory
effect.
This notion,
that a maloriented
kinetochore
sends an inhibitory
signal, has never been tested directly.
The best evidence
for a direct
involvement
of kinetochores
in the
metaphase
checkpoint
is provided
by microinjection
experiments, where injection
of anti-CENP-C
antibodies
produces
a substantial
delay in metaphase.
Chromosomes
in the injected cells are able to assemble
kinetochores
that, although
smaller than normal,
are able to direct the positioning
of the
chromosomes
at a metaphase
plate (30). However,
these
aligned chromosomes
enter anaphase
only after a lengthy delay. Additional
evidence
comes
from genetic
experiments,
where it has been shown that the presence
of supernumerary
chromosomes
with mutant
centromeres
causes a prolongation of the G2/M period
in Saccaromyces cerevisiae (32). Both
observations
could be explained
by suggesting
that the defective kinetochores
transmit
a signal
that
activates
the
metaphase
checkpoint
and causes cells to delay entry into
anaphase.
However,
it cannot
be ruled out that the checkpoint is activated
by a more subtle disruption
of kinetochoremicrotubule
interactions
resulting
from physical
damage
to
the kinetochore,
without
any need to invoke signaling
by the
kinetochores.
One particularly
striking
example
of an instance
where
unattached
chromosomes
fail to activate
a metaphase
checkpoint is found in the case of sea urchin zygotes where fusion
of the maternal
and paternal
chromosome
sets has been
prevented
by colcemid
treatment.
Under
these circumstances,
only the paternal
chromosome
set forms any attachment to a spindle
(the centrosomes
are contributed
by the
sperm);
the maternal
chromosomes
often remain unattached
in the cytoplasm.
These cells do have a functional
metaphase
checkpoint,
as disruption
of spindle
assembly
or structure
causes a significant
delay in the metaphase:anaphase
transition. Despite
this fact, the presence
of as many as 20 maternal chromosomes
with unattached
kinetochores
has no effect
on the length of the timing of anaphase
onset for the chromosomes of the paternal
nucleus
(40).
Until recently,
there were no indications
as to what the
molecular
basis for any signaling
by the kinetochore
might
be. This has now changed
with the discovery
of a phosphoepitope
that can be detected
on the kinetochores
of chromosomes
that are either unattached
or have not yet achieved
a stable bipolar orientation
at the spindle midzone
(41). This
phosphoepitope
is undetectable
on chromosomes
once they
achieve a stable bipolar
attachment
to the spindle. These observations
provide the first example
of a possible biochemical
difference
between
kinetochores
that might be transmitting
an inhibitory
signal and those that might not. In the future
it will be extremely
important
to identify
the proteins
bearing this differential
phosphorylation,
and
to determine
whether
the differences
reflect changes
in the activity
of a
specific kinase or of its counterbalancing
phosphatase.
There
is a clear need for further
experiments
to explore
the workings
of the metaphase
checkpoint.
In addition
to
characterizing
the yeast mutants,
it will be important
to design conclusive
tests of the notion
that kinetochores
of
NONHISTONE
PROTEINS
IN MITOSIS
maloriented
progression.
chromosomes
send
a signal
EVENTS
THAT
OCCUR
DURING
CHROMATID
SEPARATION
that
inhibits
mitotic
SISTER
Separation
(disjunction)
of the sister chromatids
is the landmark event defining
the onset of anaphase
and the transition
between
the preparatory
and execution
stages of mitosis.
One can imagine
two classes of mechanisms
that could bring
about the separation
of sister chromatids.
The first requires
some external
force, such as the mitotic
spindle,
to pull the
sisters apart:
the metaphase:anaphase
transition
might be
triggered
by changes
in the ability
of the chromosomes
to
resist the pull of the spindle,
by the spindle’s
ability to pull,
or both. In the second
class, separation
of the chromatids
results
solely from changes
intrinsic
to chromosomes.
Surprisingly,
the latter model has been shown to be correct.
In
a variety of cell types, synchronous
disjunction
of the sister
chromatids
can occur even in the absence of a mitotic spindle
(42, 43). This observation
indicates
that the key to anaphase
chromatid
disjunction
is to be found in the chromosomes
themselves.
The onset of anaphase
has generally
been assumed
to occur subsequent
to changes
in the relative
activities
of the
members
of the mitotic
kinase
cascade
(p342
and those
downstream
kinases
that it activates).
