全 文 ：Received 27 Feb. 2004 Accepted 12 May 2004
Supported by the State Key Basic Research and Development Plan of China (G19990116), the National Natural Science Foundation of
China and the Knowledge Innovation Program of The Chinese Academy of Sciences.
* Author for correspondence. Tel: +86 (0)10 62591431 ext. 6214; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 940-947
Wheat RAN1 Affects Microtubules Integrity and Nucleocytoplasmic
Transport in Fission Yeast System
WANG Xin, HAN Ye, CHEN Chang-Bin, CHONG Kang*, XU Zhi-Hong
(Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environment Molecular
Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in cell cycle
and whose loss affects many biological processes. We have identified cDNA of TaRAN1, a novel Ran
GTPase homologous gene in wheat (Triticum aestivum L. cv. Jingdong No. 1). The cytoplasmic microtu-
bules play an important role in cytoplasmic organization, cell division, and the correct transmission of
genetic information in fission yeast cell. Using the fission yeast system in vivo experiments, overexpression
of TaRAN1 produced defective spindle microtubules, probably resulting in chromosome missegregation we
reported previously. The microtubules of antisense TaRAN1 yeast cells were physically disrupted. This
suggested that TaRAN1 plays a role in mitotic spindle assembly and microtubule integrity and stability.
Ultrastructural analysis under transmission electron microscope (TEM) showed abnormal nuclear
membranes in the overexpression TaRAN1 yeast cells, abnormal vacuole structures and disorganized
membranes in the antisense TaRAN1 yeast cells. These results suggested that TaRAN1 was essential for
all nucleocytoplasmic transport events.
Key words: Ran GTPase; TaRAN1; microtubules; nucleocytoplasmic transport; mitosis; fission yeast
The small G protein superfamily, an evolutionarily con-
served eukaryotic GTPase, includes Rho, Rab, Rac, Arf and
Ran homologs, which modulate diverse processes, such as
cytoskeleton reorganization, microtubule organization, cell
growth, division, differentiation, morphogenesis and nuclear
transport (Paduch et al., 2001; Yang, 2002). As expected,
the physiological functions of Rho, Rab, Arf, Rac and their
associated proteins have been examined in plant (Yang,
2002). Members of the Ran family of small GTP-binding
proteins share several conservative structural and func-
tional features. Ran with an established function in animals
in transport of RNA and proteins across the nuclear pore
(Görlich and Kutay, 1999), mitotic spindle organization
(Hetzer et al., 2002), and nuclear envelope assembly has
been reported (Zhang and Clark, 2000). The function of
plant Ran, however, has not been elucidated，although
it has been identified (Ach and Gruissem, 1994). The N-
terminus of Ran GTPase-activating protein (RanGAPs) is
responsible for its targeting to the plant nuclear rim and
Ran binding protein (RanBP) transgenic Arabidopsis is
hypersensitive to auxin and arresting mitotic progress are
intriguing (Rose and Meier, 2001; Kim et al., 2001).
The microtubule cytoskeleton, as the major component
of the mitotic spindle, is essential for the precise separation
of the duplicated sister chromatids before cytokinesis (Cahill
et al., 1999; He et al., 2003). It became evident that, in ani-
mal cells, the Ran protein participates in the organization of
the division spindle during mitosis. A couple of recent pub-
lications (Kahana and Cleveland, 2001) have demonstrated
that spontaneous microtubule asters are formed in Xeno-
pus egg extracts when the conversion of Ran-GTP to Ran-
GDP (triggered by RanGAP1) is blocked by expressing ap-
propriate mutant versions of Ran. The fission yeast,
Schizosaccharomyces pombe has been a useful model for
studying cell cycle control and spindle formation in eu-
karyotic cells (Xia et al., 1996). Recent evidence suggested
that a point mutant of Ran, called spil-25, revealed a role of
the GTPase system in spindle microtubule (MT) integrity
in vivo in fission yeast that can be distinguished from its
role in nuclear transport (Fleig et al., 2000). And the gua-
nine nucleotide exchange factor (RanGEF) mutants have
defects in mitotic spindle formation and abnormal actin ring
structures at the septum (Salus et al., 2002). It, however, is
still blurry that the mechanism described above is involved
in regulating spindle formation during mitosis in plant cells
(Pay et al., 2002; Smirnova, 2003). The detected localization
pattern of plant RanGAPs in synchronized tobacco cells is
postulated the function in the regulation of nuclear trans-
port during interphase, and suggests a role for these pro-
teins in the organization of the microtubular mitotic
WANG Xin et al.: Wheat RAN1 Affects Microtubules Integrity and Nucleocytoplasmic Transport in Fission Yeast System 941
structures (Pay et al., 2002).
