REVIEWS

Role of nonhistone proteins in the chromosomal events of

mitosis
WILLIAM C. EARNSHAW’ AND ALASTAIR M. MACKAY
Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA

ABSTRACT This review is concerned with the role of of narrowed DNA minor groove. The functional significance
chromosomal nonhistone proteins in three important of SARs/MARs in vivo remains unresolved.
aspects of mitotic events: chromosome condensation, sis- The most abundant polypeptide component of the chro-
ter chromatid separation at the metaphase:anaphase tran- mosome scaffold is DNA topoisomerase II (topo II). Topo II
sition, and interactions between the chromosomes and is a widely studied enzyme that catalyzes the passage of both
cytoskeleton that occur during construction of the mitotic strands of the DNA helix through a transient gap in the
spindle and cleavage furrow. Emphasis will be given to DNA backbone. This activity allows the relaxation of super-
the potential roles of topoisomerase II and the chromo- coils, knotting and unknotting of DNA, and separation of
some passenger proteins in these events. Other important catenated circular DNA molecules. Topo II preferentially as-
aspects of mitotic events such as the regulation of the sociates with SARs in vitro (3), which suggests a mechanism
G2-’ M transition, the structural changes that affect the whereby the enzyme could contribute to chromosome
nuclear envelope and other organelles during mitosis, and higher-order structure. SARs typically are spaced approxi-
the mechanism of chromosome movement will not be con- mately 50 to 200 kb apart along the chromosomal DNA (2).
sidered here. Despite long histories of often elegant ex- Thus, interactions between SAR-binding proteins such as
perimentation, all three of our chosen subjects remain topo II or other proteins of the chromosome scaffold fraction
areas of lively, ongoing controversy. Thus, although re- could produce large ioop domains, as have been observed by
cent advances appear to have taken us many steps closer microscopy of swollen chromosomes (4-6).
to an understanding of the underlying mechanisms, we Functional evidence is beginning to accumulate in support
suspect that final answers will be some time in coming. - of the notion that chromosome scaffold components such as
Earnshaw, W. C., Mackay, A. M. Role of nonhistone pro- topo II might have a role in mitotic chromosome structure,
teins on the chromosomal events of mitosis. FASEB J. 8: as originally suggested by Laemmli et al. (1). We will discuss
947-956; 1994. a variety of evidence suggesting that topo II has a direct role
in mitotic chromosome condensation. We will follow this
Key Wordr: topoisomerase II’ chromosome passenger proteins . chro- with a discussion of the ongoing controversy over whether
rnosome condensation INCENPs #{231}ytorkeletonmetaphose:anapho.se topo II plays an additional structural role in chromosomes.
transition Next, we will discuss the chromosomal events required to
render the chromosomes competent to undergo anaphase
sister chromatid separation (disjunction). The assembly of a
ROUGHLY ONE-THIRD OF THE MASS OF MITOTIC chromosomes functional kinetochore is one aspect of chromosome struc-
is composed of DNA, one-third of histones and one-third of ture and function that is monitored by a cell cycle checkpoint
a highly heterogeneous and poorly characterized group generally known as the metaphase checkpoint. We will dis-
known as nonhistone proteins. The idea that members of this cuss the current state of knowledge concerning the workings
latter group of proteins might have a role in determining the of this important cell cycle control checkpoint.
architecture and biochemical properties of mitotic chromo- Finally, we will close with a discussion of the interplay be-
somes was developed most fully by Laemmli and co-workers tween the chromosomes and the cytoskeleton during mitosis.
(1), who established a method for the fractionation of chro- For many years it was assumed that chromosomes are just so
mosomes into a relatively soluble fraction, containing about much baggage to be hauled about by the cytoskeletal pro-
90-95% of the chromosomal proteins, and an insoluble frac- teins of the mitotic spindle. This led to the famous expression
tion composed of a subset of the nonhistone proteins. This that “the chromosome arms are like the corpse at the funeral:
insoluble fraction was termed the chromosome scaffold. It they are the reason for the proceedings but take no active
was suggested that the scaffold is composed of structural part in them (7). Now this concept has been thrown into
components that give the mitotic chromosomes their charac- question by the discovery of a class of molecules with “dual
teristic 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 ‘To whom correspondence and requests for reprints should be ad-
the biological significance of both fractions remains con- dressed, at Department of Cell Biology and Anatomy, Johns Hop-
troversial. Efforts to address this controversy have principally kins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205,
entailed identification and characterization of specific chro- USA.
mosome scaffold proteins and the identification of DNA se- 2Abbreviations: SARs, scaffold attachment regions; MARs,
matrix attachment regions; topo II, topoisomerase II; mad, mitotic
quences (termed SARs2 or MARs, scaffold or matrix attach- arrest deficient; bub, budding uninhibited by benzimadazole; cut,
ment regions) that appear to interact preferentially with the cell untimely torn; MKLP, mitosis-specific kinesinlike protein;
scaffold and nuclear matrix fractions (2). These interactions MAPs, microtubule-associated proteins; INC ENPs, inner centro-
appear to involve the binding of scaffold proteins to regions mere proteins.

