Mutagenesis vol. 26 no. 1 pp. 133–138, 2011
doi:10.1093/mutage/geq062
REVIEW
Micronucleus formation detected by live-cell imaging
Yun Huang, Michael Fenech1 and Qinghua Shi*
Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for
Physical Sciences at Microscale and Department of Biochemistry and Cell
Biology, School of Life Sciences, University of Science and Technology of
China, Hefei, Anhui 230027, People’s Republic of China and 1Commonwealth
Scientific and Industrial Research Organisation (CSIRO) Food and Nutritional
Sciences, Nutritional Genomics, Gate 13, Kintore Avenue, PO Box 10041,
Adelaide BC, South Australia 5000, Australia
*
To whom correspondence should be addressed. Laboratory of Molecular and
Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale
and Department of Biochemistry and Cell Biology, School of Life Sciences,
University of Science and Technology of China, Hefei, Anhui 230027,
People’s Republic of China; Tel: þ86 551 3600344; Fax: þ86 551 3600344;
Email: [email protected]
Received on May 30, 2010; revised on July 21, 2010;
accepted on August 27, 2010
Although micronuclei (MNi) have been extensively used to
evaluate genotoxic effects and chromosome instability, the
most basic issue regarding their formation was not
completely addressed until recently, due to limitations of
traditional experimental methods. The development of
live-cell imaging, combined with genetically engineered
chromosome labelling techniques makes it possible to
investigate the origin of a micronucleus in a single cell in
a real-time and high-throughput manner. Here, we review
all the available studies on the origins of MNi in live cells
and discuss novel findings based on this recently emerged
methodology. Some unsolved questions on MNi formation
and limitations of live-cell imaging in the investigation of
MNi have also been discussed.
Introduction
Micronuclei (MNi) have been extensively used as a cytogenetic
end point to evaluate genotoxic effects and chromosome
instability. The formation of MNi is attributed to a variety of
insults to genetic materials, which could be classified as
exogenous factors and endogenous factors (1). Exogenous
factors include radiation (2), chemical agents (3), microorganism invasion (4), etc. Endogenous factors include genetic
defects (5,6), pathological changes (7), deficiency of essential
nutritional ingredients (e.g. folic acid) (8) and injuries induced
by deleterious metabolic products (such as reactive oxygen
species) (9).
Although multiple factors have been demonstrated to increase
micronucleus (MN) formation in vivo and in vitro, the most
basic issue regarding the origins of MNi was not completely
addressed until recently, due to the limitations of traditional
experimental methods used. Based on the analysis of fixed
samples, it has been proposed that MNi could originate from
a variety of mechanisms, e.g. from displaced chromosomes
during metaphase (10) and from chromosomal fragments or
whole chromosomes that lag behind during anaphase/telophase
in bipolar (11–13) or multipolar nuclear divisions (14,15),
broken chromosomal bridges in anaphase/telophase (16,17) or
expelled nuclear buds during S phase (18). It is of great
importance to understand whether these putative mechanisms
that may explain the origin of MN really occur in live cells or
whether there are other possibilities and to what extent each
mechanism contributes to MN formation. A better understanding
of these processes will expand not only our knowledge on the
mechanisms of MN formation but also the characteristics of
damages caused by MN-inducing factors.
Development of live-cell imaging
Time-lapse imaging has proven to be an effective tool for
investigating cell migration, cell division, apoptosis, differentiation, organelle or chromosome dynamics, etc. (19,20). Live
observations on individual cells provide a unique opportunity
to track transient dynamic cellular processes that cannot be
detected in fixed specimens (19). More recently, the revolution
in development of fluorescent proteins spanning the entire
visible spectrum has resulted in the discovery of countless
dynamic intracellular events that can only be investigated by
observation over time (21). A typical modern live-cell imaging
set-up consists of the following parts: (i) an inverted fully
automated epifluorescent microscope with a high precision
programmable motorised stage; (ii) enclosed chamber systems
that mounts directly on top of a microscope stage or cage
incubator systems that encase a large part of the microscope,
which maintains adequate temperature, moisture and CO2
concentration to keep cultured cells in a physiologically normal
state during imaging; (iii) a high speed charge-coupled device
(CCD) for imaging capture; (iv) a computer for storing
the captured images and (v) computer programmes that
control the microscope illumination system, stage movement,
temperature and CCD.
