Download Plant-specific mitotic targeting of RanGAP

The Plant Journal (2005) 42, 270–282
doi: 10.1111/j.1365-313X.2005.02368.x
Plant-specific mitotic targeting of RanGAP requires
a functional WPP domain
Sun Yong Jeong1, Annkatrin Rose1, Jomon Joseph2, Mary Dasso2 and Iris Meier1,*
Plant Biotechnology Center and Department of Plant Cellular and Molecular Biology, The Ohio State University, Columbus, OH
43210, USA, and
2
Laboratory of Gene Regulation and Development, National Institute of Child Health and Development, Bethesda, MD 20892,
USA
1
Received 2 November 2004; revised 16 December 2004; accepted 14 January 2005.
*
For correspondence (fax 614 292 5379; e-mail [email protected]).
Summary
The small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, nuclear envelope (NE)
formation, and cell-cycle control. In vertebrates, these functions are controlled by a three-dimensional gradient
of Ran-GTP to Ran-GDP, established by the spatial separation of Ran GTPase-activating protein (RanGAP) and
the Ran guanine nucleotide exchange factor RCC1. While this spatial separation is established by the NE during
interphase, it is orchestrated during mitosis by association of RCC1 with the chromosomes and RanGAP with
the spindle and kinetochores. SUMOylation of vertebrate RanGAP1 is required for NE, spindle, and centromere
association. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate
RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for
its targeting to the NE in interphase. Here we show that the human and plant RanGAP-targeting domains are
kingdom specific. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate
RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary
and sufficient for this targeting. Point mutations in conserved residues of the WPP domain also abolish
targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in both
targeting events. These results indicate that plant and animal RanGAPs undergo different migration patterns
during cell division, which require their kingdom-specific targeting domains.
Keywords: RanGAP, Ran cycle, nuclear envelope, mitosis, cell plate, plant.
Introduction
Ran is a small GTPase that in vertebrates has functions in
nuclear transport, spindle formation, nuclear envelope
(NE) re-assembly, and cell-cycle control (Arnaoutov and
Dasso, 2003; Blow, 2003; Dasso, 2002; Li et al., 2003). RanGTP and Ran-GDP are interconverted by the activity of a
cytosolic Ran GTPase activating protein (RanGAP) and the
chromatin-bound nucleotide exchange factor RCC1. The
spatial distribution of Ran-GTP and Ran-GDP, established
by the spatial distribution of RanGAP and RCC1, is
important for the different functions of Ran. During interphase, nuclear Ran is predominantly GTP-bound due to
nuclear RCC1, and cytoplasmic Ran is predominantly GDPbound due to cytoplasmic RanGAP. This gradient across
the NE is functional in the directionality of nuclear import
and export, providing the information of compartment
270
identity for the appropriate loading and unloading of the
transport factors (Görlich et al., 2003, and references
therein).
During vertebrate mitosis, Ran-GTP in the vicinity of the
chromosomes displaces factors that promote microtubule
polymerization from the inhibitory effect of bound importins, suggestive of a promoting role of Ran-GTP in spindle
assembly (Gruss et al., 2001; Nachury et al., 2001; Wiese
et al., 2001). During telophase, both the local accumulation
of Ran-GTP and its hydrolysis have been implicated in the
assembly of the daughter NEs (Hetzer et al., 2000; Zhang and
Clarke, 2000). These findings indicate that the spatial positioning of RCC1 and RanGAP is equally important for the
functions of Ran in mitotic cells and that these functions rely
on the formation of a three-dimensional gradient between
ª 2005 Blackwell Publishing Ltd
Arabidopsis RanGAP1 at the cell plate 271
the two forms of Ran in the absence of a separating NE
(Kalab et al., 2002).
While vertebrate RCC1 remains chromatin-bound during
cell cycle (reviewed in Dasso, 2002), RanGAP migrates from
the NE to the vicinity of the spindle, with a sub-population
observed at the kinetochores (Joseph et al., 2002; Matunis
et al., 1996). In metazoans, RanGAP1 is conjugated with
SUMO-1, a small ubiquitin-like protein. SUMOylation is
required for NE association in interphase and for spindle
and centromere association in metaphase (Joseph et al.,
2002; Matunis et al., 1998). SUMOylated RanGAP1 binds to
the nucleoporin RanBP2/Nup358 (Matunis et al., 1998). In
metaphase cells, RanGAP1 and RanBP2/Nup358 co-localize
in the vicinity of the spindle and the kinetochores, suggesting that the same protein–protein interaction is required for targeting of RanGAP1 to its interphase and its
main mitotic location. RanGAP1 and RanBP2/Nup358 are
targeted as a single complex which is both regulated by
and essential for kinetochore–microtubule association
(Joseph et al., 2004).
Yeast homologs of RanGAP lack the C-terminal, SUMOylated targeting domain of mammalian RanGAPs and are
not associated with the NE (Melchior et al., 1993). In contrast
to higher animals and plants, the yeasts undergo closed
mitosis without breakdown of the NE. Therefore, a purely or
predominantly cytoplasmic location of yeast RanGAP would
theoretically suffice for its function in nuclear import and
export. Like vertebrate RanGAP – and unlike yeast RanGAP –
plant RanGAP is associated with the NE during interphase
(Rose and Meier, 2001). Arabidopsis RanGAP1 lacks the
SUMOylated C-terminal domain of vertebrate RanGAP, but
contains instead a plant-specific N-terminal domain, called
WPP domain after a highly conserved tryptophan–proline–
proline motif. The WPP domain is necessary and sufficient
for targeting to the plant NE (Rose and Meier, 2001).
Consistent with the presence of a different targeting domain,
the Arabidopsis genome appears to lack a homolog of
RanBP2/Nup358, suggesting that at least two modes exist
for the spatial organization of the Ran cycle in higher
eukaryotes.