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 . V

citizenship” that are chromosomal for the bulk of the cell cy- the condensation is only observed in budding and fission
cle, but become integral cytoskeletal components during the yeasts under special circumstances). It is widely assumed
closing phases of mitosis. These proteins are termed chromo- that condensation of the chromosomes into dense, discrete
some passengers (8) (Table 1). It has been proposed that the bodies allows them to be moved more easily by the mitotic
sequestration of these proteins in the cell nucleus might serve spindle. It is possible that the condensation process serves as
to keep them apart from the interphase cytoskeleton. This a rectification mechanism, during which neighboring chro-
could be important if these proteins do carry out mitosis- mosomes are untangled from one another during the transi-
specific cytoskeletal functions. Also, the association of the tion from interphase into mitosis; this would allow them to
passenger proteins with the chromosomes might serve in move as individual units during mitosis. It has also recently
part as a mechanism for the exact positioning of these pro- been proposed that condensation is needed to prevent the
teins at the spindle midplane during mitosis (8). chromosome arms from trailing too far behind the centro-
meres as they move to the spindle poles during anaphase (9).
Trailing chromosome arms could become entrapped in the
MECHANISM OF CHROMOSOME cytokinetic furrow if sufficiently extended.
CONDENSATION: THE ROLE OF DNA The mechanism of chromatin condensation is understood
TOPOISOMERASE II only poorly. Although it has long been known that mitotic
chromosome condensation is accompanied by phosphoryla-
Chromosome condensation, one of the most visually striking tion of histones Hi and H3, the role of this modification re-
of mitotic events, occurs in virtually all eukaryotes (although mains unknown. Here we will focus on recent studies of the

TABLE 1. Known chromosome passenger proteins

Molecular weight

Possible
Antigen’ SDS/PAGE5 eDNA ORF’ functions Description

MSA-36 36 kDa - Unknown Nuclear in late 02

(94) On prometaphase chromosomes
At centromere of metaphase chromosomes
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular bridge
JB antigen 38 kDa Unknown Absent or masked in interphase and prophase
(99) On prometaphase chromosomes of some cells
At centromere of metaphase chromosomes
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular bridge
TD-60 protein 60 kDa Cytokinesis? Absent or masked in interphase
(96) At centromeres from prometaphase-early anaphase
At spindle midzone, midanaphase-telophase
At cleavage furrow, midanaphase-telophase
Flanking midbody in intercellular bridge
INCENP, 133 kDa 96,000 Structural protein? Nuclear in interphase
INCENP,, 145 kDa 101,000 Anaphase onset On prometaphase chromosomes
(55, 85, 93) Cytokinesis? At centromere of metaphase chromosomes
At spindle midzone, late metaphase-telophase
At cleavage furrow, anaphase-telophase
Flanking midbody in intercellular bridge
37A5 antigens 140 kDa Unknown Masked until metaphase
(97) 155 kDa At centromere of metaphase chromosomes
At spindle midzone, anaphase-telophase
On both sides of midbody in intercellular bridge
CENP-E 300 kDa 312,000 Kinesin-like Absent in Gl, S, accumulates in cytoplasm in G2
(86, 92) Microtubule motor At kinetochores from prometaphase-early anaphase
At spindle midzone, mid-anaphase-telophase
Flanking midbody in intercellular bridge
330d -330 kDa Unknown Absent in Gl, accumulates in nucleus in S, 02
(87) (doublet) At centromeres from prometaphase-early anaphase
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular bridge
Probably identical to CENP-F
CENP-F -400 kDa Unknown Absent or masked in 01, S, nuclear in G2
(88) At kinetochores from prometaphase-early anaphase
At spindle midzone, anaphase-telophase
Flanking midbody in intercellular bridge
Probably identical to p330”
‘Reference numbers appear in parentheses. bApparent molecular weight derived from mobility on sodium dodecyl sulfate-polyacrylamide gels
(SDS-PAGE). Molecular weight calculated from deduced open reading frames (ORFs) of full-length cDNAs.