It was shown that it is possible to use phase contrast live-cell
imaging to observe MN formation in multipolar mitosis of V79
cells (15). However, for better visualisation of chromosome
dynamics, DNA/chromatin labelling techniques have been
developed and tremendously improved. After staining with
DNA-binding fluorescent dyes, MNi could be visualised in live
cells under fluorescence illumination. For instance, after
staining with nucleic acid dye Syto-16, lagging chromosomes
(LCs) during mitosis have been shown in live cells to
contribute to MN formation and generation of aneuploid cells
in normal neural progenitor cells (22). However, exposure of
cells to excitation wavelengths [mostly in the ultraviolet (UV)
region] used to visualise fluorophores may generate reactive
oxygen species or cause other light-induced stresses, which
can be toxic to cells (19,20). Most DNA dyes may also
damage genetic materials (23). Such detrimental influences
were also suggested to cause MN formation, which could
Ó The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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lead to misinterpretation of phenotypes (19). Moreover,
photobleaching of fluorophores limits their usage in long-term
imaging studies. Besides, at some instance, it is hard to
distinguish MN from other extranuclear DNAs, e.g. mitochondrial DNAs, after nucleic acid dye staining. Fortunately, the
discovery of the green fluorescent protein (GFP) from the
jellyfish Aequorea victoria has dramatically facilitated the
development of live cell fluorescence imaging technique (24).
By expressing GFP-coupled proteins, cell structures could be
tracked at high resolution in real-time manner (21). For
example, by stably expressing a human histone 2B (H2B) and
GFP-fused gene in human cells, the chromosome dynamics
have been spatio-temporally tracked during cell division
(25,26). Overexpression of H2B-GFP has no detectable
deleterious effects on cell physiology (25). As MNi are
composed of chromosome fragments or whole chromosomes,
labelling histone H2B with GFP (or one of its colour variants)
makes it possible to scrutinise MN formation in live cells.
Using this labelling technique, the mechanisms of MN
formation have been explored in a variety of cell lines cultured
under different conditions (27–30), and very interesting results
have been published. Using novel genetically engineered cell
lines with quadruple fluorescent proteins specific to chromatin,
kinetochore, nuclear envelope and spindle or centrosome, it is
also feasible to obtain more detailed mechanistic information
regarding MN formation (31,32).
DCs in the equatorial plate have also been proposed to be the
first step leading to chromosome loss and MN formation after
examining the generation of MNi in V79 Chinese hamster cells
(12). In HeLa cells, of 465 MNi whose origin was traceable,
10.97% derived from DCs in metaphase (Figure 2).
It should be pointed out that very few DCs in metaphase
could form MNi in bipolar mitosis. Of 425 cells with DCs
in metaphase, only 37 or 8.71% were observed to form MNi in
Mechanisms of MN formation detected by live-cell imaging
To identify as many mechanisms by which MNi are originated
as possible, we tracked HeLa H2B-GFP cells for spontaneous
MN formation in a high-throughput manner (29). HeLa cells
were chosen for this study because they are expected to express
all the possible mechanisms of MN generation at a higher level
than normal cells due to their defects in cell cycle checkpoints
(33,34). Besides, HeLa cells can be tracked effectively due to
their good morphology and moderate movement when
visualised microscopically. During live-cell imaging, HeLa
H2B-GFP cells were grown on glass bottom dishes. Images
were automatically acquired at multiple locations on the dish
using a Nikon TE2000E inverted microscope fitted with a 20
Nikon Plan Fluor objective, a linearly encoded stage and
a Hamamatsu Orca-ER CCD camera. Fluorescence illumination was implemented by a mercury arc lamp with two neutral
density filters (for a total 32-fold reduction in intensity). The
microscope was controlled using Simple PCI software and
housed in a custom-designed 37°C chamber with a secondary
internal chamber that delivered humidified 5% CO2. Fluorescence and differential interference contrast images were
obtained every 18–22.5 min for a period of 68.6–89.1 h.