Knowledge about the similarities and differences in the
mechanism of plant and animal RanGAP targeting (and, by
extension, establishment of the Ran gradient) will be crucial
to understand the degree of divergence between the two
kingdoms. Here we show that the human and Arabidopsis
RanGAP targeting domains are kingdom-specific. Unlike
animal RanGAP, Arabidopsis RanGAP1 appears at the
emerging cell plate in dividing plant cells and remains
associated with its growing rim. This plant-specific mitotic
targeting depends on the WPP domain and requires the
same amino acids as nuclear-rim association. It is tempting
to speculate that plants have acquired a unique targeting
domain for RanGAP in connection with its plant-specific
mitotic trafficking.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Results
Mammalian and plant RanGAP targeting signals are not
recognized in the heterologous system
The domains responsible for targeting mammalian and
plant RanGAP to the NE have no similarity, and there is
currently no evidence that the WPP domain is SUMOylated,
suggesting that mammalian and plant RanGAP targeting
mechanisms differ fundamentally. To rule out that an
unrecognized structural similarity exists between the two
domains that is recognized by a principally conserved
mechanism, we investigated whether the targeting signals
of mammalian and plant RanGAP can function in the
heterologous system.
HeLa cells were transfected with an AtRanGAP1-GFP
fusion construct (AtRanGAP1-GFP). Figure 1(a–c) shows that
the fusion protein does not accumulate at the HeLa cell NE.
When cells were co-transfected with AtRanGAP1-GFP and
human RanGAP1 fused to RFP (HsRanGAP1-RFP), and
subsequently permeabilized to diffuse soluble proteins from
the cytoplasm while retaining proteins tightly associated
with the nuclear membrane, AtRanGAP1-GFP is no longer
detectable, whereas the HsRanGAP1-RFP signal is retained
at the NE (Figure 1d–g). In a parallel experiment using
unpermeabilized cells, approximately equal amounts of
AtRanGAP1-GFP and HsRanGAP1-RFP fluorescence were
detected, demonstrating that both fusion proteins were
expressed (data not shown). These data indicate that
AtRanGAP1 has no signal for NE association in mammalian
cells.
To test whether HsRanGAP1 is targeted to the NE in plant
cells, GFP-HsRanGAP1 was transiently expressed in Arabidopsis protoplasts and tobacco BY-2 cells. GFP-HsRanGAP1,
which is targeted to the nuclear rim in HeLa cells (data not
shown), does not accumulate at the nuclear rim of either
plant cell type (Figure 1h–k). Together, these data indicate
that the WPP domain of AtRanGAP1 and the SUMO-accepting C-terminus of HsRanGAP1 are NE-targeting domains
only in their organismal context.
Specific point mutations in the WPP domain of AtRanGAP1
abolish NE targeting
Currently, only two types of proteins are known to contain
WPP domains, plant RanGAP and the plant NE-associated
protein MAF1 (Gindullis et al., 1999; Meier, 2000; Patel et al.,
2004). We took advantage of the much larger number of
available EST sequences for MAF1-like proteins than for
plant RanGAPs to identify highly conserved residues in the
WPP domain. Figure 2 shows an alignment of known MAFlike and plant RanGAP sequences. Several amino acids are
highly conserved among all sequences, including a lowerplant EST from the moss Physcomitrella patens. Mutating
272 Sun Yong Jeong et al.
(a)
Figure 1. Localization of Arabidopsis and human RanGAP1 in HeLa cells and plant cells.
Fusion constructs with GFP or RFP were transiently expressed under the control of the CMV
promoter (HeLa cells) or 35S promoter (plant
cells).
(a) AtRanGAP1-GFP in intact HeLa cell; (b) DNA
counterstained with DAPI; (c) overlay of (a) and
(b), bar equals 10 lm in a–c.
(d) AtRanGAP1-GFP in permeabilized HeLa cell;
(e) HsRanGAP1-RFP in permeabilized HeLa cell;
(f) DNA counterstained with DAPI; (g) overlay of
(d) through (f), bar equals 10 lm in d–g.
(h) AtRanGAP1-GFP in tobacco BY-2 cell; (i) GFPHsRanGAP1 in tobacco BY-2 cell, both (h) and (i)
counterstained for nucleic acids with SYTO 82
orange.
(j) AtRanGAP1-GFP in Arabidopsis protoplast; (k)
GFP-HsRanGAP1 in Arabidopsis protoplast. N,
nucleus. Bars, 10 lm.
(c)
(b)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
N
the conserved tryptophan–proline motif in AtRanGAP1
abolishes NE targeting in tobacco BY-2 cells (Rose and Meier, 2001). To investigate the role of other conserved amino
acid positions in NE targeting, a series of mutations were
introduced into the AtRanGAP1 WPP domain, replacing the
wild-type residues with alanine or, in case of alanine, with
serine (Figure 2).
Mutated proteins fused to GFP were tested for NE
targeting in transiently transformed Arabidopsis protoplasts
(Figure 3). Mutant mu2 (W18A, P19A) fails to concentrate
around the nuclear rim, in agreement with previous observations in tobacco BY-2 cells (Rose and Meier, 2001).
Mutants mu3 (T24A, R25A), mu6 (I61A, E62A), and mu7
(Y88A) also abolish NE targeting. In contrast, mu1, mu4,
mu5, mu8, mu9, mu11, and mu12 had no effect on NE
targeting, indicating that these residues, while conserved,
are not required for association of AtRanGAP1 with the NE.
AtRanGAP1 associates with the cell plate during mitosis
It has been previously shown that the tobacco antigens of an
anti-AtRanGAP1 antibody are localized in the area of the
spindle and phragmoplast microtubules in dividing cells
(Pay et al., 2002). We wished to determine more precisely
the subcellular location of AtRanGAP1 during the different
stages of the cell cycle. BY-2 cell lines stably expressing
AtRanGAP1-GFP were synchronized, counterstained with
SYTO 82 orange, and imaged by confocal microscopy. Stages of cell cycle were determined visually, using such
landmarks as chromosome condensation, chromosome
alignment in the metaphase plate, chromosome separation,
N
and reappearance of AtRanGAP1-GFP at the NE. Figure 4
shows a series of confocal images representing different
stages of cell division and comparing the localization pattern
of AtRanGAP1-GFP (Figure 4a) with that of free GFP (Figure 4b). In interphase cells, a strong association of AtRanGAP1-GFP with the NE was observed, while free GFP was
equally distributed between the cytoplasm and the nucleus.
