Plant Cell Physiol. 48(10): 1509–1513 (2007)
doi:10.1093/pcp/pcm117, available online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Short communication
Contribution of Anaphase B to Chromosome Separation in Higher Plant Cells
Estimated by Image Processing
Tomomi Hayashi 1, 3, Toshio Sano
and Seiichiro Hasezawa 1, *
1, 3
, Natsumaro Kutsuna 1, Fumi Kumagai-Sano
2
1
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa,
Chiba, 277-8562 Japan
2
Department of Science Education, Faculty of Education, Gunma University, Aramaki-cho 4-2, Maebashi, Gunma, 371-8510 Japan
Anaphase can be categorized into the two subphases
of anaphase A and B, but anaphase B has not been clearly
described in higher plant cells. In this study, we timesequentially followed the dynamics of chromosome segregation and spindle elongation in tobacco BY-2 cells using
histone–red fluorescent protein (RFP) and green fluorescent
protein (GFP)–tubulin, respectively. Construction of kymographs and determination of the positions of chromosomes
and spindle edges by image processing revealed that anaphase
B contributed to about 40% of the chromosome separation in
distance, which is comparable with that in animal cells. These
results suggest that higher plant cells potentially possess the
process of anaphase B.
Keywords: Anaphase — Fluorescent proteins — Image
processing.
Abbreviations: GFP, green fluorescent protein; GTHR,
GFP–tubulin–histone–RFP; MT, microtubule; SD-CLSM,
spinning disc-confocal laser scanning microscope; RFP, red
fluorescent protein; tdTomato, tandem-dimer Tomato.
Chromosome segregation is a fundamental process
of cell division. The stage of mitosis when chromosomes
separate in a eukaryotic cell is referred to as anaphase,
and this can be categorized into two subphases. The first
subphase, called anaphase A, is identified by the abrupt
movement of the daughter chromosomes towards the
spindle poles, driven by shortening of the kinetochore
microtubule (MTs), whereas the second phase, anaphase B,
consists of separation of the spindle poles themselves
(de Gramont and Cohen-Fix 2005, Mogliner et al. 2006).
In higher plant cells, however, bright-field observations
of dividing Allium guard mother cells and Tradescantia
stamen hair cells indicated that anaphase consisted almost
entirely of chromosome-to-pole motion, namely anaphase
A (Hepler and Palevitz 1986). Although in endosperm cells
of maize and T. americana substantial anaphase B was
observed, for a considerable period of time no clear
anaphase B was thought to exist in other plant species
(Baskin and Cande 1990). On the other hand, a recent dual
visualization of MTs and chromosomes in tobacco BY-2
cells using green fluorescent protein (GFP)–AtEB1 and the
vital dye, SYTO-82, implied the existence of anaphase B
in these cells (Dhonukshe et al. 2006).
To investigate the mode of daughter chromosome
movement and spindle MT dynamics, we established in this
study a transgenic tobacco BY-2 cell line in which we could
perform dual visualization of MTs and chromosomes in the
living cell system using GFP-labeled tubulin (Kumagai et al.
2001) and RFP-labeled histone. For the RFP, we used the
tandem-dimer Tomato (tdTomato; Shaner et al. 2004) that
has 3-fold the fluorescence quantum yield of the monomer
RFP1 (mRFP1; Campbell et al. 2002) and a fully mature
protein with 1.6-fold the brightness of DsRed (Shaner et al.
2004). The dual-visualized cells, designated BY-GTHR
(GFP–tubulin–histone–RFP), showed a similar growth
rate and mitosis progression by aphidicolin treatment to
the original BY-2 and BY-GT cells, although the peak of
mitotic index came 1 h later than that of the original cells
(Supplementary Fig. S1). Throughout cell cycle progression,
the dynamics of MTs and the cell nuclei/chromosomes
of the BY-GTHR cells could be simultaneously observed
(Fig. 1, Supplementary Fig. 2), and every statistical image
was comparable with those observed by immunocytological
analysis (Hasezawa et al. 1991).
In each living BY-GTHR cell, we monitored precisely
the dynamics of the mitotic spindle and daughter chromosomes during anaphase progression. By a combination
of dual visualization of cells and the use of a spinning
disc-confocal laser scanning microscope (SD-CLSM)
system, fluorescent images of MTs and chromosomes
could be obtained at 3 s intervals (Supplementary Fig. S3).
3
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, þ81-4-7136-3706.