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 REVIEWS
role of topo II in chromosome condensation. These studies This had been suggested by the observation that topo II is
are important because they provide the first evidence for a the most abundant component of the chromosome scaffold
role of any chromosome scaffold protein in chromosome fraction (6, 21). However, two recent studies have challenged
structure. the notion that this protein plays a structural role in chromo-
somes.
In vivo studies One study examined the association of topo II with chro-
mosomes assembled in vitro in the Xenopu.c extract system
Genetic studies of fission yeast first showed that topo II is re-
(20) and found that the enzyme can be extracted from these
quired for chromosome condensation during prophase. In-
chromosomes rather easily by increasing the concentration
dividual Schizosaccharomyces pombe chromosomes normally
of monovalent cations. Furthermore, the enzyme apparently
cannot be resolved in mitosis by light microscopy (10).
is distributed throughout the entire chromosome, whereas
However, if spindle assembly is blocked by incubating cells
chromosomal nonhistone proteins recognized by the
harboring a cold-sensitive f3-tubulin mutation at the nonper-
MPM-2 antibody are concentrated along the chromatid
missive temperature, the chromosomes become hypercon-
axes. (The MPM-2 monoclonal antibody recognizes a
densed and can be observed as separate entities (11). When
mitosis-specific phosphoepitope present on a number of cel-
such a mitotic block is performed under conditions where
lular proteins.) The MPM-2 antigens on these in vitro con-
topo II function is absent, the chromosomes appear less con-
densed chromosomes are much more resistant to extraction
densed than when the mitotic block is imposed in the
with salt than is topo II. Thus, it was concluded that topo
presence of functional enzyme (12).
II is not a scaffold protein in mitotic chromosomes, whereas
Several initial attempts to examine the role of topo II in
one or more MPM-2 antigens might be (20).
chromosome condensation in higher eukaryotes yielded in-
Two difficulties arise with this interpretation. First, topo II
conclusive results (reviewed in ref 13). However, it has re-
is definitely a major component of mitotic chromosomes in
cently been shown that injection of anti-topo II antibodies
living cells. This has been shown by indirect immunofluores-
into Drosophila embryos results in local regions where the
cence, biochemical fractionation (6, 21), and direct observa-
condensation of the chromosomes is grossly aberrant (14). So
tion of labeled protein in vivo (22 see below). Thus, the rela-
far there is no Drosophila topo II mutant, but the existence of
tive solubility of the enzyme at nearly physiological ionic
many late-larval lethal mutations affecting chromosome con-
strengths in the Xenopus extracts raises the possibility that
densation suggests that other, as yet unidentified, chro-
binding of the enzyme to chromosomes may be altered in the
mosomal proteins also participate in this process (15).
unusual buffer used in this study. Second, others have shown
that topo II is itself the major chromosomal MPM-2 antigen
In vitro studies
when appropriate protocols are used to minimize
Mitotic extracts from Xenopns eggs cause added interphase dephosphorylation (23). As the phosphorylation state of topo
nuclei to undergo mitotic chromosome condensation (16, 17), II is an important determinant of its enzymatic activity (24)
thereby providing a system where the condensing chromo- and its ability to exhibit high-affinity binding to SAR DNA
somes are accessible to direct biochemical manipulation. (25), phosphatase activity in the extracts could have
These extracts have also yielded evidence supporting a role significant consequences for both the distribution and solu-
of topo II in mitotic chromosome condensation. bility of the enzyme. Finally, the modification status of the
If chicken erythrocyte nuclei (which lack endogenous topo enzyme in embryonic extracts may differ from that in so-
II) are added to Xenopus extracts, mitotic chromosome con- matic cells, and this could affect the interaction of the en-
densation is observed (18). However, if topo II is removed zyme with chromosomes. Technical considerations could at
from the extract by immunoadsorption before addition of the least partly explain the differences between this and the
erythrocyte nuclei, then condensed chromosomes are not previous studies of topo II in mitotic chromosomes.
seen. Chromosome condensation can be restored by sup- A second recent study has suggested that only a subpopu-
plementing the depleted extract with purified S. pombe topo lation of topo II could serve a structural role in chromo-
II. These results indicate that topo II is required for chromo- somes. This study used elegant light microscope image anal-
some condensation in this in vitro system. Because of the ysis techniques to follow the cellular location of
stoichiometry of purified topo II needed to restore condensa- rhodamine-labeled topo II in living Drosophila embryos (22).
tion in the immunodepleted extracts, it was suggested that The authors found that topo II is clearly detectable in nuclei
the protein has both structural and enzymatic roles (18). and on chromosomes as they condense. However, the levels
Subsequent studies that used either naked DNA or nuclei of detectable topo II on the chromosomes unexpectedly
from Xenopus sperm as substrates for the in vitro condensa- dropped during mitosis, concomitant with an increase in the
tion reaction confirmed that topo II is involved in chromo- levels of topo II detected in the cytoplasm. This decrease oc-
some condensation in these extracts (19, 20). Treatment of curred in two stages, with about 30-60% of the enzyme leav-
the Xenopvs mitotic extracts with teniposide (VM26, which ing between prophase and metaphase, and a further 10%
inhibits the enzymatic function of topo II) or immunodeple- leaving by late telophase. Thus, it was concluded that 30%
tion with anti-topo II antibody severely inhibited chromo- of the topo II at most could serve a structural role. These
some condensation. results are in agreement with earlier studies that found
significant levels of topo II immunostaining in the cytoplasm
of mitotic Drosophila cells.
IS TOPOISOMERASE II A STRUCTURAL PROTEIN This finding does not, of course, rule out the possibility
IN MITOTIC CHROMOSOMES? that a portion of the chromosomal topo II does serve a struc-
tural role in these cells. As will be described later in this arti-
These foregoing studies provide concrete evidence for a role cle, topo II must remain on chromosomes at least until the
of topo II in mitotic chromosome condensation. Does this metaphase:anaphase transition, when it fulfills an essential
bolster the argument that chromosome scaffold components function in sister chromatid disjunction; exactly what per-
play an essential structural role in mitotic chromosomes? centage of the topo II is required for this purpose is not known.