In fact, compelling
evidence
that changes
in the kinase:phosphatase
balance
are
also essential
for the onset of anaphase
is provided
by genetic
analyses
in S. pombe, Aspergillus
nidulans,
and Drosophila
melanogaster. In all these organisms,
the presence
of a functional type I protein
phosphatase
is required
for sister chromatid disjunction
(44-48).
In flies, activity of a type 2A protein phosphatase
is also required
(49, 50).
In a landmark
advance,
it has been possible
recently
to
reconstitute
the events of anaphase
chromosome
disjunction
in vitro. Addition
of Ca2 to Xenopus egg extracts that are arrested in metaphase
can trigger
the onset of anaphase
(51).
This anaphase
appears
to be characterized
by sister chromatid disjunction
and poleward
movement.
Although
it has
not been proven
formally
that sisters disjoin
faithfully
in
these extracts,
the available
evidence
argues strongly
that the
system does mimic the chromosomal
events of anaphase
in
a cell-free
environment.
Given the prevailing
wisdom that the metaphase:anaphase
transition
is triggered
by changes
in the kinase:phosphatase
balance,
it was extremely
surprising
when studies using the
mitotic
extracts
revealed
that sister chromatid
disjunction
can occur in the presence
of high levels of Hi kinase activity
(52). In these experiments,
it was found that addition
of a
nondegradable
form of cyclin B to these extracts
does not
abolish
sister chromatid
separation,
even though
Hi kinase
activity
remains
elevated.
However,
inhibition
of cyclin proteolysis,
either with methylated
ubiquitin
or with a peptide
corresponding
to the “destruction
box” on cyclin B (53), does
inhibit
sister chromatid
separation
in the extracts
(52). The
authors
concluded
that proteolysis
of one (or more) molecule
other than cyclin B is required
for sister chromatid
disjunction. The target molecule
(or molecules)
for this proteolysis
could turn out to be related to the previously
described
CLiP
(54) or INCENP
(55) polypeptides,
or they may be novel
components.
Independent
support
for the idea that cyclin degradation
is essential
for the transition
from mitosis to G,, but not for
the initiation
of sister chromatid
separation,
comes from an
experiment
in which nondegradable
mutant
cyclin B was ex951
REVIEWS
pressed in budding
yeast. This leads to the retention
of high
HI kinase activity,
but fails to prevent
disjunction
of sister
chromatids
(56).
Even though it is attractive
to think that sister chromatids
are held together
by linker molecules
whose degradation
signals the onset of anaphase,
it cannot be excluded
that the essential substrate
for proteolysis
in the Xenopus extract experiment is the endogenous
cyclin B, which may be associated
preferentially
with the mitotic spindles assembled
in vitro. In
vivo, only cyclin B that is bound to the spindle appears
to be
degraded
during
mitotic
progression
of the Drosophila embryo (57). Similarly,
in mouse oocytes arrested
in metaphase
I, cyclin B destruction
depends
on the presence
of a spindle
(58).
The suggestion
that labile protein
linkers hold sister chromatids together
is the third explicit model that has been proposed to explain
the regulation
of sister chromatid
pairing.
Originally,
it was thought
that sister chromatids
might be
held together
by a short stretch of unreplicated
DNA. This
appears
to be ruled out by the observation
that budding
yeast
centromeres
actually
replicate
early in S phase (59), and by
the failure to detect centromere-associated
DNA replication
at the metaphase:anaphase
transition.
A second model suggested that sister chromatids
might be held together
by entangled
DNA loops (60). Such topological
entanglements
proved
difficult
to detect for plasmid
minichromosomes
in
mitotic
yeast cells (61). However,
the existence
of mutations
that reveal differences
between
the segregation
of plasmid
minichromosomes
and bona fide chromosomes
(62) again
raises the possibility
that such intertwinings
might exist in
full-sized
chromosomes.
If so, sister chromatid
separation
might require a type II topoisomerase
to separate
the entangled loops.
In fact, early studies of the replication
of SV4O virus (63)
did implicate
topo II in the separation
of daughter
DNA
molecules
after DNA
replication.
These
results
were extended by a genetic analysis
in the yeasts, which confirmed
that topo II carries out an essential
role in the separation
of
sister
chromatids
at anaphase
(64-66).
If cells attempt
anaphase
in the absence
of functional
enzyme,
the centromeres are able to move to the spindle poles, but the bulk of
the chromatin
remains
entangled
at the spindle equator
and
is entrapped
in the cleavage
furrow (33). This lethal phenotype was referred
to as cut (cell untimely
torn) (66).
Several lines of evidence
suggest that topo II also plays an
essential
role in sister chromatid
disjunction
in higher
eukaryotes.