During metaphase, a concentration in the area of the spindle
was observed for AtRanGAP1-GFP, consistent with the data
shown by Pay et al. (2002) (compare Figure 5a). However, a
similar concentration was also observed with free GFP,
indicating that a higher cytoplasmic density exists in the
area of the spindle, which might obscure a true spindle
association. At late anaphase/early telophase, a strong
association of AtRanGAP1-GFP with the position of the cell
plate was found. At that stage, free GFP was most prominent
in the area of the separating chromosomes and the cytoplasm between the chromosomes (compare Figure 4a,
panels g–i, and Figure 4b, panels g–i).
At late telophase/early cytokinesis, the most prominent
accumulation of AtRanGAP1-GFP was at the rim of the
growing cell plate (Figure 4a, panels j - l, and Video 1). At
the same stage, AtRanGAP1-GFP reappeared at the nuclear
rim, consistent with the reassembly of the NE. At comparable stages, free GFP appeared most concentrated at the
chromosomes and in the cytoplasm between the daughter
nuclei (Figure 4b, panels j–l). At the end of cytokinesis,
once the cell plate has fused with the plasma membrane,
the most prominent accumulation of AtRanGAP1-GFP was
again with the NEs of the daughter cells (Figure 4a, panels
m–o).
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Arabidopsis RanGAP1 at the cell plate 273
Figure 2. Sequence conservation of plant WPP domains and residues chosen for mutagenesis. From top: consensus strength given by height of bars, and
consensus sequence. Alignment of WPP domain sequences; gray shading indicates amino acids that match the consensus. Abbreviations for species names and
GenBank accession numbers are given at the end of each line. A.t., Arabidopsis thaliana, thale-cress; B.v., Beta vulgaris, sugar beet; C.a., Capsicum annuum,
jalapeno pepper; C.e., Canna edulis, arrowroot; G.a., Gossypium arboreum, cotton; G.m., Glycine max, soybean; H.c., Hedyotis centranthoides; H.v., Hordeum
vulgare, barley; I.n., Ipomoea nil, morning glory; L.e., Lycopersicon esculentum, tomato; L.s., Lactuca sativa, lettuce; L.j., Lotus japonicus, lotus; M.s., Medicago
sativa, alfalfa; M.t., Medicago trunculata, barrel medic; M.c., Mesembryanthemum crystallinum, ice plant; O.s., Oryza sativa, rice; P.b., Populus balsamifera, poplar;
P.p., Prunus persica, peach; S.t., Solanum tuberosum, potato; Z.m., Zea mays, maize; P. patens, Physcomitrella patens; a, see Meier (2000); b, see Gindullis et al.
(1999). Bottom: location of point mutations introduced in AtRanGAP1. , presence/absence of nuclear envelope targeting of mutant AtRanGAP1-GFP fusions.
Figure 5 shows images collected after fixing the AtRanGAP1-GFP-expressing, synchronized BY-2 cells. Under these
conditions, a larger number of cells at different mitotic
stages could be imaged. In addition, the cytoplasmic background of SYTO 82 orange staining due to association with
the organelles was strongly reduced. As early as anaphase, a
clear accumulation of signal was detected at the position of
the cell plate (Figure 5c). The signal grew outward consistent
with the location of the growing cell plate (Figure 5d–g).
Figure 5(f,g) shows initial accumulation of AtRanGAP1-GFP
around the decondensing chromatin (arrows). Figure 5(h)
shows a stage comparable to the live image in Figure 4(a,
panel j), with accumulation of the fusion protein at the
nuclear rim and the outer edges of the cell plate.
Figure 6 shows series of optical sections for 3-D reconstructions of the GFP signals in live cells at different
stages of cell plate formation. The early stage of cell plate
formation corresponding to Figure 4(a, panels g–i) shows a
disk-shaped AtRanGAP1-GFP localization (Figure 6a and
Video 2). Figure 6(b) (corresponding to Figure 4a, panels
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
j–l) shows the ring-shaped AtRanGAP1-GFP localization at
the later stage of cell plate formation (Video 3). This ringshaped localization pattern of AtRanGAP1-GFP at the late
stage of cell plate formation is indistinguishable from the
localization of the cell plate marker GFP-DRP2A (Hong et al.,
2003) at the same stage (Figure 6c and Video 4).
To confirm that the GFP fusion protein properly reflects
the localization of AtRanGAP1, an antibody was raised
against recombinant AtRanGAP1 and used in immunofluorescence experiments with root tips of 2–3-day-old
Arabidopsis seedlings. Figure 7(a) shows that the antibody recognized a single band of the correct size in a
total protein extract from WT Arabidopsis seedlings as
well as the AtRanGAP1-GFP fusion protein in an extract
from a BY-2 cell line expressing a 35S-AtRanGAP1-GFP
construct. Figure 7(b) shows double labeling with the
AtRanGAP1 antibody (FITC, green) and propidium iodide
(red) in an Arabidopsis root tip, demonstrating that
endogenous AtRanGAP1 is associated with the nuclear
rim in Arabidopsis root cells. Figure 7(c) demonstrates its
274 Sun Yong Jeong et al.
(a)
(b)
N
(c)
N
wt
(e)
N
mu1
mu2
(f)
(g)
Figure 3. Localization of Arabidopsis RanGAP1
mutants in Arabidopsis protoplasts. AtRanGAP1GFP with introduced point mutations was transiently expressed in Arabidopsis protoplasts and
imaged by confocal microscopy. wt, wildtype;
mu1–mu12, point mutations at the corresponding amino acid positions, as indicated in Figure 2. Optical sections corresponding to a central
plane through the nucleus are shown. N, nucleus. Bars, 10 lm.
(d)
N
mu3
(h)
N
N
N
mu4
mu5
(i)
(j)
N
mu6
(k)
N
mu7
N
(l)
N
N
mu8
mu9
mu11
mu12
green (Figure 7d,f) and red (Figure 7e,g) channels of a
single cell shown in Figure 7(d,e) and the cell shown in
Figure 7(c,f,g).
association with the cell plate of a dividing root cell,
consistent with the data obtained with the GFP fusion
protein. Figure 7(d–g) are separated images from the
(b)
(a)
GFP
SYTO
a
b
c
d
e
f
g
j
m
h
k
n
GFP
Merge
Merge
SYTO
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
i
l
o
Figure 4. Localization of AtRanGAP1-GFP during cell cycle in live tobacco BY-2 cells.