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 I5V IL.WT

There may be differences in the role of topo II between it could reflect the ongoing process of chromosome conden-
Drosophila and vertebrates during mitosis. First, it must be sation that continues throughout metaphase.
borne in mind that these experiments used Drosophila synci-
tial early embryos, which are known to contain a maternal
pool of topo II in addition to the injected labeled molecules. THE METAPHASE CHECKPOINT
Studies have shown topo II to be highly concentrated in mi-
totic chromosomes of vertebrates (6, 21, 23), and quantitative It is conceptually useful to consider that the five phases of
studies with avian cultured cells showed that about 70% of mitosis form two distinct groups. The first group consists of
the chromosomal topo II in prometaphase cells is associated the preparations for chromatid segregation, including chro-
with the insoluble chromosome scaffold fraction. These con- mosome condensation, assembly of a bipolar mitotic spindle,
trasting findings may be explained by the fact that ver- and attachment of the chromosomes to this spindle via their
tebrates have two isoforms of topo II, whereas only a single kinetochores (prophase, prometaphase, and metaphase in
form of this protein has been detected in Drosophila. The the conventional terminology). It is now widely accepted that
170-kDa a-form is apparently bound to chromosomes except for certain early embryos, eukaryotic cells have a cell
throughout mitosis, whereas the 180-kDa fl-form is released cycle checkpoint mechanism to monitor completion of the
(26); thus, in Drosophila, that portion of topo II that fulfills the preparation stages, particularly the correct attachment and
role of topo II may be released from chromosomes in mitosis. alignment of the chromosomes on a bipolar spindle. The
Considering both the new and old experiments, the original metaphase checkpoint has been proposed to hold the cells in
suggestion that topo II plays a role in one or more aspects prometaphase/metaphase until the last chromosome has at-
of mitotic chromosome condensation and structure appears tained a bipolar orientation or until an underlying cell cycle
to best explain the bulk of current data, although the biologi- clock overrides the checkpoint delay. The metaphase check-
cal significance of the chromosome scaffold remains an area point appears to be sensitive to both the quantity of assem-
where further experiments are needed. bled 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
CHROMOSOMAL PREPARATIONS FOR ANAPHASE its biochemical basis.
The second overall stage of mitosis includes the active
The classical literature lists several requirements that must events of chromatid separation and movement, spindle move-
be accomplished by the chromosomes to prepare them for ments, and cytokinesis (anaphase, telophase, and cytokinesis
the orderly segregation that occurs during anaphase. Essen- in the conventional terminology). This “execution” stage ap-
tial preparations that occur during interphase include repli- pears to lack quality control circuitry, and thus is entirely de-
cation of the DNA molecules, their assembly into chromatin, pendent on preparations being completed correctly before it
and repair of any DNA damage incurred since the previous begins. Studies of yeast mutants and of microinjected mam-
mitosis. Upon entry into mitosis, the chromosomes must first malian cells reveal that when cells commence mitotic execu-
condense and then subsequently form correct bipolar attach- tion without properly completing the necessary preparations,
ments to the mitotic spindle (the latter is not discussed in this the resulting division is aberrant. If cytokinesis occurs, chro-
review). Recent experiments have added at least two more mosome damage or gross aneuploidy may ensue, often result-
requirements to this list. ing in cell death. If cytokinesis fails, polyploid progeny result.
First, chromosomes must assemble a functional kinetochore The monitoring of spindle architecture by the metaphase
that is capable of interacting with microtubules and directing checkpoint has been subjected to genetic analysis in the bud-
chromosome movements. Microinjection of IgG from au- ding yeast. Although yeast chromosomes have not been ob-
toimmune patients with antibodies to the CENP antigens served to form a metaphase plate, the fact that antimicrotu-
(27) during the G, and S phases of the cell cyde blocks as- bule drugs or mutant centromeres (32) produce a mitotic
sembly of a normal trilaminar kinetochore (28). Under these delay suggests that a metaphase checkpoint is an integral
circumstances, chromosomes can still bind to microtubules, aspect of mitosis in these organisms. Furthermore, centro-
but cannot move along them. Upon entry into mitosis, these mere alignment at a metaphase plate has recently been ob-
cells delay for many hours in prometaphase or metaphase served by in situ hybridization in the fission yeast (33). Two
(29). Some injected cells eventually do enter anaphase and groups have used genetic screens to identify putative compo-
undergo a highly abnormal completion of mitosis. More re- nents of the regulatory circuitry of this checkpoint by looking
cently, it has been shown that microinjection of specific anti- for mutants that fail to arrest in mitosis when microtubule
bodies recognizing the CENP-C protein alters kinetochore assembly is inhibited. These mutants have been termed
structure, reducing both size and stability (30). These in- either mad (mitotic arrest deficient) or bub (budding unin-
jected cells also show an extensive delay at metaphase before hibited by benzimadazole). Initial screening yielded three
embarking on an abnormal completion of mitotic events. mad and seven bub mutants (34, 35).
It is not surprising that chromosomes must assemble a Whether the mad and bub mutants affect the metaphase
functional kinetochore in order to move normally in mitosis. checkpoint directly, and if so, how they do it, is unknown. Of
However, a second, much more subtle, structural change oc- the 10 mad and bub mutants, two have been partially charac-
curs in chromosomes during metaphase. This change has terized on a molecular level. Although MAD2 was at first be-
been detected only with antibodies recognizing the INCENP lieved to encode an isoprenyl transferase (36), it was recently
proteins, a doublet of 135- and 155-kDa polypeptides that shown to correspond to a separate, adjacent open reading
were the first chromosome passengers to be identified (see frame (A. Murray, personal communication). The product
below). At the onset of chromosome condensation, these pro- of the BUBJ gene appears to encode an unusual 118-kDa pro-
teins are distributed along the entire chromosome. However, tein kinase. The protein contains a carboxyl-terminal kinase
during metaphase they gradually disappear from the chro- domain and a large amino-terminal domain of unknown
mosome arms and become concentrated at the centromere function. Its physiological substrates are unknown, although
region. The mechanism underlying this redistribution is not it is known to form a complex with Bub3 protein (A. Hoyt,
understood. It could be driven by specific motor proteins or personal communication).