1509
1510
Anaphase B in higher plant cells
A
B
C
0 min
90
15
95
25
100
30
110
55
120
Fig. 1 Dynamics of microtubules and cell nuclei/chromosomes
during cell cycle progression in BY-GTHR cells. (A) G1 phase.
(B) S phase. The green and red fluorescence show microtubules
and cell nuclei visualized by GFP–tubulin and histone-RFP,
respectively. (C) Time-sequential observations of the dynamics of
microtubules and chromosomes during mitosis. 0 min, G2 phase;
15 and 25 min, prophase; 30 min, prometaphase; 55 min, metaphase; 90 min, anaphase; 95 and 110 min, telophase; 120 min,
G, phase. Bar: 10 mm.
During anaphase progression, based on daughter chromosome movement, the mitotic spindle also appeared to
extend to both ends of the cells.
To evaluate the mode of daughter chromosome movement and MT dynamics, a kymograph was constructed
from the time-sequential images (Fig. 2). Since a simple
stacking of every time-sequential image resulted in a blurred
movie that was suboptimal to kymograph construction, the
orientation and position of the chromosomes were restored
in advance (see Materials and Methods). Subsequently,
the fluorescent intensities obtained from GFP–tubulin and
histone–RFP were averaged to the y-axis (see Supplementary Fig. S4) and these averaged images were then
time-sequentially stacked (Fig. 2A). From the obtained
kymograph, the positions of the separating daughter
chromosomes were extracted at points where the fluorescence emitted from histone–RFP showed the maximum
fluorescence intensities (Fig. 2B, points C1 and C2).
Similarly, the positions of the growing edges of the mitotic
spindle were determined where the GFP–tubulin fluorescence showed 50% of the maximum intensities (Fig. 2B,
points S1 and S2). Although the spindle length should be
characterized by observation of the spindle poles, since such
structures or confidential markers to characterize the
spindle poles have not yet been determined in plant cells,
we considered the half values as the growing edges of the
spindle in this study based on an image processing
technique. After determination of these positions, the
modes of chromosome movement (g) and spindle elongation (b) were estimated (Fig. 2C, D). At the onset of
anaphase, when the sister chromatids started to separate,
their movement towards both cell poles occurred at about
2.0 mm min1, which is consistent with the observations of
Dhonukshe et al. (2006). Subsequently, their movement
gradually slowed down so that anaphase finished within
3.8 0.77 min and the daughter chromosomes were separated by a distance of 12.2 2.4 mm. The elongation of the
mitotic spindle started soon after the onset of chromosome
separation, with a final elongation from 15.1 2.4 to
19.9 3.1 mm (Fig. 2D).
In anaphase, during which we evaluated the contributions of anaphase A and B, the daughter chromosomes
moved a distance of 3.7 1.1 and 2.4 0.61 mm from the
midplane by anaphase A and B, respectively (Fig. 3A). By
calculating the changes in contribution every 30 s, we found
that daughter chromosome movement in the early phase of
anaphase (0–1.0 min) was driven mainly by the shortening
of the kinetochore MTs, namely anaphase A (Fig. 3B).
Subsequently (1.0–3.0 min), the rate of spindle elongation
that separated the daughter chromosomes (anaphase B)
increased (Fig. 3B). In this context, Dhonukshe et al. (2006)
discussed biphasic kinetics of chromosome movement, with
the first faster phase to be the sum of anaphase A and B and
the second slower phase that resembles anaphase B.
Although our calculation partially supported the above
discussion of Dhonukshe et al. (2006), we unexpectedly
found that anaphase B started at the onset of chromosomal
separation and that anaphase A still continued even with
the increasing contribution of anaphase B.
In previously reported observations of Allium and
Tradescantia plant cells, the initial pole positions were
placed near the opposite ends of the cells, and thus pole
separation was thought to be spatially difficult (Hepler and
Palevitz 1986, Gunning and Steer 1996). In contrast, in
tobacco BY-2 cells, the mitotic spindle was formed in the
middle of the cells, and hence there appeared to be ample
Anaphase B in higher plant cells
x
A
space between the spindle poles and cell wall (Fig. 1C).
In fact, mitotic spindle elongation proceeded according to
daughter chromosome movement and 40% of the chromosomal movement in the tobacco BY-2 cells was found to be
dependent on anaphase B (Figs. 2D, 3A). Thus plant cells
appear potentially to possess the process of anaphase B,
but its contribution to chromosomal segregation may
depend on cellular architecture.
