Plant Biology ISSN 1435-8603

RESEARCH PAPER

The distribution of TPX2 in dividing leaf cells of the fern

Asplenium nidus
E. Panteris, I.-D. S. Adamakis & K. Chanoumidou
Department of Botany, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece

Keywords
Asplenium nidus; cell division; cytokinesis;
microtubules; mitosis; TPX2.
Correspondence
E. Panteris, Department of Botany, School of
Biology, Aristotle University of Thessaloniki,
Thessaloniki GR-541 24, Macedonia, Greece.
E-mail: epanter@bio.auth.gr
Editor
S. Wick
Received: 2 February 2012; Accepted: 19
March 2012
doi:10.1111/j.1438-8677.2012.00615.x
ABSTRACT
Plant cell division requires the dynamic organisation of several microtubule arrays.
The mechanisms of regulation of the above arrays are under rigorous research.
Among several factors that are involved in plant microtubule dynamics, the Target-
ing Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation,
in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule
organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the
presence and distribution of a TPX2 homologue might be helpful in understanding
the patterns and regulatory mechanisms of microtubule arrays in this plant group.
In this study, a putative TPX2 homologue was identified using Western blotting in
the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is
co-localised with perinuclear preprophase microtubules and the prophase spindle,
and follows the microtubule pattern during metaphase ⁄ anaphase and telophase.
During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2
signal persists, co-localising with the phragmoplast. In early post-cytokinetic cells, a
TPX2 signal is present on the nuclear surface facing the daughter cell wall and,
thereafter it is co-localised with the fern-specific microtubule aggregation that lines
the new wall, which is possibly involved in cortical microtubule assembly.

above). Apart from A. thaliana and N. tabacum, the presence

INTRODUCTION
Cell division in higher plants requires the organisation of sev-
eral successive microtubule arrays, some of which are plant-
specific. The preprophase microtubule band, the perinuclear
prophase spindle, the acentrosomal metaphase and anaphase
spindle, the phragmoplast, the perinuclear early interphase
microtubules and the cortical interphase array follow each
other as mitosis and cytokinesis proceed in plant cells (see
among others Hasezawa & Kumagai 2002). The assembly and
organisation of the above arrays depend on several factors,
such as the c-tubulin complexes (Binarová et al. 2006; Pastu-
glia et al. 2006; see also Kong et al. 2010), the Ran GTPase
(Pay et al. 2002; Jeong et al. 2005; Zhao et al. 2008; Meier &
Brkljacic 2009) and the Aurora kinases (Van Damme et al.
2004; Demidov et al. 2005; Kawabe et al. 2005). Apart from
the above, a homologue of the Targeting Protein for Xklp2
(TPX2) has recently been identified in Arabidopsis thaliana,
Nicotiana tabacum and Trandescantia virginiana, involved in
spindle assembly during prophase (Vos et al. 2008). In the
above plants, TPX2 is intranuclear during interphase and is
exported from the nucleus during prophase, interacts with
Aurora kinases and possibly also participates in microtubule
nucleation (Vos et al. 2008; see also Petrovská et al. 2012).
After nuclear envelope breakdown, TPX2 in A. thaliana and
N. tabacum is localised at the kinetochore microtubule bun-
dles during metaphase and anaphase and is degraded during
telophase (see above). Its importance for cell division has
been further proven as nuclear envelope breakdown and
mitosis were inhibited by microinjection of anti-TPX2 (see
of TPX2 or TPX2-related protein homologues in other plant
species has also been reported (Vos et al. 2008; Evrard et al.
2009).
Microtubule organisation in ferns is characterised by some
discernible peculiarities in comparison to angiosperms. While
in the latter plants preprophase band narrowing is F-actin
mediated (Mineyuki & Palevitz 1990; Eleftheriou & Palevitz
1992), in ferns this process seems to be F-actin-independent
(Panteris et al. 1992; further unpublished observations). In
angiosperms the metaphase spindle may not emanate directly
from the prophase spindle (Zachariadis et al. 2000, 2004;
Lloyd & Chan 2006; see also Panteris et al. 2011; Panteris &
Adamakis 2012), while in ferns the former seems to be a
direct outcome of the latter (Zachariadis et al. 2004). In an-
giosperms, the reinstatement of cortical microtubules at
interphase is preceded by an array of radially arranged peri-
nuclear microtubules (see among others Wick 1985; Flanders
et al. 1990; Hasezawa & Nagata 1991), while in ferns the cor-
tical array is usually organised without the mediation of peri-
nuclear microtubules (Panteris et al. 1991, 1995).
Furthermore, a distinct population of microtubules lines the
new cell wall in post-cytokinetic cells of ferns (Jenni et al.
1990; Panteris et al. 1991; Zachariadis et al. 2003) but not of
angiosperms.
Taking into account the above features of microtubule
organisation in ferns, we studied the possible presence and
distribution of TPX2 in dividing leaf cells of the fern Aspleni-
um nidus. A polyclonal anti-AtTPX2 antibody, raised against
the conserved TPX2 signature motif (Vos et al. 2008), identi-

Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands 1
TPX2 in Asplenium nidus Panteris, Adamakis & Chanoumidou

fied a putative TPX2 homologue, the distribution of which kon D-Eclipse C1 confocal laser scanning microscope (CLSM)
exhibits important differences from that observed in A. thali- with the appropriate wavelengths and filters, and images were
ana and N. tabacum cells. Leptomycin-B treatment (Kudo recorded according to the manufacturer’s instructions. In
et al. 1998) was also applied to confirm the intranuclear some specimens, the DNA was counterstained with
localisation of TPX2 during interphase. 10 lgÆml)1 Hoechst 33258 and they were observed under a
Nikon Eclipse i80 microscope (Nikon Instruments Europe
MATERIAL AND METHODS BV, Amstelvee, the Netherlands) equipped with epifluores-
cence. Digital images were processed with Adobe Photoshop
Plant material and treatments CS2, with linear settings only.
Asplenium nidus plants were cultivated in the lab. The apical
areas of coiled young leaves were used for all the procedures, Western blotting of leaf extracts
as they comprise a large population of dividing cells. All the
chemicals and reagents were purchased from Sigma (Tauf- For Western immunoblotting of putative TPX2 isoforms,
hirchen, Germany), Merck (Darmstadt, Germany) and Appli- extracts of untreated coiled leaf areas of A. nidus were ho-
chem (Darmstadt, Germany), unless otherwise stated. mogenised at 4 C in a buffer containing 20 mm b-glycero-
Leptomycin-B, a potent inhibitor of nuclear export (Kudo et phosphate, 20 mm HEPES, pH 7.5, 20 mm NaF, 2 mm
al. 1998; see also Vos et al. 2008), was initially dissolved as a EDTA, 0.2 mm Na3VO4, 10 mm benzamidine, 5 mm DTT,
100 lM stock in absolute methanol and kept at )20 C; work- 1% (v ⁄ v) Triton X-100 and a commercial protease inhibitor
ing solution for treatment was prepared by diluting to cocktail. Lysates were incubated on ice for 10 min and then
500 nm. The lamina of the coiled leaf apical area was cut into centrifuged at 4 C for 20 min at 16,000 g. Clarified superna-
pieces ca. 4 · 4 mm. The pieces were put in small dishes with tants were collected and total protein was measured using the
500 nm leptomycin-B solution and treated for 4 h in the dark Bradford assay.
at room temperature on a rocking platform (Biorad, Hercules, Total protein extracts were boiled with 0.33 (v ⁄ v) of SDS-
CA, USA) at 35 rpm. Similar treatment with 0.5% methanol polyacrylamide gel electrophoresis sample buffer (10% w ⁄ v
was applied as a control. Oryzalin (a kind gift of Dr. Hartmut SDS, 13% v ⁄ v glycerol, 300 mm Tris–HCl, pH 6.8, 130 mm
Quader, Biozentrum, Hamburg, Germany) was dissolved as a dithiothreitol) and 0.2% (w ⁄ v) bromophenol blue. Proteins
10 mm stock in absolute acetone and stored at 4 C; working were separated by SDS-PAGE on 12% (w ⁄ v) SDS-polyacryl-
solution was 10 lm, and for control 0.1% acetone was applied. amide gels and transferred electrophoretically onto nitrocellu-
The treatment was similar to that with leptomycin-B. lose membranes (0.45 lm). Membranes were blocked in 5%
(w ⁄ v) BSA powder in TBST buffer (20 mm Tris–HCl, pH
7.5, 137 mm NaCl, 0.1% (v ⁄ v) Tween 20) overnight at 4 C,
Immunostaining and fluorescence microscopy
then washed in TBST (three times for 5 min each) and then
Control as well as leptomycin-B- and oryzalin-treated leaf incubated for 2 h at room temperature with polyclonal anti-
material was prepared as described in Panteris & Karali
(2008) with some modifications. First, the leaves or leaf pieces
were cut into ca. 2 · 2 mm samples, immediately pre-fixed
for 20 min at room temperature in 1% (w ⁄ v) paraformalde-
hyde (PFA) in PEM (50 mm PIPES, 5 mm EGTA, 5 mm
MgSO4, pH 6.8) with addition of 5% (v ⁄ v) DMSO. All the
following steps were also performed at room temperature,
unless otherwise stated. After pre-fixation, the samples were
fixed for 1 h in 4% (w ⁄ v) PFA in PEM with 5% (v ⁄ v) DMSO.
After three 10-min washes in PEM, the samples were treated
with 0.1% (w ⁄ v) NaBH4 in PEM twice for 30 min, then over-
night. After washing, the cell walls were partially digested with
2% (w ⁄ v) cellulase (Onozuka R10; Yakult Honsha, Tokyo,
Japan) and 2% (w ⁄ v) macerozyme R10 (Serva, Heidelberg,
Germany) in PEM for 1 h. After removing the enzymes, the
samples were treated with absolute methanol at )20 C for
20 min. Then, extraction and blocking with 5% (v ⁄ v) DMSO
and 1% (v ⁄ v) Triton X-100 + 3% (w ⁄ v) BSA was performed
for 1 h. The samples were incubated overnight with monoclo-
nal rat anti-a-tubulin (YOL 1 ⁄ 34, AbD Serotec, Kidlingto,
UK) and polyclonal rabbit anti-AtTPX2 (Vos et al. 2008),
diluted 1:80 and 1:3000 in PEM, respectively. Some samples
were incubated only with anti-AtTPX2. Following three 30-
min rinses with PEM, the samples were incubated overnight
with FITC-anti-rat and TRITC-anti-rabbit 1:80 in PEM. After
further washing, the samples were mounted in a drop of anti- Fig. 1. Western blot of Asplenium nidus leaf proteins with polyclonal
fade solution (glycerol:PEM 2:1 (v ⁄ v) + 0.5% (w ⁄ v) p-phe- antibody against the conserved TPX2 signature motif of Arabidopsis thali-
nylendiamine). The preparations were examined under a Ni- ana. A single band ca. 100 kDa is identified.

