The organization of the cytoskeleton during meiosis in eggplant {Solanum

melongena (L.)): microtubules and F-actin are both necessary for

coordinated meiotic division
J. A. TRAAS
1
'
2
, S. BURGAIN
1
and R. DUMAS DE VAULX
1
l
INRA, BP94, 84140 Montfcivet, France*
2
I.V.T., Wageningen, The Netherlands
Address for reprints
Summary
Because two division planes form at right angles,
male meiosis in higher plants provides striking
examples of both division control and spatial pro-
gramming.
To investigate these processes we have stained
microtubules and actin filaments during male mei-
osis in the eggplant. Our results indicate the follow-
ing.
(1) That microtubules and their nucleation sites
are involved in the establishment of polarity; this is
supported by our observation that the drug CIPC
affects spindle polarity.
(2) That actin microfilaments are involved in
spindle formation and integrity, but not in the
establishment of polarity: cytochalasin B and D
affect the organization of the spindle microtubules,
but not their polarized distribution.
(3) That microtubules radiating from the
daughter nuclei at the cell poles during interkinesis
probably establish the future division plane by
concentrating actin in that plane (cf. the proposed
role of asters in positioning the contractile ring in
animal cells).
(4) That this concentration of F-actin in the
division plane may be involved in preparing the
cytoplasm for cytokinesis and in memorizing the
division plane (much as the preprophase band
observed in polarized tissues does).
(5) That phragmoplast formation is a two-step
process. No phragmoplast forms after metaphase I,
but a four-way phragmoplast forms after meta-
phase II, indicating that mitosis and cytokinesis are
not obligatorily coupled.
These studies demonstrate that actin and micro-
tubules are jointly involved in the spatial coordi-
nation of the division process.
Key words: cytoskeleton, eggplant, F-actin, microtubules,
meiosis.
Introduction
Meiosis has been studied mainly from the 'chromosomal'
point of view and much is known about the behaviour and
configuration of the chromosomes (reviews: John &
Lewis, 1965; Sybenga, 1975; Dickinson, 1988). Yet, the
cytoplasmic mechanisms that control meiosis, determine
chromosome pairing, define with great precision the
division planes and ensure the distribution of cytoplasm
between the daughter cells are of equal importance but
remain poorly understood. Because of this, some atten-
tion has been paid to the role of the cytoskeleton during
male meiosis in higher plants and changes in microtubu-
lar and, to a lesser extent, F-actin arrays have been
described for a number of species (Van Lammeren et al.
1985; Sheldon & Dickinson, 1986; Hogan, 1987; Sheldon
& Hawes, 1988). Such studies show that, as in normal
Journal of Cell Science 92, 541-550 (1989)
Printed in Great Britain The Company of Biologists Limited 1989
somatic mitosis, microtubules function in nuclear div-
ision, whereas in cytokinesis F-actin accompanies micro-
tubules in forming the phragmoplast. However, a num-
ber of important questions remain to be answered,
especially concerning the establishment and maintenance
of the well-defined division planes by which four haploid
microspores are separated by two meiotic divisions.
Recent findings have established that F-actin plays an
important role in determining the division plane of
somatic cells (Traas et al. 1987; Lloyd & Traas, 1988;
Lloyd, 1988). Actin filaments had been thought to be
absent during mitosis, but by avoiding aldehyde fixation
(by detergent extraction or electroporation) it was dis-
covered that a network of actin persists throughout
mitosis and cytokinesis. The actin envelopes the nucleus
and, by transvacuolar filaments, connects that organelle
to the cortex. In somatic mitosis the division plane is
541
predicted by the formation of a preprophase band (PPB)
of microtubules, but it is now apparent that actin
filaments also form a cortical band, so that the PPB is no
longer considered to consist of microtubules exclusively.
Even though the PPB microtubules disappear by meta-
phase, the radial nucleus-associated actin strands remain
in the division plane, providing a memory of the division
site and helping to guide the cytokinetic apparatus out
along the pre-determined path. The significance of these
observations is that actin and microtubules combine to
set up the division plane. In view of this, we have re-
examined meiotic division, which differs from mitosis in
several important aspects: there is no PPB, for example,
and there is no known basis for the four-square position-
ing of the haploid microspores produced by two success-
ive meiotic divisions. However, in this paper we now
report the presence of an equatorial system of F-actin that
predicts the first division plane. As in mitosis, micro-
tubule-actin interactions seem to be essential for the
spatial control of male meiotic division.