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 REVIEWS
It has been suggested that as well as sensing the assembly maloriented chromosomes send a signal that inhibits mitotic
of a bipolar spindle, the metaphase checkpoint is also sensi- progression.
tive 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 EVENTS THAT OCCUR DURING SISTER
entry into anaphase for a significant time (37-39). Zirkle CHROMATID SEPARATION
(38), in a widely cited abstract, argued that this delay was
likely to be due to a signal transmitted by the kinetochores Separation (disjunction) of the sister chromatids is the land-
of the maloriented chromosome, as irradiation of the centro- mark event defining the onset of anaphase and the transition
mere region of such a chromosome with a UV microbeam between the preparatory and execution stages of mitosis.
appeared to abolish its inhibitory effect. One can imagine two classes of mechanisms that could bring
This notion, that a maloriented kinetochore sends an in- about the separation of sister chromatids. The first requires
hibitory signal, has never been tested directly. The best evi- some external force, such as the mitotic spindle, to pull the
dence for a direct involvement of kinetochores in the sisters apart: the metaphase:anaphase transition might be
metaphase checkpoint is provided by microinjection experi- triggered by changes in the ability of the chromosomes to
ments, where injection of anti-CENP-C antibodies produces resist the pull of the spindle, by the spindle’s ability to pull,
a substantial delay in metaphase. Chromosomes in the in- or both. In the second class, separation of the chromatids
jected cells are able to assemble kinetochores that, although results solely from changes intrinsic to chromosomes. Sur-
smaller than normal, are able to direct the positioning of the prisingly, the latter model has been shown to be correct. In
chromosomes at a metaphase plate (30). However, these a variety of cell types, synchronous disjunction of the sister
aligned chromosomes enter anaphase only after a lengthy de- chromatids can occur even in the absence of a mitotic spindle
lay. Additional evidence comes from genetic experiments, (42, 43). This observation indicates that the key to anaphase
where it has been shown that the presence of supernumerary chromatid disjunction is to be found in the chromosomes
chromosomes with mutant centromeres causes a prolonga- themselves.
tion of the G2/M period in Saccaromyces cerevisiae (32). Both The onset of anaphase has generally been assumed to oc-
observations could be explained by suggesting that the defec- cur subsequent to changes in the relative activities of the
tive kinetochores transmit a signal that activates the members of the mitotic kinase cascade (p342 and those
metaphase checkpoint and causes cells to delay entry into downstream kinases that it activates). In fact, compelling
anaphase. However, it cannot be ruled out that the check- evidence that changes in the kinase:phosphatase balance are
point is activated by a more subtle disruption of kinetochore- also essential for the onset of anaphase is provided by genetic
microtubule interactions resulting from physical damage to analyses in S. pombe, Aspergillus nidulans, and Drosophila
the kinetochore, without any need to invoke signaling by the melanogaster. In all these organisms, the presence of a func-
kinetochores. tional type I protein phosphatase is required for sister chro-
One particularly striking example of an instance where matid disjunction (44-48). In flies, activity of a type 2A pro-
unattached chromosomes fail to activate a metaphase check- tein phosphatase is also required (49, 50).
point is found in the case of sea urchin zygotes where fusion In a landmark advance, it has been possible recently to
of the maternal and paternal chromosome sets has been reconstitute the events of anaphase chromosome disjunction
prevented by colcemid treatment. Under these circum- in vitro. Addition of Ca2 to Xenopus egg extracts that are ar-
stances, only the paternal chromosome set forms any attach- rested in metaphase can trigger the onset of anaphase (51).
ment to a spindle (the centrosomes are contributed by the This anaphase appears to be characterized by sister chro-
sperm); the maternal chromosomes often remain unattached matid disjunction and poleward movement. Although it has
in the cytoplasm. These cells do have a functional metaphase not been proven formally that sisters disjoin faithfully in
checkpoint, as disruption of spindle assembly or structure these extracts, the available evidence argues strongly that the
causes a significant delay in the metaphase:anaphase transi- system does mimic the chromosomal events of anaphase in
tion. Despite this fact, the presence of as many as 20 mater- a cell-free environment.
nal chromosomes with unattached kinetochores has no effect Given the prevailing wisdom that the metaphase:anaphase
on the length of the timing of anaphase onset for the chromo- transition is triggered by changes in the kinase:phosphatase
somes of the paternal nucleus (40). balance, it was extremely surprising when studies using the
Until recently, there were no indications as to what the mitotic extracts revealed that sister chromatid disjunction
molecular basis for any signaling by the kinetochore might can occur in the presence of high levels of Hi kinase activity
be. This has now changed with the discovery of a phos- (52). In these experiments, it was found that addition of a
phoepitope that can be detected on the kinetochores of chro- nondegradable form of cyclin B to these extracts does not
mosomes that are either unattached or have not yet achieved abolish sister chromatid separation, even though Hi kinase
a stable bipolar orientation at the spindle midzone (41). This activity remains elevated. However, inhibition of cyclin pro-
phosphoepitope is undetectable on chromosomes once they teolysis, either with methylated ubiquitin or with a peptide
achieve a stable bipolar attachment to the spindle. These ob- corresponding to the “destruction box” on cyclin B (53), does
servations provide the first example of a possible biochemical inhibit sister chromatid separation in the extracts (52). The
difference between kinetochores that might be transmitting authors concluded that proteolysis of one (or more) molecule
an inhibitory signal and those that might not. In the future other than cyclin B is required for sister chromatid disjunc-
it will be extremely important to identify the proteins bear- tion. The target molecule (or molecules) for this proteolysis
ing this differential phosphorylation, and to determine could turn out to be related to the previously described CLiP
whether the differences reflect changes in the activity of a (54) or INCENP (55) polypeptides, or they may be novel
specific kinase or of its counterbalancing phosphatase. components.
There is a clear need for further experiments to explore Independent support for the idea that cyclin degradation
the workings of the metaphase checkpoint. In addition to is essential for the transition from mitosis to G,, but not for
characterizing the yeast mutants, it will be important to de- the initiation of sister chromatid separation, comes from an
sign conclusive tests of the notion that kinetochores of experiment in which nondegradable mutant cyclin B was ex-