Wheat is characterized by a large genome size
(approximately 17 000 Mb) and has the difficulties of plant-
let regeneration during gene transformation, thus making
the challenge to researchers to receive the successful
transgenic wheat (Chong et al., 1998; Vasil et al., 1999).
TaRAN1 (AF488730), a member of the Ran family from wheat
was cloned in our laboratory. The biochemical character-
ization of this novel TaRAN1 and its function in cell cycle
events have been identified by an approach of yeast sys-
tem (Wang et al., 2004). Here we present evidence showing
that TaRAN1 plays a role in spindle formation by maintain-
ing proper microtubule dynamics, which leads to genome
stability. In addition, novel ultrastructure phenotypes of
transgenic yeast suggested its function probably in cell
nucleocytoplasmic protein transport.
1 Materials and Methods
1.1 Yeast strains and media
Standard cell culture, media, and genetic techniques for
yeast strain were used in the experiments. Cells were grown
in either yeast extract peptone glucose (YPD) medium or
edinburgh minimal media (EMM) with appropriate
supplements. Exogenous gene expression was from the
thiamine repressible NMT1 gene promoters in plasmid
pESPM. Expression was fully repressed by addition of thia-
mine of 28 µmol/L and induced by washing in EMM lacking
thiamine. The fission yeast Leu- strain SPQ-01 was used.
1.2 Plasmid constructs
The TaRAN1 ORF (open reading frame ) from the cDNA
clone was amplified by PCR using the following primers 5-
CCG CTC GAG CGC CAG CGT CGT CCC-3 (italicized char-
acters represent XhoⅠ restriction site) and 5-CGG GAT
CCA AAT CAA GCC TTC AAC CTA A-3 (italicized char-
acters represent BamHⅠ restriction site). The PCR prod-
uct was cleaved by XhoⅠ /BamHⅠ and cloned into the
sites in pESPM. The sense and the antisense TaRAN1 ex-
pression vectors were only distinguished from different
transcriptional direction of TaRAN1 gene.
1.3 Yeast transformation and grown situation of yeast
cells screening
Expression vectors with the sense and the antisense
gene were constructed in pESPM, respectively. The con-
structs of TaRAN1 were transformed into S. pombe cells by
using lithium acetate (LiAc)-mediated method (Gietz et al.,
1992) and the transformants were selected on plates con-
taining minimal medium with thiamine at 30 oC. Cells from
the selected clones were grown to mid-exponential phase
in minimal medium containing thiamine at 30 oC. The Cells
were washed three times with the minimal medium without
thiamine to derepress the NMT1 promoter, and then incu-
bated at 30 oC for 22 h. The same amount of the transformed
mid-exponential yeast cells were restreaked onto the plates
with or without thiamine and incubated at 30 oC. The grown
situation of the yeast cells was observed after 3 d. The
pESPM vector transformant cells were used as a control.
