664
Dynamics of chromosome positioning during the
cell cycle
Daniel Gerlich and Jan Ellenberg
The arrangement and dynamics of chromosomes inside the
nucleus of mammalian cells have been studied intensively
over the last two years. Although chromosomes are
relatively immobile and occupy non-random positions in
interphase, their dynamic movements in mitosis have
traditionally been assumed to randomize this arrangement.
New methods of live cell imaging now make it possible to
follow chromosome movements directly and quantitatively in
single cells. Such studies have generated models of
chromosome positioning throughout the cell cycle and
provide a new basis to address the underlying mechanisms
in future experiments.
Addresses
Gene Expression and Cell Biology/Biophysics Programmes, EMBL,
Meyerhofstrasse 1, D-69117 Heidelberg, Germany
e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:664–671
This review comes from a themed issue on
Cell division, growth and death
Edited by Jonathon Pines and Sally Kornbluth
0955-0674/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2003.10.014
Abbreviations
FISH Fluorescence in situ hybridization
GFP Green fluorescent protein
Introduction
The cell nucleus is a highly compartmentalized structure, where many nuclear proteins and RNAs localize
to distinct bodies [1–4]. Probably the main determinant
of nuclear organization is the spatial distribution of
chromosomes that occupy confined and mostly nonoverlapping volumes inside the boundaries set by the
nuclear envelope [5]. The spatial arrangement of chromosomes is non-random and follows certain rules that we
are only at the beginning to understand [6]. When higher
eukaryotic cells divide, most non-chromatin nuclear
structures are disassembled and chromosomes serve as
a template to rebuild the architecture of new interphase
nuclei in daughter cells. In this review, we discuss the
topology and dynamics of chromosomes in higher eukaryotic cells during interphase and mitosis. Different
models for non-random chromosome positioning are
presented and evaluated.
Current Opinion in Cell Biology 2003, 15:664–671
Dynamics and topology of the interphase
genome
Chromosomes are globally static and locally mobile
The eukaryotic genome is physically separated into distinct entities, the chromosomes, a feature that becomes
cytologically evident in their highly condensed state
during mitosis. However, even decondensed interphase
chromosomes are spatially separated in the nucleus,
which can be revealed when whole chromosomes are
specifically ‘painted’ by libraries of fluorescence in situ
hybridization (FISH) probes. Most of each chromosome’s DNA is confined to a distinct nuclear volume,
the chromosome territory, and only a minor fraction
extends into adjacent nuclear regions (reviewed in
[5]). The clear spatial separation and close proximity
of large chromosome territories suggest that their motion
is restricted in the nucleus. Indeed, early cytological
studies found that chromosome configurations are maintained throughout interphase in nematode nuclei [7].
These observations have recently been confirmed with
modern in vivo imaging techniques using single chromosome visualization in live cells [8,9] and photobleaching
techniques [10,11,12].
Despite the rather immobile organization of large-scale
structures such as whole chromosomes, chromatin is
highly dynamic on a smaller scale (reviewed in [13–
15]). Labeling of individual genomic loci in live cells
revealed that they randomly diffuse within confined
regions with a radius of 0.5 mm [16–18]. This radius
varies for different loci and depends on association with
the nuclear envelope or nucleoli [16,19]. Constraining
diffusion probably ensures the integrity of chromosome
territories during interphase [13]. Thus, even though
whole territories appear as static and compact structures,
they are constantly undergoing local Brownian motion
and are readily permeable to nuclear components up to a
size of 600 kDa [20–22].
Interphase chromosomes are arranged non-randomly
The existence of distinct chromosome territories has
raised the question of whether they are arranged randomly or in reproducible patterns in interphase nuclei. An
attractive hypothesis is that a defined spatial organization
of the genome could contribute to establishing and/or
maintaining expression states of genes [1,2,23,24]. Unfortunately, studies investigating the topology of chromosome territories have been controversial (e.g. [25–28]).