(a) Confocal images of BY-2 cells expressing AtRanGAP1-GFP.
(b) Confocal images of BY-2 cells expressing free GFP. GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain).
a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. For a Z-scan through dividing
cells expressing AtRanGAP1-GFP, see Video 1.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Arabidopsis RanGAP1 at the cell plate 275
Figure 5. Localization of AtRanGAP1-GFP during cell cycle in fixed tobacco
BY-2 cells.
(a, b) metaphase; (c) anaphase; (d, e) telophase; (f, g) late telophase, early
cytokinesis; (h) late cytokinesis. Green fluorescence, GFP; red fluorescence,
SYTO 82 orange nucleic acid stain. The arrows in (c) indicate the association
of AtRanGAP1-GFP with the position of the cell plate as early as anaphase.
The arrowheads in (f) and (g) point at the initial association of AtRanGAP1GFP with the re-forming nuclear envelope. The arrows in (h) label the outward
growing association of AtRanGAP1-GFP with the rim of the cell plate (see also
Figure 6) and the arrowheads in (h) indicate the association of AtRanGAP1GFP with the newly formed nuclear envelope.
The WPP domain is necessary and sufficient for mitotic
targeting of AtRanGAP1
SUMO-1 conjugation is not only essential for targeting
HsRanGAP1 to the nuclear pores, but is also required for the
association of HsRanGAP with mitotic spindles and kinetochores (Joseph et al., 2002). In addition, RanBP2 co-localizes
with HsRanGAP1 throughout the cell cycle, indicating that
the same mechanism is involved in targeting HsRanGAP1 in
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
interphase and during mitosis. Hence, we were interested to
determine whether the same (plant-specific) signals are
also required to target plant RanGAP1 to its interphase and
mitotic locations.
To test whether the WPP domain is involved in mitotic
targeting of AtRanGAP1-GFP, deletion constructs were
fused to GFP and their localization in stably transformed
BY-2 cells was investigated. It has been previously shown
that the N-terminal 115 amino acids of AtRanGAP1 are
necessary and sufficient for targeting to the NE (Rose and
Meier, 2001). Figure 8(a) shows that this GFP fusion protein
(AtRanGAP1DC-GFP) is robustly targeted to the nuclear rim
in interphase cells (panels a–c). During metaphase, fluorescence distribution was similar to AtRanGAP1-GFP (panels
d–f). During late anaphase/early telophase a strong association with the cell plate was observed (panels g–i), indistinguishable from the localization pattern of AtRanGAP1-GFP
(compare Figure 4). In late telophase/early cytokinesis
AtRanGAP1DC-GFP accumulates at the rim of the growing
cell plate and reappears at the rims of the daughter nuclei
(panels j–l). After the new cell plate has fused with the
plasma membrane, the strongest GFP fluorescence was
again associated with the NE (panels m–o). In contrast,
AtRanGAP1DN-GFP was not found specifically associated
with any cellular structure throughout the cell cycle (Figure 8b). Somewhat higher fluorescence intensity was
observed in the cytoplasm accumulated around the rim of
the growing cell plate in telophase (Figure 8b, panels g–i).
However, the same accumulation was observed for free
GFP, indicating a general abundance of cytoplasmic proteins
in this area (compare Figure 8b, panels j–l).
To confirm that the same localization signal also targets
AtRanGAP1-GFP to the NE and the cell plate in Arabidopsis
plants, transgenic lines were created which expressed either
AtRanGAP1-GFP or AtRanGAP1DC-GFP under the control of
the 35S promoter. Root tips from 2–3-day-old seedlings were
imaged for GFP fluorescence. Figure 8(c) shows that AtRanGAP1-GFP is targeted to the NE in interphase root cells and
to the cell plate in dividing root cells. AtRanGAP1DC-GFP
showed the same localization pattern as AtRanGAP1-GFP,
demonstrating that the WPP domain is sufficient for interphase and mitotic targeting in Arabidopsis plants as well
(Figure 8d).
Specific point mutations disrupt mitotic targeting of
AtRanGAP1
We wanted to investigate whether the same point mutations
leading to disruption of NE targeting would also disrupt cellplate association of AtRanGAP1. Three point mutations were
selected that had shown no association with the NE in
transient assays (mu3, mu6, and mu7, see Figure 2). All
three point mutations showed no specific targeting
throughout cell division, behaving indistinguishably from
276 Sun Yong Jeong et al.
Figure 6. AtRanGAP1-GFP concentrates at the
newly forming cell plate in dividing tobacco BY2 cells.
(a) Three-dimensional reconstruction of diskshaped AtRanGAP1-GFP localization at the early
stage of cell plate formation, rotated around the
vertical axis by 10 degrees for each image (for
rotation movie, see Video 2).
(b) Three-dimensional reconstruction of ringshaped AtRanGAP1-GFP localization at the later
stage of cell plate formation, rotated around the
horizontal axis by 10 degrees for each image (for
rotation movie, see Video 3).
(c) Three-dimensional reconstruction of cellplate marker GFP-DRP2A localization at the later
stage of cell plate formation, rotated around the
vertical axis by 10 for each image (for rotation
movie, see Video 4).
the WPP domain deletion mutant AtRanGAP1DC-GFP (Figure 9 and Figures S1 and S2). This indicates that the
threonine–arginine pair in position 24 and 25, the isoleucine–glutamate pair in positions 61 and 62, and the tyrosine
in position 88 are required for targeting AtRanGAP1 both to
the NE and the cell plate and suggests that – as in human
cells – a common mechanism is involved in these different
targeting events of plant RanGAP throughout the cell cycle.