For example,
teniposide
(VM-26),
a potent topo II
inhibitor,
blocks disjunction
of sister chromatids
in the Xenopus extract system (51). Similarly,
microinjection
of anti-topo
II antibodies
into Drosophila embryos
appears
to cause a local
disruption
of anaphase
chromatid
disjunction
(14). Finally,
three
other
inhibitors
of topo II [etoposide
(VP-16),
mAMSA,
and ICRF-l93]
were found to disrupt
sister chromatid separation
in a variety
of mammalian
cultured
cells
(67, 68), although
this was disputed
in another
study where
lower concentrations
of a variety of inhibitors
were used (69).
Thus,
except for the study last cited (i.e., ref 69) evidence
favoring
a requirement
for topo II in sister chromatid
disjunction
appears
to be unanimous.
In addition
to topo II and the (currently
unknown)
proteolysis
targets,
other candidates
for proteins
with direct
roles in sister chromatid
disjunction
also exist. Among
them
are the products
of the Drosophila genes rough deal [phenotypelagging
chromosomes
and anaphase
bridges
(70)], lodestar
[phenotype-chromosome
tangling/anaphase
bridges
(71)],
l(1)zwlO [phenotype-premature
sister separation
in colchicine, aneuploidy
(72)], three rows [phenotype-metaphase
ar-
952
Vol. 8
September
1994
rest (73, 74)], and Jlzzy [phenotype-metaphase
arrest with a
few chromosomes
escaping
into anaphase
(75)]. Some of the
nine newly described
genes whose mutant
alleles confer cut
phenotypes
in fission yeast may also encode
proteins
with
structural
roles in this process (76).
CHROMOSOME
FUNCTION
OF
PASSENGERS
REVEAL
THE CENTROMERE
A NOVEL
It has long been realized
that the centromere
region of the
chromosomes
carries out essential
functions
during
mitosis.
With recent advances
in identification
and mapping
of the
components
involved
in chromosome
movement
during
mitosis, this list of functions
of the centromere
has risen to three
(77).
1) The centromere
serves as the attachment
point of the
chromosomes
to the mitotic spindle.
Attachment
occurs at a
specialized
buttonlike
structure,
the kinetochore.
2) The centromere
is the site of regulation
of sister chromatid pairing.
At the metaphase:anaphase
transition,
some
signal acts on the centromere
to release the last point of sister
chromatid
adhesion.
As discussed
before,
current
evidence
implicates
both topo II activity and a protease
in this release
mechanism
(52).
3) The centromere
is the location
of at least some of the
motors
that move chromosomes
during
mitosis.
This has
been demonstrated
in vivo, where the chromosomes
were
shown to move relative to spindle microtubules
(78), and in
many in vitro studies
(79-82).
In addition,
several motor
proteins
have
been
localized
to the centromere
region
(among
other
locations)
in fixed cells by indirect
immunofluorescence
(83, 84).
Studies
of the chromosome
passenger
proteins
have revealed a new and unexpected
function
for the centromere:
as
a marshaling
area or jumping-off
point for the chromosome
passenger
proteins
on their way to the overlap
zone of the
anaphase
mitotic
spindle.
All chromosome
passenger
proteins described
to date concentrate
at centromeres
during
prometaphase.
As a result, they move with the chromosomes
to the metaphase
plate. What happens
next is what defines
this class of proteins:
they transfer
from the chromosomes
to
the midzone
of the spindle
(Fig. 1). This departure
occurs
by a number
of routes. The INCENPs
appear
to be among
the first to make their move. They leave the chromosomes
and
accumulate
along
linear
tracks
transecting
the
metaphase
plate before there is any indication
of sister chromatid separation
(85). CENP-E
and CENPF/p330d
appear
to be among
the last chromosomal
passengers
to leave the
chromosomes,
with
the
former
separating
during
midanaphase
(86) and the latter by late anaphase
(87, 88).
Why do these proteins
accumulate
at centromeres
before
their transfer
to the spindle?
So far there is no answer to this
question.
One possibility
is that the proteins
must be carried
to the exact midline
of the bipolar
spindle in order to function properly
after their release from chromosomes.
Chromosome
arms are often rather
long, and may extend
a
significant
distance
laterally
away from the central
spindle.
Only the centromeres
are clustered
in a tight disk or ring at
the spindle midzone.
In fact the centromere
may be the only
portion
of the chromosomes
that lies wholly within the spindle, where the concentration
of microtubules
is highest.