These studies analyzed chromosome positions in a population of fixed cultured cells using chromosome-specific
FISH paints. Although this approach lacks an absolute
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Dynamics of chromosome positioning during the cell cycle Gerlich and Ellenberg
coordinate reference, which makes statistical analysis
difficult, two non-random principles of chromosome positioning have been reproducibly observed (reviewed in
[6]). First, chromosomes were found to be arranged
radially. Gene-rich chromosomes locate predominantly
to the nuclear interior and gene-poor chromosomes to the
nuclear envelope [29–32,33,34]. Second, certain chromosomes have preferred direct neighbors. Pairs of chromosomes that undergo translocations typical for certain
cancer types were found in close spatial proximity both
in cancer and in normal cells [35,36]. Importantly, both
modes of chromosome positioning do not apply absolutely for a given chromosome in a single nucleus; rather,
they describe a high probability for a certain position and
significant fluctuations in chromosome positioning can be
found in a population of cells [6].
665
and independent behavior of mitotic chromosomes raised
the question of whether interphase chromosome arrangements can be transmitted over cell generations. This
question has been addressed in several studies using very
different approaches.
Clonal analysis using chromosome paints
For adherent cells grown isolated in culture, sister cell
relationships can be determined by spatial proximity in
clonal colonies of two, four and eight cells. Patterns of
chromosome positions can then be analyzed by FISH
paints after fixation. If chromosome arrangements were
transmitted to descendent cells, nuclei of the same clone
should be more similar to each other than to unrelated
cells. This was indeed found in postmitotic human sister
cells [12,25,37] (Figure 1a). However, similarity between
sister cells was not perfect and differences between
clonally descendent cells increased with successive cell
divisions [12] (Figure 1a).
Are chromosome arrangements passed on
from one cell generation to the next?
The stability of interphase chromosome positions is in
striking contrast to the mitotic situation. There, chromosomes are moved rapidly over long distances by the
mitotic spindle apparatus to segregate their chromatids
and form the two daughter nuclei. The highly dynamic
Differential labeling of parental genomes
An alternative approach to chromosome paints uses differential labeling of the two parental genomes before
fertilization of an egg by a sperm. Although this strategy
Figure 1
(a)
(b) Pronuclei
First metaphase
Two-cell embryo
Four-cell embryo
(c) Labeling boundary parallel to spindle axis
0 min
12
24
28
30
54
27.5
33.5
71.6
Labeling boundary perpendicular to spindle axis
0 min
6
24
Current Opinion in Cell Biology
Methods to address chromosome positioning in dividing cells. (a) 3-D reconstructions of chromosome painted nuclei in a four-cell clone of HeLa
cells. Chromosome 10 is displayed in red and chromosome 7 in green, and the nuclear boundary is white. High similarity of chromosome
arrangements is found between sister nuclei (compare n1 with n2 or n3 with n4), but the degree of similarity is lower between cousin nuclei (compare
upper and lower row). Adapted from [12] by copyright permission of the Rockefeller University Press. (b) Visualization of differential DNA
demethylation of paternal and maternal genomes by immunofluorescence. The maternal genome is hypermethylated from before pronuclear fusion
until the four-cell stage. Maternal (labeled with methyl-DNA — green) and paternal (only labeled with DNA counterstain — blue) chromosomes
are clustered in different nuclear halves. Reprinted from [39] with permission from Nature. Copyright 2000 Macmillan Magazines Ltd. (c) Live imaging
of labeled chromosomes in mitosis (reprinted from [11] with permission from Elsevier). A normal rat kidney cell expressing H2B–CFP (cyan
fluorescent protein) and H2B–YFP (yellow fluorescent protein) was bleach-labeled on one nuclear half in prophase (red denotes bleached regions,
green unbleached regions). A time series of 3-D images was graphically reconstructed (times in minutes shown bottom left corner of each image).
Two experiments with different geometries of the bleaching pattern are shown. Scale bars denote 10 mm.