Discussion
Diverse RanGAP targeting in different kingdoms
Based on localization and the presence or absence of targeting domains, at least three classes of eukaryotic RanGAPs can be divided. The simplest form is represented by
yeast RanGAP, which has no apparent signals for targeting
to the nuclear rim or to mitotic structures, and appears to
remain cytoplasmic throughout the cell cycle (Hopper et al.,
1990). Yeast RanGAP consists of a leucine-rich repeat and an
acidic domain, which are both required for RanGAP activity
(Hillig et al., 1999; Melchior et al., 1993). Both domains are
conserved in plant and vertebrate RanGAP, but are accompanied by kingdom-specific domains necessary to anchor
RanGAP to the outer surface of the NE. We have shown here
that the plant and mammalian RanGAP targeting domains
are not functional in the heterologous cell system. No consensus sequence for SUMOylation is present on the WPP
domain and no higher molecular weight isoforms of
AtRanGAP1 have been observed in immunoblots (e.g., Figure 7a), suggesting that, unlike for human RanGAP, SUMOylation is probably not involved in subcellular targeting of
plant RanGAP. Together with the apparent lack of a plant
RanBP2 homolog, this is consistent with the hypothesis that
RanGAP is targeted by a different mechanism to the nuclear
rim in plants.
Plant RanGAP has specific mitotic locations
There are a number of differences between animal and
plant mitosis, one of the most prominent being the way in
which the new membrane division between the two
daughter cells is established. Cytokinesis in plants does
not involve contraction and fusion of a cleavage furrow.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Arabidopsis RanGAP1 at the cell plate 277
Figure 7. Immunofluorescence microscopy of
Arabidopsis root tip cells.
(a) The AtRanGAP1 antibody detects a single
band of ca. 60 kDa in a total protein extract from
Arabidopsis seedlings (lane 1, arrow) and a
single band of ca. 80 kDa in a total protein extract
of the AtRanGAP1-GFP BY-2 cell line (lane 2,
arrow). Asterisks mark a weak unspecific signal
seen in all plant protein extracts tested (lanes 1
and 2 and data not shown).
(b) The AtRanGAP1 antibody decorates the nuclear envelope in root tip cells. Green fluorescence, FITC; red fluorescence, propidium iodide
counterstaining of nuclei.
(c) The AtRanGAP1 antibody decorates a newly
forming cell plate in an Arabidopsis root tip cell.
Green, FITC; red, propidium iodide.
(d, e) Grayscale images of split channels of a
single nucleus shown in (b).
(d) green channel; (e) red channel. (f, g) Grayscale images of split channels of the dividing cell
shown in (c). (f) Green channel; (g) red channel.
Arrowheads in (c) and (f) indicate the cell plate.
Instead, a new double membrane is assembled from vesicles, which migrate along the microtubules of the
phragmoplast toward the equatorial plane where they fuse
to form the cell plate, which grows outward to eventually
fuse with the plasma membrane (Bednarek and Falbel,
2002; Hong et al., 2001).
Like mammalian RanGAP, plant RanGAP goes through a
specific pattern of re-localization during cell cycle. Tobacco
RanGAP is localized in the area of the spindle and the
phragmoplast in dividing cells (Pay et al., 2002). We have
shown here in addition that AtRanGAP1 is targeted to the
cell plate during cytokinesis and that the WPP domain is
necessary and sufficient for this targeting. During metaphase, we have observed an accumulation of AtRanGAP1GFP in the vicinity of the spindle as well (e.g., see Figure 4a,
panel d–f and Figure 5a–c). However, our assay makes it
difficult to distinguish between true spindle association and
accumulation of the fusion protein in the dense cytoplasm
that surrounds the spindle because free GFP also accumulates in this area of the cell (Figure 4b, panels d–f). We
therefore consider it is likely that AtRanGAP1 is associated
both with the vicinity of the spindle in metaphase as
reported by Pay et al. (2002) and with the cell plate during
cytokinesis as described here. Indeed, performing a microtubule precipitation experiment as described by Pay et al.
(2002), we also found that AtRanGAP1-GFP is found in the
pellet. Interestingly, the point mutant mu3, which disrupted
association of AtRanGAP1-GFP with the NE and the cell
plate, did not alter the association of the fusion protein with
the microtubule pellet. In contrast, deletion of the C-terminus of AtRanGAP1 made the remaining WPP-domain-GFP
fusion protein (AtRanGAP1DC-GFP) partition with the supernatant (data not shown). These findings suggest that the
association of plant RanGAP with microtubules might be a
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
feature that is independent of its targeting to the membrane
structures.
Association of AtRanGAP1 with the cell plate was
observed as early as anaphase (Figure 5). This is consistent
with the earliest accumulation of vesicles in the area of the
future cell plate (Segui-Simarro et al., 2004). Formation of
the cell plate involves the fusion of vesicles, which starts at
the center of the cell plate and expands centrifugally
outward as the area of the cell plate enlarges. During later
stages of cell plate growth, microtubules depolymerize at
the center of the cell plate and new ones form around the
margins where new vesicles are delivered (Segui-Simarro
et al., 2004). The localization of RanGAP1 resembles the area
of the cell plate that undergoes vesicle fusion and closely
resembles that of dynamin. Strikingly, tomato MAF1, a
representative of the only other plant protein family containing a WPP domain, also migrates from the NE to the cell
plate during cytokinesis (Patel et al., 2004). The Arabidopsis
homologs of MAF1, WPP1 and WPP2 are also associated
with the NE, indicating that the WPP domain provides
information for both NE and cell plate localization in plants.
Vertebrate RanGAP is required for the fusion of the
membrane vesicles that assemble on the decondensing
chromatin and form the new NEs (Hetzer et al., 2000; Zhang
and Clarke, 2000; Zhang et al., 2002). While nothing is
currently known about mitotic roles of the Ran cycle in
plants, it is interesting to note that the mitotic position of
AtRanGAP1 at the growing edge of the cell plate also
correlates with a membrane fusion event. There are clearly
differences in mitotic membrane trafficking between the
kingdoms. Plant Golgi stacks do not vesiculate and the
process of secretion and vesicle transport remains active
during plant mitosis (Bednarek and Falbel, 2002; Nebenführ
et al., 2000). Both Golgi vesicles and tubular ER components
278 Sun Yong Jeong et al.
(a)
(b)
GFP
SYTO
a
b
c
d
e
f
g
h
i
j
m
k
n
GFP
Merge
SYTO
Merge
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
l
o
(c)
(d)
Figure 8. The WPP domain is necessary and sufficient for AtRanGAP1-GFP localization during cell cycle.
(a) Confocal images of BY-2 cells expressing AtRanGAP1DC-GFP (WPP domain).
(b) Confocal images of BY-2 cells expressing AtRanGAP1DN-GFP (WPP domain deletion). GFP, green channel; SYTO, red channel (SYTO 82 orange nucleic acid
stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm.