Materials and methods
Plant material
Two varieties of Solatium melongena (L.) were used for the
experiments: Ronde de Valence and Doutga. Plants were grown
under greenhouse conditions. Young buds were cut from the
plants and the stage of meiocyte development was determined in
one anther of each bud. For this purpose anthers were squashed
in water and examined in an Olympus BH2 microscope
equipped with Nomarski optics. This gives a satisfactory
estimate of the stage of the four or five remaining anthers as
microspore formation is highly synchronized within each bud.
Only anthers containing meiocytes at a stage prior to division
were used. This stage is characterized by the formation of the
thick callosic wall.
Anther culture and drug treatments
Intact anthers were removed from the buds and put in 3-5 cm
Petri dishes containing 2 ml solid ' T' medium (pH 59) without
hormones (concentrations of macro- and micronutrients, vit-
amins, sucrose and agar were as described by Chambonnet &
Dumas de Vaulx, 1983). Under these conditions meiosis
proceeds normally within 18 h of culture. The following drugs
were used: chloroisopropylphenyl carbamate (CIPC) (Sigma),
colchicine (Prolabo), taxol (a generous gift from NCI, Beth-
esda), cytochalasin B and D (Sigma), and phalloidin (Sigma).
The drugs were first solubilized in dimethyl sulphoxide
(DMSO) and subsequently diluted in liquid T medium.
Colchicine was also solubilized in water. The different concen-
trations used for each drug are given in Results. The final
DMSO concentration never exceeded 1 %. For drug treat-
ments, 1 ml of the appropriate solution was added to the anthers
on 2 ml of solid medium. For control experiments 1% (v/v)
DMSO in liquid medium was used.
Fluorescence microscopy
Satisfactory staining and stabilization of microtubules and F-
actin were only obtained when the cells were first extracted in a
detergent-containing buffer essentially as described by Traas et
al. (1987) and Hussey et al. (1987). As reported in those papers
direct fixation with glutaraldehyde or formaldehyde perturbed
and fragmented cytoskeletal elements. Anthers were cut in two
and the meiocytes, tetrads or young microspores were squeezed
out and suspended in the buffer containing 50mM-Pipes
(pH6-9), SmM-EGTA, 5mM-MgSO
4
, 5% (v/v) DMSO and
0-03 % (v/v) Nonidet P40. To this extraction medium, helicase
(IBF, France), cellulase Onozuka R-10 (Yakult, Japan) and
macerase (Calbiochem, USA) were added (0-5% of each
enzyme) in order to permeabilize the thick cell wall. Immedi-
ately after cell isolation, the suspended meiocytes or tetrads
were pipetted into an Eppendorf tube and allowed to settle.
DNA and F-actin staining
After 10 min of wall and cell extraction the supernatant contain-
ing cell debris, detergent and enzymes was removed. The pellet
was resuspended in extraction buffer containing 0-5/igml~
diamino-2-phenylindole (DAPI) for DNA staining and
0-5 ^gml~ rhodaminyl lysine phallotoxine (RLP, a generous
gift from Professor Wieland, Heidelberg, FRG) for F-actin
staining (see also Traas et al. 1987; Lloyd & Traas, 1988). Cells
in RLP and DAPI were viewed immediately in an Olympus
BH2 fluorescence microscope. In order to restrict fading 2%
l,4-diazabicyclo-(2,2,2)octan (DABCO) was added to the cell
suspension.
Microtubule staining
Microtubules were visualized using immunofluorescence. As
this procedure includes a number of washing steps and long
incubations in buffer without detergent, it was necessary to fix
the extracted cells first. For this purpose the pellets of deter-
gent- and enzyme-treated cells were resuspended in 1 ml of a
buffer containing Pipes (100mM, pH6-9), EGTA (5mM),
MgSO
4
(SmM) and formaldehyde ( 8% (w/v), freshly pre-
pared). Cells were allowed to settle and washed twice in buffer
without fixative. After a final wash in water they were allowed to
attach to poly-L-lysine (M
r
> 300000, Sigma)-coated coverslips.