1.4 Transcriptional expression analysis of TaRAN1 in S.
pombe
Total RNA was isolated from yeast cells according to a
protocol of the Cold Spring Harbor Laboratory (Alfa et al.,
1993). The first-strand cDNA was synthesized using RT-
PCR Kit (TaKaRa) in a 50-µL reaction containing total RNA
of 1 µg. Reverse-transcription reactions were carried out at
42 oC for 30 min and terminated by heating to 99 oC for 5 min
and chilling to 4 oC for 5 min. The reaction mixture of 1 µL
was used as a template in the PCR. All products were loaded
on a 1% agarose gel to visualize the amplified cDNAs. RT-
PCR reactions were repeated five times. All amplified frag-
ments were verified by sequencing. Tublin primers were 5
-CAGCCAACTTTCTCAAATCAG-3 and 5 -GCCAAG
GGTCACTACAC-3. Conserved region of Ran family prim-
ers were 5-GAGAACATCCCCATTGTCC-3 and 5-
CAAACAGTTTGCAGCCCACCA-3. To ensure TaRAN1
gene-specific amplifications, PCR primers were designed
according to the sequence of non-conservative
untranslated regions of 3 terminus. TaRAN1 primers were
5 - T G C C A A G A G C A A C TA C A A - 3 a n d 5 -
ATGATCCACATATTGAGCC-3.
1.5 Fluorescence microscopy
To visualize microtubules, cells were fixed with 0.2%
glutaraldehyde and 3% paraformaldehyde in the PEM buffer
(50 µmol/L PIPES (pH 6.9), 5 mmol/L EGTA, 5 mmol/L
MgSO4) for 90 min (Alfa et al., 1993). They were washed
three times with PEM. Cell wall was digested by suspend-
ing the cells at a density of 5× 107-1× 108 cells/mL in
PBS buffer containing the following degradative enzymes,
i.e., 0.3% Cellulase “Onozuka” R-10 (Yakult. Co., Japan),
0.3% Pectolyase Y-23 (Yakult. Co., Japan) and 0.3% Lyticase
(L-4025, Sigma). The cells were incubated at 37 oC until
about 80% of them were judged, by a decrease in refractility
under phase contrast optics, to have lost their cell walls.
Usually, 20-40 min is optimal to digesting the cells. They
were washed three times with 1×PBS + 0.3% BSA. And
then incubated with an anti-b-tublin monoclonal antibody
(1:100), as previous described (Hagan and Hyams, 1988).
Goat anti-mouse IgG conjugated with Fluorescein (FITC)
was from Sigma. To view DNA, cells were stained with
Propidium iodide (1 µg/mL PI). The microtubules in the cell
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004942
were measured by adjusting the focal plane up and down
while observing and stained cells were examined with a
fluorescence microscope (Zeiss, Germany).
1.6 Electron microscopy
Observation of electron microscopy was performed ac-
cording to Doye et al. (1994). Exponentially growing cells
were washed in K-phosphate buffer (pH 6.5) with 0.5 mmol/L
MgCl2. Cells were resuspended in the same buffer supple-
mented with 2% formaldehyde and 2% glutaradehyde, and
incubated for 90 min at 4 oC. Cells were then washed three
times in Na-acetate of 0.1 mol/L (pH 6.1) at 4 oC and postfixed
for 15 min at 4 oC with 2% Osmium Tetroxyde in 1:2 Na-
acetate buffer. Cells were then rinsed three times in H2O,
stained with 1% Uranyl-Acetate for 1 h at room tempera-
ture in the dark, and washed again three times in H2O, and
dehydrated in 70% and 90% ethanol for 15 min. After
pelleting in a microtube, the cell pellet was incubated twice,
for 10 min each time, in 100% ethanol twice, for 10 min each
time, in propylenoxyde, then incubated overnight in a 1:1
mix of Epon and propylenoxide. The next day, the pellet
was incubated in pure epon for 1 h. Then epon was ex-
changed for fresh epon, and the tube incubated at 60 oC for
3 d to induce epon polymerization. Thin sections of embed-
ded cells were produced with an ultramicrotome (Leica,
Deerfield, IL), stained with lead and uranyl for 30 min, and
observed under a electron microscope (JEM-1230, JEOL
Ltd., Japan).