(c) AtRanGAP1-GFP localization in primary root tip cells of transgenic Arabidopsis plants. Arrows indicate the cell plate of a dividing cell.
(d) AtRanGAP1DC-GFP localization in primary root tips of transgenic Arabidopsis plants. Arrows indicate the cell plate of a newly divided cell.
are recruited to the division plane during mitosis and
cytokinesis (Cutler and Ehrhardt, 2002). Members of the
Arabidopsis Rab family have demonstrated roles in intracellular membrane trafficking and it was hypothesized that
Rab GTPases would, in conjunction with SNARE proteins,
provide specificity for membrane fusion events (for reviews,
see Stenmark and Olkkonen, 2001; Zerial and McBride,
2001). The data presented here provide an opening into
investigating whether the Ran cycle, too, has a role in the
formation of the cell plate.
The WPP domain is required for mitotic RanGAP targeting in
plants
We have proposed previously that both in vertebrates and
plants the subcellular targeting of RanGAP is more relevant
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Arabidopsis RanGAP1 at the cell plate 279
GFP
SYTO
Merge
a
b
c
d
e
f
membrane re-organization events discussed here (NE breakdown and reformation; phragmoplast-type cell plate formation) occur together in many algal taxa that are basal to the
lineage of the land plants, such as Coleochaetales (Marchant
and Pickett-Heaps, 1973), Charales (Pickett-Heaps, 1967), and
Zygnematales (Fowke and Pickett-Heaps, 1969a,b; LopezBautista et al., 2003). Therefore, an unrecognized mechanistic connection might exist in plants between these two
membrane rearrangement events.
Experimental procedures
g
h
i
j
k
l
m
n
o
Figure 9. Point mutations in AtRanGAP1-GFP abolish targeting during cell
cycle. Confocal images of BY-2 cells expressing AtRanGAP1-GFP with a point
mutation at position 3 (see Figure 2 and Experimental procedures). GFP,
green channel; SYTO, red channel (SYTO 82 orange nucleic acid stain).
a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l,
cytokinesis; m–o, after completion of cytokinesis. Bars, 10 lm. For corresponding Figures of AtRanGAP1-GFP with point mutation at positions 6 and 7,
see Figures S1 and S2.
for its function in mitosis than for its association with the
nuclear pore in interphase (Joseph et al., 2002; Rose and
Meier, 2001). This is based on the correlation of specific
RanGAP targeting domains with the occurrence of open
mitosis in higher eukaryotes. SUMO modification appears to
be confined to metazoan RanGAP and is involved in the
process that spatially regulates the RanGAP position during
the cell cycle (Joseph et al., 2002). We have shown here that
the different subcellular addresses of plant RanGAP during
interphase and cell cycle also require the same signal sequence. However, this signal fundamentally differs from the
one utilized by vertebrate RanGAP.
The use of a different signal correlates with the unique,
plant-specific positioning during cytokinesis at the cell plate.
It is conceivable that the specific targeting mechanism of
higher plant RanGAP has evolved in parallel with the
occurrence of phragmoplast-type open mitosis. Plant and
animal RanGAP might therefore have acquired different
targeting domains binding to different protein interaction
partners suitable for their subcellular trafficking ‘needs.’
Consistent with this hypothesis we have identified a colocalizing interaction partner of the WPP domain, which is a
protein unique to plants (unpublished data). Indeed, the two
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Constructs
pRTL-AtRanGAP1-GFP (Rose and Meier, 2001) and pDsRed-hRanGAP1 (Joseph et al., 2002) were described previously. For cloning of
the AtRanGAP1 coding sequence into a mammalian expression
vector, the NcoI fragment was filled in and ligated into the SmaI site
of pEGFP-N2 (Clontech, Palo Alto, CA, USA) to obtain pEGFPAtRanGAP1. This provides expression of AtRanGAP1 as a fusion
protein with GFP at its C-terminus. For cloning of the HsRanGAP1
coding sequence into a plant expression vector, the coding sequence
was PCR-amplified using the primers 5¢-GAAGATCTATGGCCTCGGAAGACATTG-3¢ and 5¢-GAAGATCTGACCTTGTACAGCGTCTGC3¢ and cloned into the BglII site of pRTL2-mGFPS65T (von Arnim
et al., 1998) using the internal BglII sites of the PCR primers to obtain
pRTL-HsRanGAP1-GFP. This provides expression of HsRanGAP1 as
a fusion protein with GFP at its N-terminus. For stable BY-2 cell
transformation, the fragments of AtRanGAP1-GFP, AtRanGAP1DCGFP, AtRanGAP1DN-GFP (Rose and Meier, 2001), AtRanGAP1 (mu3,
mu6 and mu7)-GFP (see below) and free GFP (mGFPS65T) were
digested with XhoI and partially with XbaI (RanGAP1 has an internal
XbaI site) and cloned into XhoI and SpeI in the binary vector
pFGC1008 (http://www.arabidopsis.org). The same constructs were
used to transform Arabidopsis plants.
Site-directed mutagenesis
Point mutations were introduced into AtRanGAP1-GFP as described
previously (Rose and Meier, 2001) using the following mutagenic
primers and their respective reverse primers (introduced nucleotide
changes are underlined) – mu1: 5¢-CAGAACCGTGTTTTGGCAGTGAAGATGTGG-3¢; mu2: 5¢-GTCAGTGAAGATGGCGGCACCGAGTAAGAG-3¢; mu3: 5¢-CGAGTAAGAGTGCCGCTCTCATGCTTGTTG-3¢;
mu4: 5¢-CTCATGCTTGTTGAGGCGATGACCAAGAAC-3¢; mu5: 5¢-CTTCTCCAGGAAGGCCGCTCTTTTGTCTG-3¢; mu6: 5¢-CGCCAAGCGCGCTGCAGATTTGGCCTTTG-3¢; mu7: 5¢-CTGCTGTTCACGTCGCTGCTAAAGAATCC-3¢; mu8: 5¢-CACGTCTATGCTAAAGCATCCAGCAAGCTC-3¢; mu9: 5¢-CTATGCTAAAGAATCCGCCAAGCTCATGCTTG-3¢;
mu11: 5¢-GTCTGTTGAAGAGTCTGAGCAAGACGCCAAG-3¢; mu12:
5¢-GGCTGAGCAAGACTCCAAGCGCATTGAAG-3¢.