Cells were then prepared for immunofluorescence using a
monoclonal anti-tubulin antibody (MAS 077, Sera Lab) and a
fluorescein isothiocyanate (FITC)-labelled second antibody
following standard procedures. The culture supernatant con-
taining the primary antibody (the YL 1/2 anti-yeast tubulin
originally prepared by Kilmartin et al. (1981)) was diluted 1: 50
(v/v) in Pipes (50mM; pH6-9) and 3 % (w/v) bovine serum
albumin (BSA). Citifluor (with glycerol; City University,
London) was used as an antifading agent. Preparations were
observed in an Olympus BH2-RFL microscope with exciter
filters BP-490 (continuous spectrum near 490nm), BP-545
(546 nm) and BP-405 (405 and 435 nm) for blue, green and
violet light. They were used with the appropriate barrier filters.
Results
Microtubules during meiosis
The different microtubular arrangements during meiosis
are represented in Fig. 1. No differences were found
between the two varieties of eggplant. At interphase I,
microtubules form a complex network extending from
the nucleus to the plasma membrane (Fig. 1A,B). The
microtubular network remains until prometaphase
although the number of cytoplasmic microtubules gradu-
ally decreases (Fig. 1A-E). At the same time the amount
of fluorescence surrounding the nuclear envelope in-
creases. When the chromosomes are fully condensed their
position at the inside the nuclear envelope appears to co-
localize with the concentrations of tubulin on the outside
(Fig. 1C,D). After breakdown of the nuclear envelope
542 jf. A. Traas et al.
microtubules invade the nucleus (Fig. IF) and an eccen-
tric spindle is formed with its pointed poles extending
into the cortical cytoplasm (Fig. 1G). After chromosome
division the two daughter nuclei, lying at one side of the
cell close to the plasma membrane, move to the two cell
poles (Fig. 1H). At this stage new microtubules already
start to grow out from the nucleus and at interphase II a
dense array radiates out from its surface to the plasma
membrane (Fig. II, J). However, no phragmoplast is
formed. As metaphase II approaches, these arrays are
partially depolymerized and microtubules only radiate
out from the nuclear poles. At metaphase II the two
spindles poles again associate with the cell cortex
(Fig. IK). In telophase, microtubules radiate out from
the daughter nuclei (Fig. 1L,M) but this time a four-way
phragmoplast is formed (Fig. IN). The cell wall is
formed centripetally. After cytokinesis microtubules re-
form a random interphase array (Fig. 10).
F-actin during meiosis (Fig. 2)
In interphase, F-actin forms a network filling the cyto-
plasm, extending from the nuclear envelope to the plasma
membrane (Fig. 2A). In prophase I, this network also
concentrates around the nucleus (Fig. 2B), but in con-
trast to the microtubules this network remains present in
the cytoplasm during meiosis. In metaphase I, actin also
co-distributes with the spindle microtubules (Fig. 2C).
In interphase II, when the microtubules radiate out from
the two nuclei, a dense web of F-actin invades the tubule-
free zone and forms a phragmoplast-like disk
(Fig. 2D,E). In many cells this equatorial web is more
dense at the cell cortex, this dense zone forming a ring
that indicates the future division site. This structure
disappears completely before the spindles are formed,
although actin bundles remain present throughout the
cytoplasm. During the second division, F-actin again co-
distributes with the spindle microtubules (Fig. 2F). In
telophase II, during phragmoplast formation, microfila-
ments are again concentrated in the future division plane,
before the microtubules (Fig. 2G). Later they are associ-
ated with the radiating microtubules (Fig. 2H). After
cytokinesis, the actin filaments reorganize into a random
array (Fig. 21).
Drug treatments
Pollen mother cells (PMCs) normally developed into
microspores in the cultured anthers within 18-24 h. 1 %
DMSO did not influence cell division markedly.
The results obtained using the different drugs are
summarized in Table 1. Taxol and phalloidin have both
been reported to affect cell division in plant cells (Weer-
denburg et al. 1986; Palevitz, 1980). However, in our
hands taxol (1-25 jUM) and phalloidin (5-20/ i gmP
1
) did
not influence meiosis significantly. Since cytoskeletal
organization was also unaffected it remains uncertain
whether these drugs effectively reached the meiocytes.
Therefore, only the results obtained using CIPC (50 ^M
to 1 raM), colchicine (50 fiM to 1 mM) and cytochalasin B
and D (10/iM to 100 /ZM) will be discussed in detail, as
they all had specific effects on meiosis.
The effect of CIPC on meiosis. CIPC is known to affect
the splitting or replication of spindle poles in diverse
organisms (Oliver et al. 1982; Clayton & Lloyd, 1984;
review: Gunning & Hardham, 1982) regardless of the
morphology of the polar organizers. This drug was
therefore used here in an attempt to disturb the symmetry
of meiosis. CIPC disturbed the meiotic divisions, causing
the formation of micronuclei in about 90% of the cells.