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Current Opinion in Cell Biology 2003, 15:664–671
666 Cell division, growth and death
does not reveal the identity of each individual chromosome, it can visualize distinct patterns of labeled chromosome subsets. The paternal genome can be labeled by
feeding labeled nucleotide analogues to male mice such
that they are incorporated in replicating sperm DNA
during spermatogenesis. Mating of such mice with untreated females yields zygotes that carry both a labeled
and unlabeled copy of each chromosome, which has been
used to follow chromosome positioning during the first
embryonic cell divisions [38]. Similarly, visualization of
differential DNA demethylation of paternal and maternal
genomes by immunofluorescence can be used to follow
parental genomes during embryogenesis [39].
Immediately after fertilization, labeled chromosomes are
isolated in the male pronucleus, and even after fusion
with the unlabeled female pronucleus and congression to
the common metaphase plate of the first mitosis, paternal
and maternal chromosomes remain clearly separated
(Figure 1b). Importantly, spatial separation of parental
genomes was still observed in the daughter nuclei after
the first and second embryonic cell division [38,39]
(Figure 1b), demonstrating that chromosome positions
are not randomized during early embryonic mitosis. A
drawback of replication labeling of parental genomes is
the loss of the label in subsequent cell divisions that
occurs as a result of the incorporation of unlabeled
nucleotides, which makes comparison between cells
beyond the four-cell stage difficult [38]. This drawback
unfortunately also applies to the visualization of differential parental DNA demethylation [39].
Genome in situ hybridization in interspecific hybrids
To follow parental chromosome positioning beyond the
four-cell embryo, a more permanent mark is needed. This
can be achieved by generating hybrid zygotes from two
species that are different enough to selectively reveal
paternal and maternal genomes with species-specific
genome-wide FISH probes. Such genomic in situ hybridization (GISH) has been used to analyze the distribution
of parental genomes in embryonic and somatic cells in a
variety of plant and animal cells. These studies showed
that parental genomes are spatially separated in the
somatic cells of many plant hybrids (reviewed in [40])
and to some extent in hybrids derived from two mouse
species [38].
Together, these studies suggest that global patterns of
chromosome arrangements are transmitted to subsequent
cell generations to the extent that nuclei of direct descendents resemble each other more than unrelated cells.
However, although similar, the patterns are not identical
— some positional changes are observed, particularly
when cells were followed over more than two cell generations. This is in agreement with the concept of a
probabilistic order, where chromosomes occupy preferential but not strictly predetermined positions [6,35,36].
Current Opinion in Cell Biology 2003, 15:664–671
Tracking chromosomes through mitosis
Comparison of chromosome arrangements between
clones of fixed cells only provides indirect mechanistic
evidence for non-random chromosome positioning. The
complex dynamics of mitotic chromosomes can only be
addressed directly in live cells. GFP fusion proteins to
core histones are widely used to visualize chromatin
dynamics in live cells [41]. However, GFP-tagged histones do not allow the discrimination by light microscopy
of single chromosomes in interphase, or even during all
stages of mitosis, because of the high compaction of
chromosomes in metaphase and anaphase. To circumvent
this problem, recent studies have used pattern photobleaching of GFP–histone-labeled cells [11,12]. In this
approach, artificial landmarks on chromatin are created by
photobleaching selected nuclear regions. These bleached
patterns remain visible for several hours because of the
slow exchange of core histones on chromatin [10] and
bleached and unbleached chromosomes, especially if
enhanced with a non-bleached reference channel, can
therefore be followed through mitosis (Figure 1c) [11,12].
3D time-lapse imaging in live cells (i.e. 4D imaging,
reviewed in [42]) of bleach-labeled chromosomes confirmed that no major rearrangements of chromosome
positions occur during interphase [11,12]. More interestingly, when the arrangement of chromosomes in a
nucleus in which half of the chromosomes were
bleach-labeled in late G2 was followed through mitosis,
postmitotic daughter nuclei showed striking separation of
labeled from unlabeled chromosomes into two clusters,
suggesting a high degree of transmission of positional
order through mitosis [11] (Figure 1c). In a different
study, Walter et al. [12] left only a small peripheral
chromatin region fluorescent after bleaching most of
the nucleus. Again, in many daughter nuclei, fluorescent
chromosomes were found clustered in a small nuclear
sub-region. However, both studies also observed some
spatial rearrangements of chromosomes. In particular, the
labeling scheme used by Walter et al. was suitable to
report on significant local changes in direct chromosome
neighborhoods because of the small number of labeled
chromosomes. Together, these studies suggest that chromosomes move preferentially to positions in postmitotic
daughter nuclei that are similar, but not identical, to the
mother nucleus (discussed in [43–45]).