2 Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands
Panteris, Adamakis & Chanoumidou TPX2 in Asplenium nidus

AtTPX2 diluted 1:10,000 in TBST buffer containing 5%
(w ⁄ v) BSA powder. Following two 5-min washes in TBSTXS
(1x TBS, 1% (w ⁄ v) BSA, 1% (w ⁄ v) Triton X and 1% (w ⁄ v)
SDS) one wash with a 0.8% (w ⁄ v) NaCl solution and finally
an incubation of 5 min with a 5% (w ⁄ v) non-fat milk solu-
tion in TBST, the membranes were incubated for 2 h at
room temperature with horseradish peroxidase-conjugated
secondary antibodies (1:2500) diluted in TBST buffer con-
taining 5% (w ⁄ v) non-fat milk powder. After washing in
TBSTXS buffer (three times for 5 min) and one wash with a
0.8% (w ⁄ v) NaCl solution, proteins were detected using
enhanced chemiluminescence (Cell Signalling, Beverly, MA,
USA).
RESULTS AND DISCUSSION
A putative TPX2 homologue in A. nidus
A single band at ca. 100 kDa was identified by Western blot-
ting of the leaf extract (Fig. 1), suggesting that the putative A.
nidus TPX2 homologue may be rather smaller than those of
A. thaliana, N. tabacum and T. virginiana, which have a mass
of 120 kDa (Vos et al. 2008). Also, A. nidus TPX2 seems to
be more stable than those of the above angiosperms, which
by Western blotting were labelled as bands mainly at 65 kDa,
probably due to their high lability (see above). Interestingly,
the molecular mass of the putative A. nidus TPX2 homologue
is similar to those of Xenopus laevis at ca. 100 kDa (Witt-
mann et al. 2000) and human at ca. 98 kDa (Garrett et al.
2002) cells.
The distribution of TPX2 signal in dividing leaf cells
The CLSM observations revealed that anti-AtTPX2 stained
specific microtubule arrays throughout cell division. In prep-
rophase ⁄ prophase cells, TPX2 signal was co-localised with
perinuclear microtubules (Fig. 2A–C and K–M; Figures S1
and S3). In cells, the prophase nuclei of which exhibited pro-
trusions to the preprophase band, characteristic of fern cells
at this stage (Panteris et al. 1991; see also Figures S1 and S2),
the TPX2 signal also followed the pattern of perinuclear
microtubules (Fig. 2K–M and Figure S1). In metaphase ⁄ ana-
phase cells, the TPX2 signal was prominent on the kinetocho-
re microtubule bundles and interzonal microtubules
(Fig. 2D–I, Figure S3). At late anaphase, a distinguishable
TPX2 signal could be observed at the spindle midzone (Fig. 2
H and L), at the area of interzonal microtubule overlap
(Fig. 2G–M). This might suggest a role for TPX2 in phrag-
moplast organisation in A. nidus.
The TPX2 signal was also present in the initiating (Fig. 3A–
C) and expanding phragmoplast (Fig. 3D–F; see also Fig-
ure S3). After completion of cytokinesis, the daughter nuclei of