Autofocusing was performed every 90 min using the
fluorescence channel. Using this system, we have followed
.10 000 HeLa H2B-GFP cells for at least two cell cycle time
for the formation of MNi (29). Almost 500 MNi traceable by
live-cell imaging were analysed, and seven mechanisms by
which MNi formed have been identified as follows (Figure 1).
Mechanism 1 (M1)
MNi were derived from displaced chromosomes (DCs)
in metaphase. Chromosomes that abnormally aligned at metaphase plate were observed to lag behind and decondense to form
MNi in interphase daughter cells, while other chromosomes
segregated towards spindle poles during anaphase and telophase.
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Fig. 1. The mechanisms of MN formation in HeLa H2B-GFP cells detected by
live-cell imaging [from Rao et al. (29)]. Selected images from time-lapse
records show different mechanisms of MN origination. (M1) An MN
originates from a DC in metaphase; (M2) an MN originates from a LC
generated in anaphase of bipolar and multipolar (M2*) mitosis, respectively;
(M3) MNi originate from fragments of a broken CB; (M4) an MN originates
from the MN in mother cell; (M5) MNi originate from nuclear fragments
appeared during mitosis with CB; (M6) an MN originates from extruded
chromosomes that fused into one daughter cell; (M7) an MN appears after
chromosome clusters decondensed into interphase nuclei following an
apparently normal cell division. Chromosomal material is shown in green, and
time is presented as hour:minute at top-left. The insets show fluorescent
channel of corresponding area. The arrows point to DC, LC, CB, nuclear
fragments, extruded chromosomes and MN. The lower insets in the images
(M4–M7) show explanatory diagrams for each image; bar 5 10 lm.
Fig. 2. The contributions of different mechanisms to spontaneous MN
formation in HeLa cells [from Rao et al. (29)]. M1–M7: Mechanism 1–
Mechanism 7; N.D., not determined.
MN formation
resulting daughter cells. In the vast majority of mitoses, the DCs
disappeared either before anaphase onset (82.12% or 349/425)
or during anaphase (8.24% or 35/425) or decondensed to form
nuclei of similar size to the main nuclei (0.94% or 4/425).
Mechanism 2 (M2)
MNi derived from LCs during anaphase–telophase of bipolar
and multipolar divisions. Normally, when all the chromosomes
aligned at the metaphase plate, sister chromatids started to
separate and move towards spindle poles in twoclusters of
chromosomes. However, in some bipolar or multipolar mitoses,
a few chromosomes moved more slowly than other chromosomes or remained at the cell equator during anaphase–
telophase. When the chromosomes in the clusters decondensed
to form interphase daughter nuclei, a few LCs remained outside
of the chromosome clusters/nuclei and decondensed separately
to form MNi. MNi deriving from LCs have been observed in
PtK1, MRC5 and embryonic cortical cells (22,27) by using
time-lapse imaging. In HeLa cells, this mechanism is a major
contribution to spontaneously arising MNi, with 63.66% MNi
whose origin was traceable generated from this pathway
(Figure 2). However, it should be kept in mind that in bipolar
mitosis, most LCs did not form MNi in daughter cells. We
observed anaphase–telophase LCs in 610 bipolar mitoses, but
MNi formed in resulting daughter cells in only 164 or 26.88%
of such mitoses. In the remainder mitoses, LCs disappeared
either before (50.82%) or after (22.30%) the completion of
chromosome decondensation.
Mechanism 3 (M3)
MNi were derived from fragments of broken chromosomal
bridges (CBs). CBs were observed in some divisions from
anaphase to early interphase of daughter cells and are likely to
originate from dicentric chromosomes that have their centromeres pulled to opposite poles of the dividing cell. With cell
cycle progression, CBs could be broken by different mechanisms, and the fragments were observed to remain outside of
the daughter nuclei and consequently formed MNi. In our
previous study, 20% of 465 MNi whose origin was traceable
were found to originate from this mechanism (Figure 2).
Furthermore, in 33.33% (10/30) mitoses with single CBs, more
than one MNi were generated in daughter cells from pieces of
broken CBs. This is consistent with previous observations that
a CB could break at several sites and produce more than two
chromosomal fragments (28,35).