Models for mitotic chromosome positioning
Mitotic chromosome dynamics
During open mitosis in many higher eukaryotes, the
spatial order of the nucleus is reversibly broken down
and reconstituted in daughter cells after cell division.
To distribute the genetic information equally, it is essential to package the genome into very compact structures,
the metaphase chromosomes. Metaphase chromosomes
are shaped during condensation in prophase without
changing their positions relative to each other [11,46].
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Dynamics of chromosome positioning during the cell cycle Gerlich and Ellenberg
Metaphase chromosomes can then move as individual
units during mitosis. A first step in nuclear disassembly is
the breakdown of the nuclear envelope, which is facilitated by mechanical tearing in mammalian cells [47]. This
defines the onset of prometaphase. Chromosomes are
already condensed at this stage and become sequentially
attached to spindle microtubules at their kinetochores,
probably in a stochastic manner [48]. Chromosomes attached in a bipolar manner then congress towards the
central spindle, forming the metaphase plate. Once all
chromosomes are aligned, anaphase is initiated by degradation of the physical link between sister chromatids [49].
Sister chromatids move individually towards opposite
poles of the spindle to form daughter nuclei, which
become sealed by a newly formed nuclear envelope in
telophase. Subsequent isometric decondensation of chromatin fully reconstitutes interphase nuclei in daughter
cells [50,51]. Different models have been proposed to
relate these complex dynamics of individual chromosomes to the question of how the spatial arrangements
of all chromosomes in the nucleus can be transmitted
from one cell generation to the next.
Physical linkage between chromosomes/mitotic preset
On the basis of observations in FISH studies that showed
non-random positioning of chromosomes in metaphase
rosettes in human cells [26], it was suggested that physical
linkage between neighboring chromosomes during mito-
667
sis might help to preserve relative positions (Figure 2a).
Several observations are consistent with this model. First,
during chromatin condensation and decondensation, no
major relative positional changes occur [46,51]. Second,
during congression of chromosomes to the metaphase
plate, relative neighborhoods are still maintained
[11,52]. Third, symmetrical chromosome positions were
observed in postmitotic sister cells, suggesting a dependence on the metaphase configuration, the ‘mitotic preset’ [37]. Importantly, micromanipulation experiments
showed that chromosomes are indeed physically attached
to each other during metaphase [53]. The molecular basis
for such a physical link is unknown.
Physical linkage would provide an intuitive explanation
of how direct chromosomal neighborhoods could be preserved throughout mitosis. However, it is difficult to
imagine how the radial order of all chromosomes could
be transmitted via links, as the metaphase plate is essentially a flat structure and relative positions along the
spindle axis are thus lost in mitosis, allowing only a very
limited positional preset of a three-dimensional nucleus.
In addition, a recent study could not confirm a nonrandom metaphase rosette configuration [28].
Tethering of chromosomes by the nuclear envelope
A different model of chromosome positioning proposes
that relative chromosome positions are randomized during
Figure 2
(a)
Physical linkage
G2
(b)
Telophase
G1
Chromosome-specific interactions with nuclear envelope
G2
(c)
Metaphase
Metaphase
Telophase
G1
Chromsome-specific anaphase onset
G2
Metaphase
Telophase
G1
Current Opinion in Cell Biology
Models of mitotic chromosome positioning. (a) Physical linkage between chromosomes in interphase and mitosis for conservation of
neighborhood relations. (b) Mitotic chromosome dynamics randomize chromosome positioning. Chromosome-specific interactions with the nuclear
envelope then establish radial positioning during early G1. (c) Positional information along the spindle axis is lost after chromosomes have congressed
to the flat metaphase plate. Chromosome-specific anaphase onset re-establishes chromosome positions along this axis during early anaphase.