A B C

D E F

Fig. 2. CLSM leaf sections depicting mitotic cells after

a-tubulin (A, D, G, K) and TPX2 (B, E, H, L) immuno-
staining, and their combination (C, F, I, M). All the
images are at external periclinal views. The intensely
fluorescent cortical spots in (B) and (E), as well as in
other images, are due to autofluorescence of organ- G H I
elles. A–C: Preprophase ⁄ prophase cells. TPX2 signal (B)
is prominent around the prophase nucleus (asterisks in
A and B), coincident with perinuclear microtubules (A;
see overlap in C). TPX2 signal (B) can also be observed
around the nucleus of an early preprophase cell
(arrows point to the profiles of wide preprophase band
in A), co-localising with perinuclear microtubules (A).
D–M: Metaphase (D–F) and anaphase (G–M) cells.
TPX2 signal can be observed on kinetochore microtu-
bule bundles (compare E, H, L with D, G, K; see also K L M
overlap in F, I, M). TPX2 is also present on interzonal
microtubules (arrows in G and H) and appears aggre-
gated at their midzone (arrows in L). Also note the co-
localisation of TPX2 with perinuclear microtubules in
the prophase cell (asterisk) in (K–M). Scale
bars = 10 lm.

Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands 3
TPX2 in Asplenium nidus
A B C
D E F
G H I Fig. 3. CLSM leaf sections depicting cytokinetic (A–F)
K L M follow the overall distribution of microtubules (cf. G)
A. nidus leaf cells were surrounded by tangentially arranged
microtubules (Fig. 3G), as also reported by Zachariadis et al.
(2003). In these cells, the TPX2 signal was also present on the
surface of daughter nuclei, however restricted only to the area
facing the new cell wall (Fig. 3H and I). In A. thaliana and
N. tabacum, TPX2 is degraded by telophase (Vos et al. 2008),
co-localising only with a subset of microtubules in the early
phragmoplast in A. thaliana (Petrovská et al. 2012) but not
with those of expanding phragmoplasts. In contrast, according
to our observations, the putative A. nidus TPX2 homologue
does not disappear from the cytoplasm during telophase, but
persists during cytokinesis. This seems similar to the distribu-
tion of TPX2 in X. laevis cells, in which TPX2 co-localises with
the midbody (Wittmann et al. 2000; O’ Brien & Wiese 2006), a
structure analogous to the phragmoplast of plant cells (Otegui
et al. 2005), while during early G1 TPX2 is present at the
nuclear rim (O’ Brien & Wiese 2006).
Asplenium nidus TPX2 and cortical microtubule initiation
Post-cytokinetic cells of A. nidus exhibited a cortical microtu-
bule aggregation lining the new wall (Fig. 3K), as is common
in ferns (see Introduction). The TPX2 signal was also
co-localised with these microtubules (Fig. 3L and M; see also
Figure S3), but not with other cortical microtubules.
4 Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands
Panteris, Adamakis & Chanoumidou
and post-cytokinetic (G–M) cells after a-tubulin (A, D,
G, K) and TPX2 (B, E, H, L) immunostaining, and their
combination (C, F, I, M). A–C: TPX2 (B) is present in
the phragmoplast (A; see overlap in C) of this early
cytokinetic cell. D–F: Surface view of expanding phrag-
moplast (D), in which TPX2 signal can be observed
(E; see overlap in F). G–I: Post-cytokinetic cell, the
daughter nuclei of which (asterisks in G) are sur-
rounded by microtubules. TPX2 signal (H) does not
but is restricted on the nuclear surface facing the
daughter wall (see also overlap in I). K–M: Post-cytoki-
netic cell, the daughter wall of which (arrows in K) is
lined with a microtubule aggregation (see intense fluo-
rescence in K). TPX2 signal (L), coincident with these
microtubules (cf. K) can also be observed (see overlap
in M). Scale bars = 10 lm.
The above cortical microtubule aggregation has been
related with putative localised microtubule organising centre
(MTOC) activity, contributing to interphase microtubule ini-
tiation (see Introduction). TPX2 seems to be involved in
microtubule nucleation in cells of both plants and vertebrates
(Vos et al. 2008 and references therein). In accordance, the
post-cytokinetic localisation of A. nidus TPX2 under the new
cell walls may reflect its involvement in microtubule assem-
bly. In addition, the localisation of TPX2 on the nuclear face
proximal to the new cell wall (Fig. 3H and I) might represent
a preparatory step, probably followed by transport of TPX2
to the cortical cytoplasm lining the new wall to participate in
microtubule assembly there.
In post-cytokinetic cells mildly affected by oryzalin, the
microtubule aggregation under the new cell wall was
restricted to the area between the daughter nuclei (Fig. 4A,
cf. Fig. 3K), where the TPX2 signal was also co-localised
(Fig. 4B and C). In heavily affected cells, almost devoid of
microtubules (Fig. 4D), the TPX2 signal could not be
observed (Fig. 4E and F). It seems, therefore, that TPX2
localisation under the new cell wall may depend on the pres-
ence of intact microtubules.
It was recorded that Aurora 1 is aggregated at the cell plate
(Van Damme et al. 2004; Demidov et al. 2005; Petrovská
et al. 2012) and that AtTPX2 interacts with Aurora kinases (Vos
Panteris, Adamakis & Chanoumidou TPX2 in Asplenium nidus

A B C

D E F

Fig. 4. CLSM leaf sections depicting oryzalin-treated post-cytokinetic cells after a-tubulin (A and D) and TPX2 (B and E) immunostaining, and their combi-
nation (C and F). A–C: Cells mildly affected by oryzalin. Remnants of microtubule aggregation (arrow in A) line the new wall of the post-cytokinetic cell,
restricted between the daughter nuclei. TPX2 signal (arrow in B) is co-localised with these microtubules (see also overlap in C). Note also the presence of
TPX2 signal (arrowhead in B) in the phragmoplast (arrowhead in A) of an affected cytokinetic cell. D–F: Heavily affected cells. No TPX2 signal (E) can be
observed in the remnants of cortical microtubules (arrowheads in D) restricted between the daughter nuclei in post-cytokinetic cells (see also overlap in F)
Scale bar = 10 lm.