It should be mentioned that broken CBs evolving into MNi
in daughter cells was observed in only 12.66% of mitoses with
broken CBs in HeLa cells and 70.00% in human oral cancer
cells (28). The different cell lines used and the different number
of CBs examined [271 CBs in HeLa and 45 in oral cancer cells
(28)] may account for this variation. In most (207/237) mitoses
with broken CBs, the chromosomal fragments did not form
MNi in HeLa cells. They were observed to disappear either
immediately or a few frames after the CB broke. This is similar
to the phenomenon observed by a live-cell imaging study of
COLO320 cells in which the broken CBs shrunk towards the
closer nucleus very rapidly (36). Thus, quick shrinking of
broken CBs towards main nuclei may be responsible for the
low production of MNi in such mitoses in HeLa cells.
Mechanism 4 (M4)
MNi were inherited from mother cells. While chromosomes in
the main nucleus condensed, congregated and aligned at
metaphase plate from prophase to metaphase, the MN did not
join the metaphase plate and remained displaced in the
cytoplasm. When the chromosomes that aligned at metaphase
plate separated and segregated into daughter cells during
anaphase–telophase, the genetic material in MN did not
separate but moved, as a whole, with a cluster of chromosomes
into onedaughter cell and later decondensed separately to form
a new MN. In HeLa cells, of 465 MNi whose origin was
traceable, 3.01% originated via this pathway (Figure 2). This
finding can be useful in interpreting observations in traditional
MN assays, e.g. an MN with multiple X centromeric signals
may inherit from an MN in mother cell following multiple
rounds of DNA replication and cell divisions (37).
Mechanism 5 (M5)
MNi originated from the nuclear fragments that appeared
during mitosis with CBs. In this mechanism, some nuclear
material disjoined from segregated chromosome clusters
(namely nuclear fragments) during anaphase–telophase and
then become MN. They were not left behind as seen in M2 but
moved either ahead of or beside the normally segregating
chromosome clusters. Additionally, these nuclear fragments
were already present before CBs broke, making this mechanism different from M3. Of 465 MNi whose origin was
traceable by live-cell imaging, 3.01% derived through this
pathway in HeLa cells (Figure 2).
Mechanism 6 (M6)
MNi originated from chromosomes that were extruded from
a mitotic cell. DCs from metaphase plate were suddenly
extruded from the cell to form a minicell when cleavage furrow
formation started. Later, the minicell fused with onedaughter
cell, with the chromosomes in it decondensed separately to
form an MN. In HeLa cells, of 465 MNi whose origin was
traceable, 4.09% derived from chromosomes that were
extruded from a mitotic cell (Figure 2).
Mechanism 7 (M7)
MNi appeared after chromosomes decondensed to form
interphase nuclei following a normal mitosis. When performing
live-cell imaging using H2B-GFP HeLa cells, a cell was
observed to undergo mitosis without a detectable abnormality
and produced two daughter cells. While the chromosomes in
the daughter cells decondensed and formed interphase nuclei,
a GFP-positive dot appeared adjacent to the main nucleus in
cytoplasm. The position of the dot relative to the daughter
nucleus changed continuously, from attached to detached and
vice versa. Thus, the dot was identified as an MN, which also
existed temporarily and interchangeably as a nuclear bud.
Nuclear budding has been proposed as one of the mechanisms
for MN formation, based on the study of correlation between
the frequencies of nuclear buds and MNi in S phase of fixed
cell samples from cultures in which gene amplification was
induced (18). It was suggested that amplification of damaged
DNA in S phase results in nuclear budding, and buds could be
expelled from the nucleus and form MNi (18,38). However, we
did not observe such a mechanism in our live-cell imaging
studies because most (18/21) of the MNi that first appeared in
interphase appeared in G1, but not S phase. By live-cell
imaging of HeLa cells, the G1 phase was measured to last 9–19
h with the cell cycle duration being 19–29 h by Sakaue-Sawano
et al. (39). The average duration of the cell cycle in our HeLa
H2B-GFP cells, based on 975 normal mitoses, was 28.3 h
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Y. Huang et al.