Positions along the metaphase plate are maintained by essentially linear congression and segregation (not shown).
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Current Opinion in Cell Biology 2003, 15:664–671
668 Cell division, growth and death
mitosis and reestablished in daughter nuclei by specific
interactions of some chromosomes with the reforming
nuclear envelope (Figure 2b). Support for this model
came from comparing quiescent cells and cells that had
reentered mitosis after quiescence [31]. Chromosomes
were found to occupy random radial positions in quiescent cells. Only after cells had passed through a mitotic
division was the typical peripheral localization of genepoor chromosomes observed. Heterochromatin proteins
that have been shown to interact with nuclear-membrane
proteins [54,55] might represent the molecular link that
tethers gene-poor chromosomes rich in heterochromatin
to peripheral positions when they contact the nuclear
envelope by random movement during early G1. However, attempts to validate this model in cells lacking one
such nuclear membrane protein have failed to detect the
expected changes in radial chromosome positions [32].
On the other hand, increased chromatin dynamics were
observed in live cells during early G1 [12], which could
reflect repositioning movements when the nuclear envelope reforms. However, during this stage in the cell cycle,
nuclei from adherent cells also flatten, because cells
reattach to the substrate, and grow as a result of DNA
decondensation. These events involve increased chromatin dynamics that do not contribute to positional
exchanges of chromosomes [11,50,51].
Although the model of chromosome-specific interactions
with the nuclear envelope can explain how a radial order
is established, it does not provide any explanation for the
observation of preferred neighborhoods for certain chromosomes, which should end up anywhere a given radial
distance from the center. Furthermore, it cannot explain
why chromosome arrangements in clonally descendent
cells resemble each other more than in unrelated cells
(see above).
Chromosome-specific timing of segregation
Live-cell imaging of bleach-labeled chromosomes suggested that non-random relative chromosome positions
are established during early anaphase [11] (Figure 2c).
Two nuclear halves differentially labeled by photobleaching were used to analyze chromosome positioning
relative to the mitotic spindle. Experiments in which the
labeling boundary was oriented parallel to the spindle axis
showed that positions along the plane of the metaphase
plate are largely maintained in daughter nuclei (Figure 1c,
top row). Given the essentially linear movements of
chromosomes along spindle microtubules during congression and segregation, this was not unexpected. By contrast, experiments with the labeling boundary oriented
perpendicular to the spindle axis demonstrated that
although spatial order along the spindle axis is lost during
congression to the flat metaphase plate, this order is reestablished during chromosome segregation, such that
postmitotic daughter nuclei were again similar to the
mother nucleus (Figure 1c, bottom row). Centromere
Current Opinion in Cell Biology 2003, 15:664–671
tracking during anaphase has shown that the timing of
the initiation of poleward chromosome movements correlates with the position along the spindle axis in the
daughter cell [11]. This leads to the model that chromosome-specific timing of segregation could determine their
positions along the spindle axis.
This model is supported by early studies on the timing of
centromere separation in metaphase spreads (reviewed in
[56]). There, chromosomes with large amounts of pericentromeric heterochromatin reproducibly separated late
in anaphase. In addition, this sequential separation of
centromeres could be perturbed by drugs that prevent the
formation of constitutive heterochromatin in both fixed
[57] and live-cell experiments [11]. Additional support
for the idea that the amount of pericentromeric heterochromatin could influence the timing of anaphase onset
came from experiments with artificial chromosomes in
yeast, in which increasing amounts of pericentromeric
heterochromatin delayed segregation [58].
In contrast to the two other models discussed above,
chromosome-specific timing of segregation is consistent
with both modes of non-random chromosome positioning
(i.e. both radial positioning and preferred neighborhoods).
This model predicts that interphase positions mainly
depend on centromeric composition rather than on chromosomal arm sequences. Importantly, this model does not
exclude the possibility of chromosome-specific interactions
with the nuclear envelope, which might further modify
postmitotic chromosome positioning during early G1.