et al. 2008; Petrovská et al. 2012). Especially, Petrovská et al.
(2012) discussed the combined function of At Aurora 1, AtT-
PX2 and c-tubulin in regulating microtubule assembly. It is
tempting, therefore, to assume that the presence of TPX2
under the daughter cell wall in A. nidus post-cytokinetic cells
might reflect such an interaction between Aurora kinases,
TPX2 and c-tubulin, which is also localised there (see Panter-
is et al. 2000), probably triggering transient MTOC activity.
As a result, cortical microtubules are initiated on the daugh-
ter cell wall surface of A. nidus, as is also the case in other
ferns (see Introduction). In contrast, in A. thaliana and
N. tabacum, in which TPX2 is degraded by telophase, such
an interaction does not occur, and interphase microtubules
originate from the nuclear surface (see Introduction).
Intranuclear origin of TPX2 in A. nidus cells
The expected intranuclear TPX2 signal (Vos et al. 2008)
was not credibly discernible in A. nidus interphase cells due
to the intense autofluorescence of the nucleoplasm in this
plant (see Fig. 2B, E and H). In order to confirm the in-
tranuclear origin of TPX2, we treated A. nidus leaf cells
with leptomycin-B, a potent inhibitor of nuclear export
(Kudo et al. 1998). In affected leaf areas, most cells exhib-
ited interphase appearance, while few preprophase cells
could be observed (Fig. 5). Apart from the above cells, no
other mitotic stage was observed as a result of treatment
duration (4 h): cells that were at mitosis by treatment initi-
ation had already accomplished cell division by treatment
completion, while few new mitotic cells emerged. Since lep-
tomycin-B does not selectively inhibit the nuclear export of
TPX2, a direct correlation of TPX2 absence from the cyto-
plasm with mitotic arrest would be highly speculative. In
the few preprophase cells, preprophase microtubule bands
and perinuclear microtubules were prominent, although the
latter did not assume bipolar organisation (Fig. 5A and D).
In the above cells, no TPX2 signal could be observed
around the nucleus (Fig. 5B, C, E and F, Figure S4; cf.
Fig. 2B and C, Figure S1). This supports that in A. nidus,
similarly to A. thaliana and N. tabacum (Vos et al. 2008),
TPX2 has to be exported from the nucleoplasm on the
nuclear surface.

A B C

D E F

Fig. 5. CLSM sections of leptomycin-B-treated leaf

segments, after a-tubulin (A and D) and TPX2 (B and
E) immunostaining, and their combination (C, F). Prep-
rophase cells in (A) and (D) exhibit preprophase bands
(arrows) and perinuclear microtubules. No TPX2 signal
can be observed around the nuclei of these leptomy-
cin-B-affected preprophase cells (B and E; see also
overlap in C and F). Scale bars = 10 lm.

Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands 5
TPX2 in Asplenium nidus Panteris, Adamakis & Chanoumidou

University) kindly provided access to the microscopes used

CONCLUSIONS
We identified a putative TPX2 homologue in the fern A.
nidus. Although not yet molecularly characterised, it
seems similar in size to TPX2 homologues of vertebrates
(ca. 100 kDa; Fig. 1). In addition, similar to its vertebrate ho-
mologues, it persists during the telophase ⁄ interphase transi-
tion. In particular, its co-localisation with cortical
microtubules under the daughter cell wall in post-cytokinetic
cells implies its involvement in localised MTOC activity. Since
a microtubule aggregation under the daughter cell wall has
also been reported in post-cytokinetic cells of bryophytes (Ap-
ostolakos & Galatis 1992), it might be interesting to investi-
gate whether TPX2 exhibits a similar localisation, establishing
a common feature among land cryptogamic plants.
ACKNOWLEDGEMENTS
We are grateful to Professor Anne-Catherine Schmit (Institut
de Biologie Moléculaire des Plants, CNRS, Strasbourg,
France) for the anti-AtTPX2 antibody. Dr. Penelope Mavra-
gani (School of Biology, Aristotle University) and Dr. Anasta-
sia Tsingotjidou (Faculty of Veterinary Medicine, Aristotle
in this study.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Figure S1. CLSM sections through the nuclei (A–C) and
projections of 20 sections (D–F) of A. nidus leaf protoderm
after a-tubulin (A and D) and TPX2 (B and E) immuno-
staining, and their combination (C and F).
Figure S2. CLSM sections of prophase cells after a-tubulin
immunostaining.
Figure S3. Fluorescence microscopy images of A. nidus
dividing cells after a-tubulin (A and D) and TPX2 (B and E)
immunostaining, and DNA counterstaining with Hoechst
33,258 (C and F).
Figure S4. Projection of 20 CLSM sections of the cell
depicted in Fig. 5D–F.
Please note: Wiley-Blackwell is not responsible for the con-
tent or functionality of any supporting materials supplied by
the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.