(range 22.1–35.3 h), thus MNi first appearing within 10 h
after anaphase onset were thought to arise in G1 phase and not
likely to originate through nuclear budding in S phase. Of the
21 MNi that originated via this mechanism, 18 first appeared
between 2.6 and 10.1 h after anaphase onset and were thought
to form before commencement of S phase. In our study, 4.52%
of 465 MNi whose origin was traceable originated through this
mechanism (Figure 2).
Our real-time imaging analyses have shown that spontaneously arising MNi originate via multiple mechanisms in HeLa
cells, and the contribution of each mechanism to spontaneously
arising MNi is quite different (Figure 2). Of 465 MNi whose
origin was traceable, 43.66 and 20.00% originated from LCs in
bipolar and multipolar mitosis, respectively (M2), while
10.97% derived from DCs (M1) and 9.25% from broken
CBs (M3) in bipolar mitosis. MNi arising through the four
novel mechanisms (M4–M7) only accounted for 14.63% of the
overall MNi. This relative contribution of each MN formation
mechanism to spontaneously arising MNi is similar to that
observed after low concentration (150 lM) of hydroxyurea
(HU) treatment (30), where most of MNi in daughter cells
arose via mechanisms 1–3.
Obviously, long-term live-cell imaging has allowed us to
answer the following questions: (i) Did the cell undergo bipolar
or multipolar division? (ii) Were mono-, bi- or multi-nucleated
daughter cells generated from a specific division? (iii) Did the
daughter cells contain MNi, and/or if the MN was inherited
from the mother cell or generated in the observed mitosis? This
technique also enables us to identify and trace chromosome
abnormalities such as DCs, LCs and CBs during cell division
and determine in detail how the misplaced chromosomes
evolve into MNi. By using this technology, we and others
have not only confirmed the previously proposed mechanisms,
M1–M3 but also found four novel mechanisms, M4–M7, for
MN formation and found that these multiple mechanisms could
operate concurrently in HeLa cells in the formation of MNi.
Furthermore, this technique could illustrate which stage of
cell cycle and which cytoplasmic or nuclear structures might
be perturbed during MNi generation and allows further
understanding of the possible targets and mechanisms of
MNi-inducing agents. Data from live-cell imaging study are
very helpful to justify results obtained from MN assay using
fixed samples.
Unsolved questions
Although live-cell imaging has greatly furthered our understanding of MN formation, there are still many questions
left unanswered. There is also much room for improvement in
live-cell imaging and associated techniques when used for the
study of MN formation.
Firstly, although real-time imaging studies have shown that
most spontaneously arising MNi originate via DCs, LCs and
broken CBs in HeLa cells, whether an MN appearing in
daughter cell is exactly from a specific DC, LC or fragment of
broken CB observed during mitosis is a frequently asked
question during image analysis. This is mainly due to the
change in morphology and position of DCs or LCs in mitosis
and failure in tracing DCs or LCs continually during long-term
live-cell imaging.
Cytotoxicity of excitation light prohibits the continual
acquisition of images within a long period. It is known that
cells may be damaged with prolonged exposure to light of short
136
wavelength such as UV (19), and we have observed that
excitation wavelengths for GFP or red fluorescent protein are in
fact detrimental to cells in our experiments. Persistent exposure
of cells for .30 min could prolong cell cycle and increase cell
death (our unpublished results). More sensitive fluorescence
detection systems are required to allow use of lower light
intensity and thus minimise toxicity. Alternatively, photoxicity
may be minimised by using quenching agents in the culture
medium and reducing photosensitisers; for example, folate
concentration could be increased to quench the toxicity of
UV and riboflavin reduced to minimise its photosensitising
effects (40).
Performing live-cell imaging as usual on cells with a specific
chromosome rather than all chromosomes fluorescently
labelled could be one way to answer the question whether it
is the specific DC or LC that gives rise to the MN in daughter
cells, when the mother cell contains more than one DC or LC
but daughter cell has only one MN. Actually, segregation of
the single chromosome has been successfully tracked by
integrating bacterial lac operator repeats into a single
chromosome of cells expressing a lac repressor-GFP fusion
protein that associates with the repeats(41). However, the
occurrence of DCs at metaphase, LCs during anaphase–
telophase or MNi from a specific chromosome is very low,
thus high-throughput technologies in image acquisition and
analysis are required to obtain data of statistical power.