2 Results
2.1 Transcriptional expression analysis of TaRAN1 in S.
pombe
A series of yeast lines were identified by using an ap-
proach of quantitative RT-PCR analysis. Different specific
primers were used to check the abundance of exogenous
TaRAN1 and transcripts of conserved region of Ran GTPase
family in all transgenic yeast cells. The results showed that
transcriptional messages of the exogenous TaRAN1 gene
were detected in transformed TaRAN1 yeast lines, but were
absent in yeast lines transformed by an empty vector.
Furthermore, the transcriptional level of conserved region
of Ran GTPase family was improved in the sense and
antisense TaRAN1 transgenic yeast lines (Fig.1). Therefore,
cells with high transcription levels of the latter two groups
were really affected by the presence of the transgene.
2.2 The grown situation of the transgenic TaRAN1 yeast
cells
To test function of TaRAN1 in cell division, a system of
S. pombe was used in the experiment. The constructs of
sense and antisense TaRAN1, as well as the pESPM empty
vector as a control, were transformed into the Leu- strain
SPQ-01. Cells transformed with all three constructs grew
normal on minimal medium with thiamine. Cells with the
antisense of TaRAN1, showed a slow growth on the plate
of without thiamine (Fig.2).
2.3 TaRAN1 affects formation and function of the mitotic
spindle
We analyzed microtubules in transgenic yeast cells by
indirect immunofluorescence. In ones transformed by empty
vector or yeast cells of wild-type control, the cytoplasmic
microtubules (MTs) were aligned along the long axis of the
cell almost reaching the cell tip (Fig.3A, C). The sense
TaRAN1 cells had aberrant spindle structures up to 54.7%,
which were seen in neither the vector control cells (Fig.3E
and Table 1) nor wild-type cells. These aberrant structures,
such as short v-shaped spindles, probably resulted in the
improperly separation of DNA (Fig.3E). These data indi-
cated that the overexpression of TaRAN1 in transgenic cells
resulted in spindle assembly defects similar to those seen
in the spil-25 mutant (Fleig et al., 2000). These defects
probably resulted in a subsequent loss of chromosome seg-
regation fidelity we ever observed (Wang et al., 2004). The
antisense TaRAN1 strain of 29% contained more vague
nuclear. And their spindles disappeared (Fig.3G). In
comparison, the percentages of analogous cells in wild-
type strain, empty vector, or the sense TaRAN1 cells were
less than 0.5%. These data also suggested that the antisense
TaRAN1 cells exhibited a substantial mitotic delay. In
general, these results suggested that the presence of the
transgene might specifically affect microtubules (MTs)
function.
2.4 The TaRAN1 transgenic cells had abnormal vacuole
and nuclear membrane ultrastructure
Ultrastructural analysis under transmission electron
Fig.1. An examination of all transgenic yeast lines by quantita-
tive RT-PCR analysis. Tublin RT-PCR was included as a loading
control. These results repeated five times and had the same trend.
a, b and c represent specific region of TaRAN1 gene, conserved
region of Ran GTPase family genes and the yeast tublin gene,
respectively.
WANG Xin et al.: Wheat RAN1 Affects Microtubules Integrity and Nucleocytoplasmic Transport in Fission Yeast System 943
microscope (TEM) revealed striking nuclear membrane ab-
normalities in the sense and antisense TaRAN1 cells that
were never observed in control cells (Figs.4-10). Nuclear
membranes were distorted and formed outpouches that ap-
peared to contain cytoplasm in the sense TaRAN1 cells.