REFERENCES
Apostolakos P., Galatis B. (1992) Patterns of microtu-
bule organization in two polyhedral cell types in
the gametophyte of the liverwort Marchantia palea-
cea Bert. New Phytologist, 122, 165–178.
Binarová P., Cenklová V., Procházková J.,
Doskočilová A., Volc J., Vrlı́k M., Bögre L. (2006)
c-Tubulin is essential for acentrosomal microtubule
nucleation and coordination of late mitotic events
in Arabidopsis. The Plant Cell, 18, 1199–1212.
Demidov D., Van Damme D., Geelen D., Blattner
F.R., Houben A. (2005) Identification and dynam-
ics of two classes of Aurora-like kinases in Arabid-
opsis and other plants. The Plant Cell, 17, 836–848.
Eleftheriou E.P., Palevitz B.A. (1992) The effect of
cytochalasin D on preprophase band organization
in root tip cells of Allium. Journal of Cell Science,
103, 989–998.
Evrard J.-L., Pieuchot L., Vos J.W., Vernos I., Schmit
A.-C. (2009) Plant TPX2 and related proteins. Plant
Signaling and Behavior, 4, 69–72.
Flanders D.J., Rawlings D.J., Shaw P.J., Lloyd C.W.
(1990) Re-establishment of the interphase microtu-
bule array in vacuolated plant cells, studied by con-
focal microscopy and 3-D imaging. Development,
110, 897–904.
Garrett S., Auer K., Compton D.A., Kapoor T.M.
(2002) hTPX2 is required for normal spindle mor-
phology and centrosome integrity during vertebrate
cell division. Current Biology, 12, 2055–2059.
Hasezawa S., Kumagai F. (2002) Dynamic changes
and the role of the cytoskeleton during the cell
cycle in higher plant cells. International Review of
Cytology, 214, 161–191.
Hasezawa S., Nagata T. (1991) Dynamic organization
of plant microtubules at the three distinct transi-
tion points during the cell cycle progression of syn-
chronized tobacco BY-2 cells. Botanica Acta, 104,
206–211.
Jenni V., Cattelan H., Roos U.-P. (1990) Immunoflu-
orescence and ultrastructure of mitosis and cell
division in the fern Athyrium filix-femina. Botanica
Helvetica, 100, 101–119.
Jeong S.Y., Rose A., Joseph J., Dasso M., Meier I.
(2005) Plant-specific mitotic targeting of RanGAP
requires a functional WPP domain. The Plant Jour-
nal, 42, 270–282.
Kawabe A., Matsunaga S., Nakagawa K., Kurihara D.,
Yoneda A., Hasezawa S., Uchiyama S., Fukui K.
(2005) Characterization of plant Aurora kinases
during mitosis. Plant Molecular Biology, 58, 1–13.
Kong Z., Hotta T., Lee Y.-R.J., Horio T., Liu B. (2010)
The c-tubulin complex protein GCP4 is required for
organizing functional microtubule arrays in Arabid-
opsis thaliana. The Plant Cell, 22, 191–204.
Kudo N., Wolff B., Sekimoto T., Schreiner E., Yoneda
Y., Yanagida M., Horinouchi S., Yoshida M. (1998)
Leptomycin B inhibition of signal-mediated nuclear
export by direct binding to CRM1. Experimental
Cell Research, 242, 540–547.
Lloyd C.W., Chan J. (2006) Not so divided: the com-
mon basis of plant and animal cell division. Nature