The
notion
that proximity
to microtubules
is important
for the
transfer
of the passenger
proteins
is supported
by the observation that these proteins
remain
associated
with the centromeres in cells where drug treatment
has caused spindle disassembly
(55, 87, 88).
The FASEB Journal
EARNSHAW AND MACKAY
REVIEWS
Figure 1. The chromosome
passenger proteins are both chromosomal
tightly associated with the chromosomes
at the beginning of mitosis, but
the metaphase:anaphase
boundary. As anaphase and telophase progress,
(red) is seen in transiently
transfected mammalian
LLC PK cells (93) in
at the conclusion
of telophase (E). When expressed at high levels in
microtubules
(ii). DNA (stained with DAPI) is visualized in blue. Bar,
Immunoelectron
microscopy
of the
INCENPs
and
CENP-E
in anaphase
cells shows that these proteins
are intimately
associated
with an amorphous
deposit
of electron
dense
material
that enshrouds
the antiparallel
interpolar
NONHISTONE
PROTEINS IN MITOSIS
and cytoskeletal components.
The INCENP
proteins (red) are
associate with microtubules
(green) in the spindle midzone near
they concentrate
in the stem body material. Chicken INCENP11
interphase (A), prophase
(B), metaphase
(C), anaphase
(D), and
interphase
cells, INCENP,,
binds to and bundles cytoplasmic
10 atm.
microtubules
of the central spindle.
This material,
known as
stem body matrix,
may be involved
in the physical
integration of the two half spindles.
With one exception,
all the
identified
matrix
components
are chromosome
passenger
953
REVIEWS
proteins.
The
CHO-l
antigen
(89)
recently
renamed
MKLP-1
(mitosis-specific
kinesinlike
protein
1) (90) shows
no association
with the nucleus
during
interphase
or with
chromosomes
during
mitosis (90, 91), and is thus not a passenger
protein.
However,
its distribution
throughout
anaphase
and telophase
is identical
to that described
previously for the passenger
proteins
(concentration
in the spindle
midzone
and association
with the midbody
in the intercellular bridge during
cytokinesis).
How do the passenger
proteins
associate
with the spindle?
In the case of CENP-E,
the largest known member
of the superfamily
of kinesin-related
proteins
(92), cytoskeletal
interactions
may be essential.
Biochemical
experiments
have
shown
that the protein
can interact
with microtubules
in
vitro via its amino-terminal
motor domain
and also via a distal ATP-insensitive
site (92). Thus, CENP-E
is predicted
to
cross-link
adjacent
microtubules.
The INCENPs
also appear
to associate
with cytoplasmic
microtubules,
at least in transfected
interphase
cells that
greatly
overexpress
the proteins
(93). It is not yet known
whether
this binding
involves direct interaction
between
the
INCENPs
and
microtubules
or whether
it is mediated
via
one or more MAP (microtubule-associated
protein).
The 42
amino-terminal
amino acids of the INCENPs
are required
for these proteins
to transfer
from the chromosomes
to the
spindle
(93). It is surprising
that this region of the protein
apparently
is not required
for recombinant
INCENPs
to associate with and bundle cytoplasmic
microtubules
during interphase.
Although
it is not yet known whether
this region
corresponds
to a spindle
targeting
signal or a chromosome
release switch, the relatively
small size of the region should
be a substantial
asset in future characterization
of its role in
the movements
of the INCENPs
during
mitosis.
Experiments in which mammalian
cells are transiently
transfected
with chicken INCENP
constructs
indicate
that movement
to
the centromere
may be required
for transfer
to the spindle.
Deletion
of the 42 amino-terminal
amino
acids of the INCENPs
appears
to prevent
the concentration
of the protein
at centromeres
(A. M. Mackay
and W. C. Earnshaw,
unpublished results).
FUNCTION
PROTEINS
OF
THE
CHROMOSOME
PASSENGER
The protean
redistribution
of the chromosome
passenger
proteins
during
mitosis has suggested
that they could be involved in a variety of functions.
These include:
1) regulation
of sister chromatid
pairing;
2) structural
integration
of the
two half spindles;
3) motor function
during
spindle
elongation (anaphase
B); 4) stabilization
of the plus ends of
microtubules
in the central
spindle;
5) the earliest stages of
chromosome
movement
toward
the spindle
poles (anaphase
A); and 6) localization
or assembly
of the cleavage
furrow.