The nuclear membrane of antisense TaRAN1 cells looked
more vague and incomplete. Furthermore, the antisense
TaRAN1 cells had abnormal vacuole structures. It contained
multiple electron-dense membrane-bound compartments as
well as disorganized membranes that were associated with
Fig.3. Abnormal spindles in mitotic TaRAN1 transgenic yeast cells. Cells pregrown at 30 oC to log phase in the presense of thiamine were
shifted to the medium absense of thiamine for 24 h. The spindles were examined by immunostaining. A, C, E and G are tublin, recognized
with the ß-tublin antibody, or DNA morphology, visualized with PI simultaneously (B, D, F, H). Normal cytoplasmic microtubules
(MTs) are aligned along the long axis of the cell reaching the cell tips in the empty vector transgenic yeast lines (A and B). Normal
elongated spindle of the anaphase was in mitotic cell in the empty vector transgenic lines (C and D). Aberrant spindle structures with v-
shaped were in the sense TaRAN1 transgenic cells (E and F). Absence of complete spindle structures was in the antisense TaRAN1
transgenic cells (G and H). Bar =10 mm. Tub, tublin; PI, propidium iodide.
Table 1 Relative abundance of various forms of spindles corresponded to Fig.3
Growth
Distribution of various spindle ( % )*
Genotype
media
Long axis-shaped v-shaped spindle None
spindle (Fig.3D) (Fig.3E) (Fig.3G)
Vector +thiamine 100 < 0.5 < 0.5
-thiamine 100 < 0.5 < 0.5
Sense TaRAN1 +thiamine 100 < 0.5 < 0.5
-thiamine 44.1 54.7 < 0.5
Antisense TaRAN1 +thiamine 100 < 0.5 < 0.5
-thiamine 69 < 0.5 29
*, the various spindles are corresponded to those in Fig.3.
Fig.2. The grown situation of the Schizosaccharomyces pombe transformed with sense and antisense TaRAN1. The strain SPQ-01
transformed with pESPM vector alone, sense TaRAN1-pESPM, or antisense TaRAN1-pESPM respectively was grown on minimal
plates with or without thiamine. Serial dilutions of cells (1:10) were spotted on different plates. These plates were incubated at 30 oC for
3 d before being photographed. Antisense TaRAN1 suppresses the growth rate of the yeast cells.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004944
the nucleus or present in the cytoplasm. Therefore, the
altered morphology resulting from transgenic TaRAN1 gene
may reflect impaired nuclear-related transport.
3 Discussion
Microtubules are polymers of a- and b-tubulins that are
highly conserved in eukaryotic cells (Hyman and Karsenti,
1996). They play critical roles in establishing the spatial
distribution of molecules and organelles and in chromo-
some segregation. All of them require microtubules to un-
dergo remodeling in a cell cycle-dependent manner (Cai
and Hu, 1996). Like higher eukaryotes, the fission yeast
Schizosaccharomyces pombe has a complex and dynamic
microtubule cytoskeleton. The microtubule cytoskeleton
is important for cell polarity, namely, in maintaining a bipo-
lar cell extension and elongated cell morphology. In wild-
type yeast cells, interphase microtubules extend along the
long axis of the cell, almost reaching the cell tips (Hagan
and Hyams, 1988). Tublin mutants (Toda et al., 1984), mi-
crotubule destabilizing drugs (Walker, 1982), or mutant such
as mal3+ (a conserved family of microtubule-binding
protein) and spil-25 (Beinhauer et al., 1997; Fleig, 2000)
that give rise to abnormally short interphase microtubules
all lead to aberrantly shaped cells. Several recent studies
demonstrated that in vitro RanGTP promotes microtubule
formation when added to mitotic Xenopus egg extracts
(Carazo-Salas et al., 1999; Kalab et al., 1999; Wilde and
Zheng, 1999). But the related spindle formation function of
Figs. 4-10. Ultrastructural analysis of TaRAN1 transgenic cells. Transgenic cells (24 h after thiamine removal) were fixed and processed
for experiment of electron microscopy. 4, 8. Empty vector transgenic yeast cell as control (including lognitudinal and transverse sections).