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 REVIEWS
pressed in budding yeast. This leads to the retention of high rest (73, 74)], and Jlzzy [phenotype-metaphase arrest with a
HI kinase activity, but fails to prevent disjunction of sister few chromosomes escaping into anaphase (75)]. Some of the
chromatids (56). nine newly described genes whose mutant alleles confer cut
Even though it is attractive to think that sister chromatids phenotypes in fission yeast may also encode proteins with
are held together by linker molecules whose degradation sig- structural roles in this process (76).
nals the onset of anaphase, it cannot be excluded that the es-
sential substrate for proteolysis in the Xenopus extract experi-
ment is the endogenous cyclin B, which may be associated CHROMOSOME PASSENGERS REVEAL A NOVEL
preferentially with the mitotic spindles assembled in vitro. In FUNCTION OF THE CENTROMERE
vivo, only cyclin B that is bound to the spindle appears to be
degraded during mitotic progression of the Drosophila em- It has long been realized that the centromere region of the
bryo (57). Similarly, in mouse oocytes arrested in metaphase chromosomes carries out essential functions during mitosis.
I, cyclin B destruction depends on the presence of a spindle With recent advances in identification and mapping of the
(58). components involved in chromosome movement during mi-
The suggestion that labile protein linkers hold sister chro- tosis, this list of functions of the centromere has risen to three
matids together is the third explicit model that has been pro- (77).
posed to explain the regulation of sister chromatid pairing. 1) The centromere serves as the attachment point of the
Originally, it was thought that sister chromatids might be chromosomes to the mitotic spindle. Attachment occurs at a
held together by a short stretch of unreplicated DNA. This specialized buttonlike structure, the kinetochore.
appears to be ruled out by the observation that budding yeast 2) The centromere is the site of regulation of sister chro-
centromeres actually replicate early in S phase (59), and by matid pairing. At the metaphase:anaphase transition, some
the failure to detect centromere-associated DNA replication signal acts on the centromere to release the last point of sister
at the metaphase:anaphase transition. A second model sug- chromatid adhesion. As discussed before, current evidence
gested that sister chromatids might be held together by en- implicates both topo II activity and a protease in this release
tangled DNA loops (60). Such topological entanglements mechanism (52).
proved difficult to detect for plasmid minichromosomes in 3) The centromere is the location of at least some of the
mitotic yeast cells (61). However, the existence of mutations motors that move chromosomes during mitosis. This has
that reveal differences between the segregation of plasmid been demonstrated in vivo, where the chromosomes were
minichromosomes and bona fide chromosomes (62) again shown to move relative to spindle microtubules (78), and in
raises the possibility that such intertwinings might exist in many in vitro studies (79-82). In addition, several motor
full-sized chromosomes. If so, sister chromatid separation proteins have been localized to the centromere region
might require a type II topoisomerase to separate the entan- (among other locations) in fixed cells by indirect im-
gled loops. munofluorescence (83, 84).
In fact, early studies of the replication of SV4O virus (63) Studies of the chromosome passenger proteins have rev-
did implicate topo II in the separation of daughter DNA ealed a new and unexpected function for the centromere: as