The mutations were designed to replace residues in the wild-type
sequence with either alanine (mu1–9) or serine (mu11 and 12) and
were confirmed by sequencing.
Mammalian cell culture, transfection and fluorescence
microscopy
HeLa cells were cultured in DMEM medium (BioFluids, Rockville,
MD, USA) supplemented with 10% FBS (Gemini-bioproducts,
280 Sun Yong Jeong et al.
Calabasas, CA, USA) at 37C in a humidified incubator with 5% CO2.
Cells grown on cover slips were co-transfected with pEGFP-AtRanGAP1 and pDsRed-hRanGAP1 using Effectene Transfection Reagent
(Qiagen, Valencia, CA, USA) following the manufacturer’s protocol.
Cells were fixed with 3.7% paraformaldehyde in PBS for 20 min
(Figures 1a–c) or permeabilized with 0.005% digitonin (SigmaAldrich, St Louis, MO, USA) in transport buffer [110 mM KOAc,
20 mM HEPES, pH 7.3, 2 mM Mg(OAc)2, 0.5 mM EGTA, 2 mM DTT,
1 lg ml)1 each of leupeptin, pepstatin, and aprotinin] for 5 min and
fixed with 3.7% paraformaldehyde (Figure 1d–g) 48 h after transfection. Samples were stained with DAPI for visualizing DNA and
mounted in Vactashield anti-fade mounting medium (Vector
Laboratories, Burlingame, CA, USA). Slides were examined with a
Zeiss Axioskop fluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA) and images were collected and analyzed with
Openlab software.
Plants and plant cell cultures
Arabidopsis plants (Jeong et al., 2003) and tobacco BY-2 suspension culture cells (Rose and Meier, 2001) were cultured as described
previously. Green Arabidopsis suspension culture cells were cultured in Gamborg’s B-5 Basal medium with minimal organics
(Caisson Laboratories, Rexburg, ID, USA) supplemented with 2%
(w/v) sucrose, 5 lM 2,4-dichloro-phenoxyacetid acid, and 0.05%
(w/v) MES, pH 5.7. Cultures were maintained by shaking at 200 rpm
in constant light at 24C and subcultured weekly by 1:10 dilution
with fresh medium.
Arabidopsis protoplast transformation
Four- to five-day-old Arabidopsis suspension culture cells were
harvested by centrifugation for 10 min at 300 g, washed once with
the same volume (0.4 M mannitol/20 mM MES, pH 5.5), and incubated at room temperature on a platform shaker at 150 rpm in 1%
cellulase, 0.1% pectolyase (Karlan, Santa Rosa, CA, USA) in the
same volume (0.4 M mannitol/20 mM MES, pH 5.5). After completion of protoplast formation (2–5 h, monitored via microscopy),
protoplasts were harvested through centrifugation for 5 min at
150 g, washed once with 1/2 volume ice-cold W5 medium (154 mM
NaCl, 5 mM KCl, 125 mM CaCl2, 5 mM glucose, pH 6), resuspended
in ice-cold W5 medium at a concentration of 3–5 · 106 cells ml)1,
and incubated on ice for 2 h. Immediately prior to transformation,
the protoplasts were harvested at 4C and resuspended in ice-cold
MaMg solution (0.4 M mannitol, 15 mM MgCl2, 5 mM MES, pH 5.6).
20 lg DNA, 300 ll protoplast suspension and 300 ll of 40% PEG6000 (Calbiochem, La Jolla, CA, USA) in 0.1 M Ca(NO3)2, 0.4 M
mannitol, pH 8.0, were mixed by gentle inversion at room temperature for 30 min. The suspension was slowly diluted with 8 ml W5
medium and the protoplasts harvested via centrifugation for 5 min at
50 g. After removal of the supernatant, the transformed protoplasts
were resuspended in 4 ml Gamborg’s medium containing 0.4 M
mannitol and incubated in the dark for 48 h prior to microscopy.
BY-2 cell transformation
Transient transformation of BY-2 cells was performed using a biolistic DNA delivery method essentially as described previously for
NT-1 cells (Gindullis et al., 1999). For stable transformation of BY-2
cells, all constructs of AtRanGAP1-GFP, AtRanGAP1DC-GFP,
AtRanGAP1DN-GFP, AtRanGAP1 (mu3, mu6 and mu7)-GFP and
free GFP (mGFPS65T) in pFGC1008 were transformed into
Agrobacterium tumefaciens (strain LBA4404) and colonies were
selected with 8.5 lg ml)1 chloramphenicol and 50 lg ml)1 streptomycin. Agrobacterium-mediated transformation of BY-2 cells was
performed as described by Hong et al. (2001). Transformed calli
were selected on MS agar plates containing 25 lg ml)1 of hygromycin and 250 lg ml)1 of carbenicilline after 4 weeks. The selected
calli were transferred to new MS agar plates containing the same
concentration of antibiotics as above. After 1 week, calli were
transferred into MS liquid media containing 20 lg ml)1 of hygromycin to establish suspension cultures.
BY-2 cell synchronization
Synchronization of BY-2 cells was performed essentially as described by Nagata and Kumagai (1999) with the following modifications. Seven-day-old BY-2 cells were transferred to 45 ml of fresh
MS medium and 5 lg ml)1 of aphidicolin (Sigma-Aldrich) in DMSO
was added. Twenty-four hours after incubation in the dark, cells
were collected by filtering through sterile miracloth and washed
with 200 ml of fresh MS medium. Cells were transferred into 100 ml
of fresh MS medium and were continuously cultured. About 50%
of M-phase cells, about 30% of late G2 phase and about 10% of G1
phase could be identified after 10 h release from aphidicolin. For
improved synchronization, 5 h after release from aphidicolin, 50 ml
of the culture was transferred to a new flask and 1.54 lg ml)1 of
propyzamide (Sigma-Aldrich) in DMSO was added. After 4 h of
continued culture in the presence of propyzamide, cells were
washed as described above and transferred to fresh medium. Two
hours after release from propyzamide, more than 60% of
M-phase cells, about 20% of late G2 phase and about 10% of G1
phase were typically detected.