5, 7. Antisense TaRAN1 yeast cell. 6, 9, 10. Sense TaRAN1 yeast cell. 9, 10. Magnification of Fig.6. Antisense TaRAN1 cells have no large,
complete vacuole and instead contain multiple electron-dense, membrane-bound compartments as well as disorganized membranes
(white arrows). Note invaginations of cytoplasm in the nucleus (black arrow); distorted nucleus membranes (black arrowhead) in sense
TaRAN1 yeast cells. Bar = 0.48 mm.
WANG Xin et al.: Wheat RAN1 Affects Microtubules Integrity and Nucleocytoplasmic Transport in Fission Yeast System 945
homologs of Ran gene in plant was never reported before
this report.
If nuclear Ran acts as some type of signal for the cell to
progress through mitosis, the depletion of Ran would halt
this process and arrest cell division, as seen in the fission
yeast system in vivo experiments. Consistent with above
phenomenon, the nuclear area of the antisense TaRAN1
cells became more vague, and formal nucleomixis (Wang et
al., 2004). Furthermore, in the antisense TaRAN1 yeast cells,
spindles were physically distrupted and growth rate of them
was decreasing. Interestingly, we found that cell lines of
the antisense TaRAN1 were hypersensitive to TBZ (Wang
et al., 2004), which correlates with the observation that
their microtubules bundles were hardly formed (Fig.3G).
Morphological observation showed that the TaRAN1
transgenic cells exhibited the defective chromosome seg-
regation and reminiscent of a G2 cell cycle delay (Wang
et al., 2004). It is possible that the abnormal spindle micro-
tubules of the TaRAN1 transgenic cells (Fig.3E) resulted in
the defective chromosome segregation (Wang et al., 2004).
This is consistent with a model in which Ran plays a direct
role in mitotic spindle formation (Salus, 2002).
The requirement for Ran in nuclear transport has been
extensively studied in animal (Görlich and Kutay, 1999). A
family of related Ran-GTP binding protein acts as receptors
for both nuclear import and export. Import receptors asso-
ciate with their cargo in the cytosol, where Ran-GTP con-
centrations are low, and dissociate from their cargo after
entering the nucleus and binding Ran-GTP. Conversely,
export receptors bind their cargo in complexes containing
Ran-GTP within the nucleus and dissociate from their cargo
after transit to the cytosol and GTP hydrolysis. The TEM
analysis, however, has not been used to study Ran mu-
tants in yeast in any other reports. The novel and abnormal
ultrastructural phenotypes of the sense and antisense
TaRAN1 cells may suggest that cells do not form appropri-
ate nuclear transportation linking the nucleus and cyto-
plasm during interphase. The role of nuclear shuttling re-
mains an intriguing possibility that awaits for further to
test.
The molecular mechanisms underlying these obviously
important cellular processes are poorly understood in plant
cells. To gain information about the biological role of the
plant RanGTPase system, a central factor of these pro-
cesses in plant cells is naturally one of our major interests.
Our results not only suggested that nuclear Ran was re-
quired for proper spindle formation, cell cycle and cell divi-
sion in vivo, but also that the Ran protein was involved in
nuclear-related transport probably, which was the first re-
port about the function of Ran gene in plant. Ran is a con-
served small GTP-binding protein in eukaryotic cells. It is
probable that they can function in plant cells to influence
spindle functioning and vacuole transportation. Most likely,
Ran interacts with a variety of proteins during mitosis, ei-
ther individually or as a complex, and identification of these
proteins will be the next step to define the role of Ran pro-
tein in the cell cycle.
Acknowledgements: Authors thank Miss BAI Yue and
Mr. YUAN Cheng for initiating these studies. We are grate-
ful to Dr. YUAN Ming and Dr. REN Dong-Tao (China Agri-
cultural University) for their generous gift of anti-b-tublin
monoclonal antibody and thank Dr. BAO Shi-Lai (Institute
of Genetics and Developmental Biology, The Chinese Acad-
emy of Sciences) for helpful discussion.
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