molecules after DNA replication. These results were ex- a marshaling area or jumping-off point for the chromosome
tended by a genetic analysis in the yeasts, which confirmed passenger proteins on their way to the overlap zone of the
that topo II carries out an essential role in the separation of anaphase mitotic spindle. All chromosome passenger pro-
sister chromatids at anaphase (64-66). If cells attempt teins described to date concentrate at centromeres during
anaphase in the absence of functional enzyme, the centro- prometaphase. As a result, they move with the chromosomes
meres are able to move to the spindle poles, but the bulk of to the metaphase plate. What happens next is what defines
the chromatin remains entangled at the spindle equator and this class of proteins: they transfer from the chromosomes to
is entrapped in the cleavage furrow (33). This lethal pheno- the midzone of the spindle (Fig. 1). This departure occurs
type was referred to as cut (cell untimely torn) (66). by a number of routes. The INCENPs appear to be among
Several lines of evidence suggest that topo II also plays an the first to make their move. They leave the chromosomes
essential role in sister chromatid disjunction in higher eu- and accumulate along linear tracks transecting the
karyotes. For example, teniposide (VM-26), a potent topo II metaphase plate before there is any indication of sister chro-
inhibitor, blocks disjunction of sister chromatids in the Xeno- matid separation (85). CENP-E and CENPF/p330d appear
pus extract system (51). Similarly, microinjection of anti-topo to be among the last chromosomal passengers to leave the
II antibodies into Drosophila embryos appears to cause a local chromosomes, with the former separating during
disruption of anaphase chromatid disjunction (14). Finally, midanaphase (86) and the latter by late anaphase (87, 88).
three other inhibitors of topo II [etoposide (VP-16), m- Why do these proteins accumulate at centromeres before
AMSA, and ICRF-l93] were found to disrupt sister chro- their transfer to the spindle? So far there is no answer to this
matid separation in a variety of mammalian cultured cells question. One possibility is that the proteins must be carried
(67, 68), although this was disputed in another study where to the exact midline of the bipolar spindle in order to func-
lower concentrations of a variety of inhibitors were used (69). tion properly after their release from chromosomes. Chro-
Thus, except for the study last cited (i.e., ref 69) evidence mosome arms are often rather long, and may extend a
favoring a requirement for topo II in sister chromatid dis- significant distance laterally away from the central spindle.
junction appears to be unanimous. Only the centromeres are clustered in a tight disk or ring at
In addition to topo II and the (currently unknown) pro- the spindle midzone. In fact the centromere may be the only
teolysis targets, other candidates for proteins with direct portion of the chromosomes that lies wholly within the spin-
roles in sister chromatid disjunction also exist. Among them dle, where the concentration of microtubules is highest. The
are the products of the Drosophila genes rough deal [phenotype- notion that proximity to microtubules is important for the
lagging chromosomes and anaphase bridges (70)], lodestar transfer of the passenger proteins is supported by the obser-
[phenotype-chromosome tangling/anaphase bridges (71)], vation that these proteins remain associated with the centro-
l(1)zwlO [phenotype-premature sister separation in colchi- meres in cells where drug treatment has caused spindle dis-
cine, aneuploidy (72)], three rows [phenotype-metaphase ar- assembly (55, 87, 88).