Quantitative hypothesis testing with computer models
and simulations
Validation of any hypothetical mechanism of non-random
chromosome positioning requires a situation where it is
not active. This is not always easy to achieve experimentally, if, for example, several redundant protein–protein
interactions are involved, as could be the case for nuclear
envelope interactions. In this case, computer models and
simulations can be very helpful to predict chromosome
configurations under control and perturbed conditions in
silico. This approach was taken to generate reference data
for in vivo studies of chromosome positioning [11] and is
illustrated in Figure 3.
The simulations predicted transmission of chromosome
positions along the plane of the metaphase plate, whereas
positional information along the spindle axis should be
lost after congression, which would lead to randomized
chromosome positions along this axis in daughter nuclei.
Although the first prediction matched experimental observations, the second was in striking contrast. Because
of this, the model was modified by introducing chromosome-specific anaphase onset. Simulations with this new
model were in agreement with the experiments, which
demonstrated that such a property would in principle be
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Dynamics of chromosome positioning during the cell cycle Gerlich and Ellenberg
669
Figure 3
(a)
Interphase
(b)
Chromosome
territory
Congression
(c)
Segregation
Chromosome
FVolume
Kinetochore
exclusion
FNE Binding
FBrownian
FMT
FMT
FMT
Spindle pole
FMT
FNE Elastic repulsion
Plasma membrane
Nuclear
envelope
(d)
Current Opinion in Cell Biology
Computer model and simulations of mitotic chromosome dynamics. (a) Interphase chromosomes modeled as spheres that occupy non-overlapping
territories in the nucleus with sizes according to their DNA content [59]. Random chromosome movements occur by Brownian motion (FBrownian),
but are limited by exclusive volume interaction with neighboring chromosomes (FVolume exclusion) and interaction with the nuclear envelope
(FNE Elastic repulsion, FNE Binding). (b) Long-range dynamics after attachment of chromosomes to both spindle poles by microtubules. The
microtubule-dependent force (FMT) towards the more distant spindle pole is larger, leading to displacement towards the metaphase plate.
(c) After removal of cohesion between sister chromatids, they are pulled towards opposite spindle poles. (d) Simulation of mitotic chromosome
dynamics (reprinted from [11] with permission from Elsevier).
sufficient to explain preferred positioning along this axis.
Importantly, simulations with random anaphase onset
(equivalent to inactivity of the proposed mechanism)
could be compared with potential experimental perturbation of specific anaphase onset by induction of heterochromatin decondensation [11]. Computer models
would also be helpful to evaluate the other two models
of mitotic chromosome dynamics, assuming either chromosome-specific nuclear envelope attachments or physical linkage between chromosomes.
A second key task is to define the functional consequences of preferred chromosome positions. Correlation
between changes in gene expression state and nuclear
relocation has been described for several loci (reviewed in
[1,2,23,24]). Functional relevance is also suggested by the
observation of clustering of chromosomes that frequently
undergo tumor-specific translocations [35,36]. It will be
important to follow changes in chromosome positioning
during embryonic development and differentiation in live
cells and to interfere with potential specific chromosomal
rearrangements to assay their functional consequences.
Conclusions and future directions
Even though it is becoming clear that chromosomes
occupy preferred positions in the interphase nucleus,
we are still far from understanding how these patterns
are established and maintained in dividing cells. The
models for mitotic chromosome positioning discussed
here are still at a highly speculative stage and will have
to address the underlying molecular mechanisms. Quantitative assays of chromosome positioning in live cells
have now set the stage for thorough testing of the
required factors. This will probably include downregulation of genes by RNA interference, perturbation by
specific drugs or analysis of chromosomes with modified
sequence composition. Predictions from computer simulations will help us to evaluate any mechanism of chromosome positioning quantitatively.
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Acknowledgements
D. G. is supported by an EMBO longterm fellowship. J. E.
acknowledges support from the Human Frontiers Science Programme
(RGP0031/2001-M).
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