Confocal microscopy of plant cells
SYTO 82 orange (Molecular Probes, Eugene, OR, USA) was used to
stain nucleic acids as described previously (Rose and Meier, 2001),
using either 500 nM SYTO 82 for 45 min or 1 lM of SYTO 82 for
5–10 min. Digitized confocal images were acquired on a PCM2000/
Nikon Eclipse E600 confocal laser scanning microscope as previously described (Rose and Meier, 2001). For preparing Z-series of
images and 3-D movies, the software ‘Simple PCI’ (Compix Imaging
System, Cranberry Township, PA, USA) was used. For Z-series,
images were scanned in 0.75 lm interval optical sections using a
40X objective lens and 2X rolling average image processing. For 3-D
movies, optical sections were stacked with a setting of 0.3 lm for
the pixel size (X–Y) and 0.75 lm for the step size (Z). The image field
(X–Y) and range of optical sections (Z) were chosen for the best
representation of the cell plates.
Immunofluorescence microscopy
Fixation of BY-2 cells and Arabidopsis root tips, and immunolabeling of Arabidopsis root tips were carried out essentially as described by Woo et al. (1999) and Smertenko et al. (2004) with the
following modifications. To fix BY-2 cells expressing AtRanGAP1GFP, synchronized cells were incubated with a fixative containing
3.7% of paraformaldehyde, 0.2% picric acid and 5 mM EGTA in PBS
buffer for 1 h at room temperature and the fixative was washed out
with PBS three times for 10 min each. The cells were kept in PBS at
4C before observation of GFP fluorescence.
Two-day-old Arabidopsis seedlings were grown in MS liquid
media by shaking at 200 rpm in constant light at 24C. Seedlings
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
Arabidopsis RanGAP1 at the cell plate 281
were fixed for 30 min at room temperature in 4% paraformaldehyde
in 0.1 Pipes, pH 6.8, 5 mM EGTA, 2 mM MgCl2 and 0.4% Triton X-100.
The fixative was removed by three 10-min washes in PBS. Cells were
treated with 1% cellulase, 0.1% pectolyase (Karlan), 0.4 M mannitol,
20 mM MES, 5 mM EGTA, 1 mM PMSF and 5 ll ml)1 of protease
inhibitor cocktail (Sigma-Aldrich) for 20 min to digest cell walls. Cells
were washed three times for 10 min each with PBS, attached to polyL-Lys-coated slides and semi-dried. Cells were permeabilized with
1% Triton X-100 in PBS for 10 min and washed three times for 10 min
each. Blocking was carried out with 0.05% Tween-20, 1% BSA in
PBS for 1 h. Cells were labeled with the primary antibody (rabbit
polyclonal anti-AtRanGAP1) diluted 1:100 in PBS containing 0.05%
Tween-20 and 1% BSA overnight at 4C. The specimens were then
washed three times for 10 min each with PBS and incubated for 1 h
at room temperature with FITC-conjugated sheep anti-rabbit secondary antibody (Sigma-Aldrich) diluted 1:200 in PBS containing 0.05%
Tween-20 and 1% BSA. The specimens were washed with PBS two
times for 20 min and incubated with 10 lg ml)1 of propidium iodide
in water for 10 min and washed with PBS two times for 20 min. The
specimens were mounted with mounting medium (Sigma-Aldrich)
and the slides were placed in the dark for at least 1 h before
observation under the confocal microscope.
Immunoblot analysis
Immunoblot analysis was performed as described in Jeong et al.
(2003). The anti-AtRanGAP1 antibody was used at a 1:5000 dilution
and anti-GFP rabbit polyclonal (Molecular Probes) was used at a
1:1500 dilution.
Acknowledgements
We greatly acknowledge the help by Dr Tomasz Calikowski and
Xianfeng Xu in developing the AtRanGAP1 antibody. We would like
to thank Dr Desh Pal S. Verma and Dr Zonglie Hong for the Arabidopsis cell suspension culture and for providing a BY-2 cell line
expressing GFP-DRP2A. We thank Dr Biao Ding for generous user
time of his confocal microscope. Financial support by the National
Science Foundation (MCB-0079577, MCB-0343167, and MCB209339) and the US Department of Agriculture (Plant Growth and
Development 2001-01901) to I.M. is greatly acknowledged.
Supplementary Material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2368/
TPJ2368sm.htm
Video 1 (1.6 MB). Scan through BY-2 cells stably expressing
AtRanGAP1-GFP (green), counterstained for nucleic acids with
SYTO 82 orange (red). The center cell is in early cytokinesis (note
the RanGAP1-GFP signal at the growing cell plate), the cells on the
left and right are in late cytokinesis and show the typical interphase
pattern of AtRanGAP1-GFP concentration at the NE.
Video 2 (2.5 MB). Rotation of a 3-D reconstruction of the cell plate in
early cytokinesis of a BY-2 cell stably expressing AtRanGAP1-GFP.
Video 3 (2.8 MB). Rotation of a 3-D reconstruction of the cell plate in
late cytokinesis of a BY-2 cell stably expressing AtRanGAP1-GFP.
Video 4 (2.3 MB). Rotation of a 3-D reconstruction of the cell plate in
late cytokinesis of a BY-2 cell stably expressing GFP-DRP2A.
Figure S1. Point mutations in AtRanGAP1-GFP abolish targeting during cell cycle. Confocal images of BY-2 cells expressing
AtRanGAP1-GFP with a point mutation at position 6 (see Figure 2
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 270–282
and Experimental procedures). GFP, green channel; SYTO, red
channel (SYTO 82 orange nucleic acid stain). a–c, interphase; d–f,
metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o,
after completion of cytokinesis. Bars, 10 lm.
Figure S2. Point mutations in AtRanGAP1-GFP abolish targeting
during cell cycle. Confocal images of BY-2 cells expressing AtRanGAP1-GFP with a point mutation at position 7 (see Figure 2 and
Experimental procedures). GFP, green channel; SYTO, red channel
(SYTO 82 orange nucleic acid stain). a–c, interphase; d–f, metaphase; g–i, telophase to early cytokinesis; j–l, cytokinesis; m–o, after
completion of cytokinesis. Bars, 10 lm.
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