952 Vol. 8 September 1994 The FASEB Journal EARNSHAW AND MACKAY
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 REVIEWS

Figure 1. The chromosome passenger proteins are both chromosomal and cytoskeletal components. The INCENP proteins (red) are
tightly associated with the chromosomes at the beginning of mitosis, but associate with microtubules (green) in the spindle midzone near
the metaphase:anaphase boundary. As anaphase and telophase progress, they concentrate in the stem body material. Chicken INCENP11
(red) is seen in transiently transfected mammalian LLC PK cells (93) in interphase (A), prophase (B), metaphase (C), anaphase (D), and
at the conclusion of telophase (E). When expressed at high levels in interphase cells, INCENP,, binds to and bundles cytoplasmic
microtubules (ii). DNA (stained with DAPI) is visualized in blue. Bar, 10 atm.

Immunoelectron microscopy of the INCENPs and microtubules of the central spindle. This material, known as
CENP-E in anaphase cells shows that these proteins are inti- stem body matrix, may be involved in the physical integra-
mately associated with an amorphous deposit of electron tion of the two half spindles. With one exception, all the
dense material that enshrouds the antiparallel interpolar identified matrix components are chromosome passenger

NONHISTONE PROTEINS IN MITOSIS 953

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 REVIEWS
proteins. The CHO-l antigen (89) recently renamed results). Unfortunately, this phenotype could result equally
MKLP-1 (mitosis-specific kinesinlike protein 1) (90) shows well from either a direct involvement of these proteins in the
no association with the nucleus during interphase or with structural events of sister chromatid separation or from in-
chromosomes during mitosis (90, 91), and is thus not a pas- volvement in one of the preparative processes that are sensed
senger protein. However, its distribution throughout by the metaphase checkpoint.
anaphase and telophase is identical to that described previ- One other dominant negative mutant of the INCENPs has
ously for the passenger proteins (concentration in the spindle also been constructed by deleting a small portion of the
midzone and association with the midbody in the intercellu- carboxyl-terminal domain (A. M. Mackay and W. C. Earn-
lar bridge during cytokinesis). shaw, unpublished results). Expression of this protein in cul-
How do the passenger proteins associate with the spindle? tured cells produces a variety of phenotypes, most of which
In the case of CENP-E, the largest known member of the su- appear to result from failures in cytokinesis. It is tempting
perfamily of kinesin-related proteins (92), cytoskeletal inter- to suggest that this may indicate a direct role for these pro-
actions may be essential. Biochemical experiments have teins in cytokinesis, a model consistent with the observation
shown that the protein can interact with microtubules in that the INCENPs are among the earliest proteins known to
vitro via its amino-terminal motor domain and also via a dis- concentrate at the site where the cleavage furrow will subse-
tal ATP-insensitive site (92). Thus, CENP-E is predicted to quently form (98). However, the lack of checkpoint controls
cross-link adjacent microtubules. during the execution stages of mitosis makes it difficult to as-
The INCENPs also appear to associate with cytoplasmic sign a direct role for the INCENPs in cytokinesis on the basis
microtubules, at least in transfected interphase cells that of these observations alone. As described earlier, failure of
greatly overexpress the proteins (93). It is not yet known cytokinesis is a common outcome when cells execute mitosis
whether this binding involves direct interaction between the after incomplete or defective preparations.
INCENPs and microtubules or whether it is mediated via In future experiments it will be important to identify the
one or more MAP (microtubule-associated protein). The 42 binding partners of the chromosome passenger proteins on
amino-terminal amino acids of the INCENPs are required chromosomes and on the spindle (and membrane, where ap-
for these proteins to transfer from the chromosomes to the plicable). This, as well as a characterization of the mechan-
spindle (93). It is surprising that this region of the protein isms by which these proteins change their binding allegiance
apparently is not required for recombinant INCENPs to as- at the metaphase:anaphase transition, should eventually lead
sociate with and bundle cytoplasmic microtubules during in- to a molecular understanding of the nature of the collabora-
terphase. Although it is not yet known whether this region tion between the chromosomes and the cytoskeleton during
corresponds to a spindle targeting signal or a chromosome mitotic execution.
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. Experi- CONCLUSIONS
ments in which mammalian cells are transiently transfected
In the 17 years since the initial presentation of the chromo-
with chicken INCENP constructs indicate that movement to
the centromere may be required for transfer to the spindle. some scaffold hypothesis, evidence has gradually accumu-
lated to suggest that chromosomal nonhistone proteins play
Deletion of the 42 amino-terminal amino acids of the IN-
a variety of roles in the chromosomal events of mitosis.
CENPs appears to prevent the concentration of the protein
Although certain aspects of the model remain controversial,
at centromeres (A. M. Mackay and W. C. Earnshaw, unpub-
lished results). by focusing attention on the chromosomal nonhistone pro-
teins, the model has clearly led to a significant enhancement
of our understanding of the biology of mitotic chromosomes.
FUNCTION OF THE CHROMOSOME PASSENGER Further advances in this area will no doubt continue to clar-
PROTEINS ify the role of the scaffold as additional nonhistone proteins
are cloned and characterized by cell biological and genetic
The protean redistribution of the chromosome passenger means.
proteins during mitosis has suggested that they could be in-
Work on mitosis from the authors’ laboratory has been funded by
volved in a variety of functions. These include: 1) regulation
of sister chromatid pairing; 2) structural integration of the National Institutes of Health grants GM30985 and GM35212. We
would like to acknowledge many colleagues for communicating
two half spindles; 3) motor function during spindle elonga-
results before publication, as well as Kip Sluder and our colleagues
tion (anaphase B); 4) stabilization of the plus ends of
M. Eckley, A. Pluta, J. Tomkiel, and C. Yang for comments on the
microtubules in the central spindle; 5) the earliest stages of
chromosome movement toward the spindle poles (anaphase manuscript. We apologize to those colleagues whose articles could
not be cited due to space limitations.
A); and 6) localization or assembly of the cleavage furrow.
Unfortunately, this list of possibilities is constrained all too
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