Unfortunately,
this list of possibilities
is constrained
all too
little by available
data. At present,
no functional
data are
available
concerning
the roles of MSA-36
(94), JB antigen
(95), TD-60
protein
(96), the 37A5 antigens
(97), and
CENP-F/p330”
(87, 88) in vivo (Table 1). Presumably
these
data will emerge
as the various
cDNAs
are cloned
and
specific reagents
become
available.
There are preliminary
indications
that CENP-E
and INCENPs
may be involved
in the separation
of sister chromatids.
Microinjection
of antibodies
to CENP-E
was found
to retard
the metaphase:anaphase
transition
(86). Expression of a mutant
INCENP
can affect the transit
of cells to
anaphase
(A. M. Mackay
and W. C. Earnshaw,
unpublished
954
Vol. 8
September 1994
results).
Unfortunately,
this phenotype
could result equally
well from either a direct involvement
of these proteins
in the
structural
events of sister chromatid
separation
or from involvement
in one of the preparative
processes
that are sensed
by the metaphase
checkpoint.
One other dominant
negative
mutant
of the INCENPs
has
also been constructed
by deleting
a small portion
of the
carboxyl-terminal
domain
(A. M. Mackay
and W. C. Earnshaw, unpublished
results).
Expression
of this protein in cultured cells produces
a variety of phenotypes,
most of which
appear
to result from failures
in cytokinesis.
It is tempting
to suggest that this may indicate
a direct role for these proteins in cytokinesis,
a model consistent
with the observation
that the INCENPs
are among the earliest proteins
known to
concentrate
at the site where the cleavage furrow will subsequently
form (98). However,
the lack of checkpoint
controls
during the execution
stages of mitosis makes it difficult to assign a direct role for the INCENPs
in cytokinesis
on the basis
of these observations
alone. As described
earlier,
failure of
cytokinesis
is a common
outcome
when cells execute mitosis
after incomplete
or defective
preparations.
In future experiments
it will be important
to identify
the
binding
partners
of the chromosome
passenger
proteins
on
chromosomes
and on the spindle (and membrane,
where applicable).
This, as well as a characterization
of the mechanisms by which these proteins
change their binding
allegiance
at the metaphase:anaphase
transition,
should eventually
lead
to a molecular
understanding
of the nature of the collaboration between
the chromosomes
and the cytoskeleton
during
mitotic
execution.
CONCLUSIONS
In the 17 years since the initial presentation
of the chromosome scaffold
hypothesis,
evidence
has gradually
accumulated to suggest that chromosomal
nonhistone
proteins
play
a variety
of roles in the chromosomal
events
of mitosis.
Although
certain
aspects of the model remain
controversial,
by focusing
attention
on the chromosomal
nonhistone
proteins, the model has clearly led to a significant
enhancement
of our understanding
of the biology of mitotic chromosomes.
Further
advances
in this area will no doubt continue
to clarify the role of the scaffold as additional
nonhistone
proteins
are cloned and characterized
by cell biological
and genetic
means.
Work on mitosis from the authors’ laboratory has been funded by
National Institutes of Health grants GM30985 and GM35212. We
would like to acknowledge
many colleagues
for communicating
results before publication,
as well as Kip Sluder
and our colleagues
M. Eckley, A. Pluta, J. Tomkiel, and C. Yang for comments
on the
manuscript.
We apologize to those colleagues whose articles could
not
be
cited
due
to
space
limitations.
REFERENCES
1. Laemmli,
U. K., Cheng,
S. M., Adolph,
K. W., Paulson,J.
R., Brown,
J. A., and Baumbach,
W. R. (1978) Metaphase
chromosome
structure:
the role of nonhistone
proteins.
Cold Spring Harbor Sym. Quasi. Biol. 423,
351-360
2. Gasser,
S. M., Amati,
B. B., Cardenas,
M. E., and Hofmann,
J. F.-X.
(1989) Studies
on scaffold attachment
sites and their relation
to genome
function.
Jot. Rev. Cytol. 119, 57-96
3. Adachi,
Y., K#{228}s,
E., and Laemmli,
U. K. (1989) Preferential
cooperative
binding
of DNA topoisomerase
II to scaffold-associated
regions.
EMBO
J. 13, 3997-4006
4. Earnshaw,
W. C., and Laemmli,
U. K. (1983) Architecture
of metaphase
chromosomes
and chromosome
scaffolds.
j
Cell Biol. 96, 84-93
The FASEB Journal
EARNSHAW
AND
MACKAY
REVIEWS
5. Earnshaw,
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14.
W. C., and Heck, M. S. S. (1985) Localization
of topoisomer-
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