Reviews Molecular Cell Biology, 7, 147–152.
Meier I., Brkljacic J. (2009) Adding pieces to the puz-
zling nuclear envelope. Current Opinion in Plant
Biology, 12, 752–759.
Mineyuki Y., Palevitz B.A. (1990) Relationship
between preprophase band organization, F-actin
and the division site in Allium. Fluorescence and
morphometric studies on cytochalasin treated cells.
Journal of Cell Science, 97, 283–295.
O’ Brien L.L., Wiese C. (2006) TPX2 is required for
postmitotic nuclear assembly in cell-free Xenopus
laevis egg extracts. Journal of Cell Biology, 173, 685–
694.
Otegui M.S., Verbrugghe K.J., Skop A.R. (2005) Mid-
bodies and phragmoplasts: analogous structures
involved in cytokinesis. Trends in Cell Biology, 15,
404–413.
Panteris E., Adamakis I.-D.S. (2012) Aberrant micro-
tubule organization in dividing root cells of p60-
katanin mutants. Plant Signaling and Behavior, 7,
16–18.
Panteris E., Karali D.S. (2008) The role of new micro-
tubule assembly and MAP65 in microtubule bundle
formation in pavement cells of Asplenium nidus.
Journal of Biological Research, 10, 139–147.
Panteris E., Galatis B., Apostolakos P. (1991) Patterns
of cortical and perinuclear microtubule organiza-
tion in meristematic root cells of Adiantum capillus
veneris. Protoplasma, 165, 173–188.
Panteris E., Apostolakos P., Galatis B. (1992) The
organization of F-actin in root tip cells of Adian-
tum capillus veneris throughout the cell cycle. A
double label fluorescence microscopy study. Pro-
toplasma, 170, 128–137.
Panteris E., Apostolakos P., Galatis B. (1995) The
effect of taxol on Triticum preprophase root cells:
preprophase microtubule band organization seems
to depend on new microtubule assembly. Protoplas-
ma, 186, 72–78.
Panteris E., Apostolakos P., Gräf R., Galatis B. (2000)
Gamma-tubulin colocalizes with microtubule arrays
and tubulin paracrystals in dividing vegetative cells
of higher plants. Protoplasma, 210, 179–187.
Panteris E., Adamakis I.-D.S., Voulgari G., Papado-
poulou G. (2011) A role for katanin in plant cell
division: microtubule organization in dividing root
cells of fra2 and lue1 Arabidopsis thaliana mutants.
Cytoskeleton, 68, 401–413.
Pastuglia M., Azimzadeh J., Goussot M., Camilleri C.,
Belcram K., Evrard J.-L., Schmit A.-C., Guerche P.,
Bouchez D. (2006) c-Tubulin is essential for micro-
tubule organization and development in Arabidop-
sis. The Plant Cell, 18, 1412–1425.
Pay A., Resch K., Frohnmeyer H., Fejes E., Nagy F.,
Nick P. (2002) Plant RanGAPs are localized at the
nuclear envelope in interphase and associated with
microtubules in mitotic cells. The Plant Journal, 30,
699–709.
Petrovská B., Cenklová V., Pochylová Ž., Kourová H.,
Doskočilová A., Plı́hal O., Binarová L., Binarová P.
(2012) Plant Aurora kinases play a role in mainte-
nance of primary meristems and control of endore-
duplication. New Phytologist, 193, 590–604.