In budding and fission yeast, although sister chromatid
separation without spindle elongation occurred at the onset
of anaphase, chromosome segregation was largely accomplished by the movement of the spindle pole bodies, and the
contribution of anaphase B to chromosome segregation
reached 470% (Straight et al. 1997, Nabeshima et al. 1998,
Khodjakov et al. 2004). In contrast, that in animal and
plant cells, including tobacco BY-2 cells, was between 30
and 50%, but only about 10% in a Tradescantia stamen
hair cell (Supplementary Fig. S5; Hepler and Palevitz 1986,
Murray et al. 1996, Haraguchi et al. 1997, Desai et al. 1998,
Maddox et al. 2002, Cimini et al. 2006). Furthermore,
elongation of the mitotic spindle was 44-fold in a budding
yeast cell but only 1.1- to 1.5-fold in higher plant and
animal cells (Supplementary Fig. S5). Based on these
observations, it appears that anaphase B may dominate in
yeast cells which initially possess a short spindle that has
ample space to elongate, whereas anaphase A may dominate in plant and animal cells that form a large spindle in
which the chromosomes can move sufficiently. In particular, the large spindle of Tradescantia stamen hair cells may
limit the contribution of anaphase B to chromosome
separation in these cells (Gunning and Steer 1996).
The contribution of anaphase B in tobacco BY-2 cells
is found to be about 40%, comparable with that in animal
cells (Fig. 3A). The driving force of anaphase B has been
attributed to the sliding of overlap MTs in the midzone and
the pulling of the spindle to the cell cortex by astral MTs
(de Gramont and Cohen-Fix 2005). As bipolar kinesins are
known to be responsible for the sliding of these overlap
MTs (Sharp et al. 2000), plant MT motor proteins that have
not yet been characterized may assert a comparable sliding
force. In addition, although astral MTs have been rarely
q
p
t
b
B
g
a2
Intensity (A.U.)
a1
p
C
S1
C1
C2
q
S2
Distance from metaphase
chromosome position (mm)
12
S2
8
a2
4
C2
0
b
−4
g
−8
C1
a1
−12
S1
0
1
2
3
4
Time (min)
D
24
b
a1+a2
g
Distance (mm)
20
1511
16
12
8
4
0
0
1
2
3
4
Time (min)
Fig. 2 Evaluation of spindle elongation and daughter chromosome movement. (A) A kymograph constructed from fluorescent
images of GFP–tubulin and histone-RFP during anaphase progression. The images were taken at 3 s intervals and stacked over a
270 s period (90 images). (B) A fluorescent intensity profile
obtained from the line p–q in (A). The green and pink lines
represent fluorescent intensities of GFP–tubulin and H2B-RFP,
respectively. The edges of the spindle were defined as the positions
where the GFP fluorescence was half the value of each of the two
peaks (S1, S2). The positions of chromosomes were determined as
those where the RFP fluorescence gave the maximum fluorescent
intensities (C1, C2). From the intensity profile, the a1 and a2, b and
g values represent the distances between the daughter chromosomes and the spindle edges, the spindle length and the distance
between daughter chromosomes, respectively. (C) Changes in
positions of the spindle edges and chromosomes quantified from
the procedure in (B). The green and pink lines show the positions of
the spindle edges and chromosomes. (D) Changes in distance
between the daughter chromosomes and spindle edges (a1 þ a2),
the spindle length (b) and the distance between the daughter
chromosomes (g) calculated from (C).
1512
Anaphase B in higher plant cells
7
B
6
5
4
anaphase A
3
2
1
anaphase B
Distance of chromosome
movement at every 30 sec. (mm)
Distance of chromosome
movement in 4 min. (mm)
A
2
anaphase A
anaphase B
1.5
1
0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
Time after start of anaphase (min)
0
Fig. 3 Contribution of anaphase A and B to chromosome movement. (A) Contribution of anaphase A and B to the total distance of
chromosome movement. (B) Changes in contribution calculated from every 30 s interval. The values show the distances between
the midplane and chromosomes, and time 0 min represents the onset of anaphase. The data shows the mean values and SDs
of 15 independent cells.
mentioned in plant cells, since some aster-like polar MTs
were observed in GFP–EB1-transformed Arabidopsis suspension cells and tobacco BY-2 cells (Chan et al. 2005,
Dhonukshe et al. 2005), such MT structures may contribute
to the driving force of anaphase B in plant cells.