Usually 7-11 nuclei per cell were formed (Fig. 3C,E),
although a minority of the cells had fewer nuclei, the
latter often of unequal size. As judged from DAPI
staining, one or two divisions could occur in the presence
of the drug, although in most cells the multiple nuclei
formed during the first division (Fig. 3A). CIPC was
effective at concentrations of 50 jitA and higher. At 0-1 mM
virtually no normal divisions were found.
The effect of CIPC on the cytoskeleton. In interphase,
loose networks of short microtubule bundles, having a
fragmented appearance, were observed (Fig. 3D). At the
onset of cell division tubulin staining was concentrated
around the chromosomes (Fig. 3A). Incomplete, or
multipolar spindles were observed at metaphase. After
Table 1. Effects of inhibitors on meiosis
Inhibitor (concn) Effect on cell division Effect on microtubules Effect on F-actin
CIPC
Colchicine (50/iM-l mM)
Cytochalasin B or D
(10-2
Cytochalasin B or D
(20-100 IM)
Formation of micronuclei
Arrest of development
After 24 h, fragmentation of nuclei
Increased % of abnormal division
planes
Arrest of cell development
F-actin networks of short bundles
with fragmented appearance
No rearrangement in phragmoplast
Formation of non- or multipolar
spindles
No formation of phragmoplast
Interphase arrays of short
disorganized microtubules
Depolymerization of microtubules No direct effect on F-actin
networks, but actin
reorganization is arrested
Microtubule reorganization is
perturbed
No spindle or phragmoplast
formation
F-actin networks fragmented
Phalloidin and taxol treatments are not summarized here, as these drugs did not seem to affect the cytoskeleton or cell division. Therefore we
could not determine whether the inhibitors reached the meiocytes.
Eggplant cytoskeleton during meiosis 543
544 J. A. Traas et al.
Fig. 1. Microtubular organization during meiosis. Bar,
10 fim. A-O, are at the same magnification. A,B. Two focal
planes of an interphase cell. Microtubules at the cell cortex
(A) and in the cytoplasm (B) form random networks.
C,D,E. Cortical (D) and cytoplasmic (E) view of a prophase
cell. Cytoplasmic microtubules start to depolymerize and
tubulin staining is concentrated around the nucleus. At this
stage chromosomes are attached to the inner membrane of the
nuclear envelope (C: DAPI staining). Concentrations of
tubulin seem to correspond with the position of the
chromosomes (pointers). F. Late prophase. Microtubules
have invaded the nucleus and surround the chromosomes. At
this stage spindle fibres grow out to the membrane (out of
focus). G. Metaphase I. Spindle with its pointed poles
extending into the cell cortex (pointers). Note that the
spindle is eccentric. H. Telophase I. The daughter nuclei
move to the two opposite cell poles. Microtubules start to
radiate out from the nuclear surface. I J . Interkinesis.
Microtubules radiate out from the nuclei many of them run
from nuclear envelope to plasma membrane. A cortical view
shows that only very short bits or only the ends of the
microtubules are attached to the membrane (J). Between the
nuclei there is a microtubule-free zone. K. Metaphase II.
Two spindles in parallel planes are formed simultaneously.
L,M,N. Telophase Il/cytokinesis. Microtubules radiate out
from the nuclei to form the phragmoplast. In L the cell was
slightly squashed and flattened, which makes it easier to
interpret microtubular organization at this stage. In N the
newly formed wall that forms centripetally is visible. O. Early
tetrad with random microtubular arrays.
Fig. 2. F-actin during meiosis,
DAPI staining not shown. Bars:
10 lira. A. Random interphase
network. B. Network in of cell
in prophase. Note increased
fluorescence around the nucleus.
C. Metaphase I. Actin is
associated with the spindle and
forms a network throughout the
cytoplasm. D. Interkinesis.
Actin is present throughout the
cytoplasm, although it is
concentrated between the
nuclei, indicating the future
plane of division. Pointers mark
the positions of the nuclei.
E. Interkinesis. Lower
magnification of the actin disk
in different cells. RLP staining
is brighter at the cell periphery
(arrows), showing that in many
cells the transcellular network is
more dense at the cell cortex,
this dense zone has the
appearance of a ring.