Secondly, in HeLa cells, untreated or treated with low-dose
HU, multiple mechanisms have been identified to contribute
MNi formation, and the contribution of each mechanism to
MNi formation is very different. Which mechanisms and how
much a specific mechanism contribute to MN formation in
other cell lines? Does the chromosome composition of cells
(e.g. diploid, tetraploid and aneuploid), genetic (e.g. functional,
deficient p53, etc.) and epigenetic modifications affect the
contribution of each MN formation mechanism and how?
Thus, further studies in other cell lines with different genetic
and epigenetic composition will be needed to verify whether
the observed mechanisms apply to all cell types.
Thirdly, there are three types of MNi, containing whole
chromosomes, acentric fragments or both whole chromosomes
and acentric fragments. The discrimination of these types of
MNi would be helpful to understand the property of damage in
cells and MNi-inducing reagents. MNi containing acentric
fragments are generated from mis-repair of chromosome breaks
(42) or from broken CBs; acentric chromosome fragments
become MNi because they lag during chromosome aggregation
to metaphase plate and/or during segregation towards spindle
poles during anaphase–telophase. However, these have not
been carefully examined in live cells because methods have not
yet been used to identify the presence of centromeres, which
would distinguish MNi originating from whole chromosomes
from those originating from acentric fragments.
Fourthly, the molecular basis and regulation underlying
each MN formation mechanism remain largely unknown. For
example, numerous studies have indicated that increased
MN formation is associated with the abnormalities in
microtubule assembly and disassembling, kinetochore structure
and function, attachment of microtubules to kinetochores, as
well as deregulation of cell cycle progression, etc. (1,27,43,44).
However, in a specific cell, which abnormality and how it
results in the formation of MN have not been explored.
Furthermore, differences between chromosomes may
occur with respect to their tendency to be involved in MN
MN formation
formation; for example, the inactive X chromosome in
females may have a greater tendency for generating MN than
the active X chromosome if the inactive chromosome is less
likely to assemble kinetochore due to its inactive chromatin
state (45).
Summary and the prospective
By using live-cell imaging and genetically engineering
chromosome labelling technique, the process of MN formation
has been carefully tracked in live cells. Three previously
proposed mechanisms of MN formation (M1–M3) have been
confirmed, and four novel pathways (M4–M7) have also been
observed. These mechanisms operate concurrently in HeLa
cells in the formation of spontaneously arising MNi and lowdose HU-induced MNi, with different relative contributions to
the overall MNi production. Many questions regarding MNi
formation remain unanswered. For instance, Do these mechanisms effect in other cell lines, and how? Does chromosome
ploidy as well as genetic and epigenetic modifications of
specific nuclear DNA (e.g. hypomethylation of alpha-satelllite
DNA) affect the contribution of each MN formation mechanism? Are there any difference in mechanism for the generation
of MNi containing whole chromosomes and those containing
acentric fragments? Technically, there is room for improvement in live-cell imaging when applied to the study of MNi
formation, e.g. fluorescently labelling a single chromosome
would allow one to trace it and answer if it displaced in
metaphase, lagged in anaphase–telophase and evolve into an
MN in resulting daughter cells. In addition, as indicated above,
it has now become feasible to use engineered cells that express
histone, kinetochore, spindle, centrosome and nuclear membrane proteins that fluoresce with different colours to obtain
detailed mechanistic information regarding MN formation.
Furthermore, these advances together with development of
three-dimensional image capture and a computer programme to
automatically analyse live-cell imaging movies for MNi
formation will be very helpful in facilitating data capture to
elucidate mechanisms by which endogenous or extraneous
factors cause MN.
Funding
National Natural Science Foundation of China (30671168,
30725013, 30711120571, 30721002); National High Technology
Research and Development Program of China (2006AA02Z4B4);
Doctoral Fund of Ministry of Education of China (20070358022).
Acknowledgements
Conflict of interest statement: None declared.
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