In this study, we have combined a dual visualization
system in living cells using fluorescent proteins with image
processing in order to clarify the existence of anaphase B in
tobacco BY-2 cells. Although the precise determination of
MT dynamics requires markers to monitor the minus ends
of MTs and kinetochores, the system developed here will
certainly serve as a powerful tool for analyzing MT motor
activities and kinetochore dynamics.
Materials and Methods
A tobacco BY-2 cell line (Nicotiana tabacum L. cv. Bright
Yellow 2) was maintained by weekly subculture in a modified
Linsmaier and Skoog medium supplemented with 2,4-D
(LSD medium), in which KH2PO4 and thiamine HCl were
increased to 370 and 1 mgl1, respectively. This basal medium
was supplemented with 3% sucrose and 0.2 mg l1 2,4-D, and then
adjusted to pH 5.8 before autoclaving (Nagata et al. 1992). The cell
suspension was cultured on a rotary shaker at 130 r.p.m. and
278C in the dark.
The tobacco BY-GTHR cell line was established by
transformation of the tobacco BY-GT cell line (Kumagai et al.
2001) with histone–RFP. The tobacco histone H2B cDNA was
PCR amplified from a tobacco BY-2 cDNA library using primers
designed from a corresponding expressed sequence tag sequence
deposited in the TAB (Transcriptome Analysis of BY-2) database
(clone No. 35066r1, http://mrg.psc.riken.jp/strc/index.htm). The
open reading frame region of the tobacco histone H2B and a
PCR-amplified tdTomato (Shaner et al. 2004), as an RFP, were
used to replace the GFP and fimbrin-ABD2 regions, respectively,
of the cauliflower mosaic virus 35S-sGFP (S65T)-FimABD2 vector
(Sano et al. 2005), resulting in an in-frame fusion of the N-terminus
of histone H2B and C-terminus of tdTomato. The resulting
histone–RFP region was first subcloned into the pENTR/D-TOPO
vector (Invitrogen, Carlsbad, CA, USA) and then into the pGWB2
binary vector (a gift from Dr. T. Nakagawa) using the Gateway
system (Invitrogen).
The histone–RFP vector construct was transformed into
Agrobacterium tumefaciens strain, LBA4404, and then into the
tobacco BY-GT16 cell line as described by Mayo et al. (2006).
For time-sequential observations, the BY-GTHR cells were
transferred into f35 mm Petri dishes with f14 mm coverslip
windows at the bottom (Matsunami Glass Ind. Ltd, Osaka,
Japan). The dishes were placed onto the inverted platform of
a fluorescence microscope equipped with an SD-CLSM system
(CSU 10, Yokogawa, Tokyo, Japan) and a cooled CCD camera
(Cool-SNAP HQ, PhotoMetrics, Huntington Beach, Canada).
To construct the kymographs, time-sequential images were
processed by our originally developed ‘KBI plug-ins’ running in
the ImageJ software. First, the orientation of chromosomes in
every time-sequential image was restored by an AutoHorizoner
plug-in. The positions of chromosomes were also restored using
a StackReg plug-in (Thévenaz et al. 1998, http://bigwww.epfl.
ch/publications/thevenaz9801.html). Subsequently, images of 100
pixel width horizontal to the midplane (the double arrowhead
in Supplementary Fig. S4A that was equivalent to 11.6 mm of the
objects) were averaged along the y-axis direction to one pixel in
height. Finally, the kymographs were constructed by stacking
the averaged images time-sequentially with a Kymograph plug-in.
The positions of the separating daughter chromosomes and the
growing edges of the mitotic spindle were extracted with a
HorizGetPeaks plug-in by determination of the positions described
in the text. The KBI plug-ins can be downloaded for free from
http://www.biol.s.u-tokyo.ac.jp/users/hasezawa/kbi/ij_plugins/
index.html.
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.oxford
journals.org.
Anaphase B in higher plant cells
Acknowledgments
We thank Dr. Tsuyoshi Nakagawa (Shimane University)
and Dr. Roger Y. Tsien (University of California at San Diego) for
the pGWB2 vector and tdTomato, respectively. This study was
financially supported in part by a Grant-in-Aid for Scientific
Research (Grant No. 18370015) and a Grant-in-Aid for Scientific
Research on Priority Areas (Grant No. 19039008) from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan, to S.H.
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(Received June 22, 2007; Accepted August 29, 2007)