6 Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands
Panteris, Adamakis & Chanoumidou
Van Damme D., Bouget F.-Y., Van Poucke K., Inzé D.,
Geelen D. (2004) Molecular dissection of plant cyto-
kinesis and phragmoplast structure: a survey of GFP-
tagged proteins. The Plant Journal, 40, 386–398.
Vos J.W., Pieuchot L., Evrard J.-L., Janski N., Bergdoll
M., de Ronde D., Perez L.H., Sardon T., Vernos I.,
Schmit A.-C. (2008) The plant TPX2 protein regu-
lates prospindle assembly before nuclear envelope
breakdown. The Plant Cell, 20, 2783–2797.
Wick S.M. (1985) Immunofluorescence microscopy of
tubulin and microtubule arrays in plant cells. III.
Transition between mitotic ⁄ cytokinetic and inter-
phase microtubule arrays. Cell Biology International
Reports, 9, 357–371.
Plant Biology ª 2012 German Botanical Society and The Royal Botanical Society of the Netherlands
Wittmann T., Wilm M., Karsenti E., Vernos I. (2000)
TPX2, a novel Xenopus MAP involved in spindle
pole organization. Journal of Cell Biology, 149,
1405–1418.
Zachariadis M., Galatis B., Apostolakos P. (2000)
Study of mitosis in root-tip cells of Triticum turgi-
dum treated with the DNA-intercalating agent ethi-
dium bromide. Protoplasma, 211, 151–164.
Zachariadis M., Quader H., Galatis B., Apostolakos
P. (2003) Organization of the endoplasmic reticu-
lum in dividing cells of the gymnosperms Pinus
brutia and Pinus nigra, and of the pterophyte
Asplenium nidus. Cell Biology International, 27,
31–40.
TPX2 in Asplenium nidus
Zachariadis M., Quader H., Galatis B., Apostolakos P.
(2004) An inhibitor of the ATP-dependent endo-
plasmic reticulum Ca2+-pump affects spindle orga-
nization in dividing cells of the angiosperm
Triticum turgidum but not in species of gymno-
sperms and pteridophytes. Journal of Biological
Research, 2, 3–19.
Zhao Q., Brkljacic J., Meier I. (2008) Two distinct,
interacting classes of nuclear envelope-associated
coiled-coil proteins are required for the tissue-spe-
cific nuclear envelope targeting of Arabidopsis Ran-
GAP. The Plant Cell, 20, 1639–1651.
7