F. Telophase II. The
chromosomes have just moved
to the spindle poles. Note the
presence of F-actin in the
spindle. The F-actin disk seen
in interkinesis has completely
disappeared.
G,H. Phragmoplast formation.
First F-actin becomes
concentrated in the division
planes (G). Later it is more
intimately associated with the
microtubules (H). I. Early
tetrad with random
microfilament networks.
cell division there was no phragmoplast formation and an
irregular network of short bundles was re-formed, inter-
connecting the daughter nuclei. The F-actin system was
similarly affected. Often short filament bundles were
observed, giving the networks a fragmented appearance.
These networks remained present during cell division
and no obvious changes in their organization could be
observed (Fig. 3F,G).
The effect of colchicine on meiosis. Colchicine at con-
centrations of SOfiM and higher blocked cell development
Eggplant cytoskeleton during meiosis 545
Fig. 3. CIPC treatment. Bars: 10/im. A,B- Microtubule (A) and DNA (B) staining of a metaphase cell. A multi- or non-polar
spindle-like structure has been formed. C. DAPI staining of CIPC-treated cells showing the presence of micronuclei.
D,E. Tubulin (D) and DNA (E) staining of a cell after division. A random network of short microtubules interconnects the
micronuclei. F,G. Actin (F) and DNA (G) staining of a cell after cell division. At least four nuclei have formed, but there is no
phragmoplast.
Fig. 4. Colchicine treatment. Bar, 10^m. A-D are at the same magnification. A,B. Tubulin (A) and DNA (B) staining of a
cell in interkinesis. All microtubules have disappeared and only an irregular, tubulin-containing structure remains. C,D. Actin
and DNA-staining of a (pro)metaphase cell. F-actin network is present, but there is no concentration of actin around the
chromosomes.
and usually no microspores were formed in the presence
of the drug. Longer treatments with colchicine (36-48 h)
caused the fragmentation of a number of nuclei that were
probably in division. However, in contrast to CIPC-
treated cells, micronuclei were never observed.
The effect of colchicine on the cytoskeleton. Colchicine
caused the depolymerization of the microtubular array in
most cells. However, irregular, tubulin-containing struc-
tures remained present (Fig. 4A,B). In a number of cells
fragmented networks could still be observed. F-actin
networks were present at all stages although no spindle or
phragmoplast-like structures were observed (Fig. 4C,D).
After 2448 h the networks became increasingly frag-
mented.
The effect of cytochalasin B and D on meiosis. At
concentrations higher than 20 ^M cytochalasin B or D
caused arrested cell development. However, at lower
concentrations (10/iM) the formation of abnormal div-
546 Jf. A. Tracts et al.
eft
Fig. 5. Cytochalasin B treatment (30fiM). Bars: lO^Jm. A,B. Tubulin and DNA staining in a prophase cell. The chromosomes
start to condense (B) and niicrotubule organization seems normal. C,D. Tubulin and DNA in a metaphase I cell. No spindle is
present. Microtubules run more or less parallel to each other from one pole of the cell to the other. They seem to grow out from
putative nucleating centres at the cortex. E. Microtubules in a cell after cell division. There is no phragmoplast formation.
F. F-actin staining, showing that the bundles are fragmented. G,H. DAPI staining of a number of cells with abnormal division
planes formed in the presence of cytochalasin B at 10[1M.
ision planes and of dyads was observed in about 5 % of
the cells (Fig. 5G,H). This was higher than the 1%
(mostly dyads) usually observed in controls.
The effect of cytochalasin B and D on the cytoskeleton.
Cytochalasin, at concentrations of 10jJM and higher,
disturbed the reorganization of the microtubular system
during meiosis. In pre-meiotic cells and cells in prophase
normal microtubular networks were observed (Fig. 5A).
In dividing cells, however, spindles were usually absent
(Fig. 5C). Phragmoplasts were never observed
(Fig. 5E). Instead, dividing cells usually possessed a
number of thick microtubule bundles that in metaphase/
telophase were orientated more or less parallel to each
other (Fig. 5C). The F-actin bundles were always highly
fragmented (Fig. 5F).
Discussion
The way in which plant cell division is coordinated is one
of the major aspects of plant morphogenesis. Because of
this, much attention has been paid to the cytoskeleton,
since it plays a key role in establishing polarity and in
determining the division planes with great precision
(reviews: Lloyd, 1987; Traas, 1989). Descriptions of the
microtubular component of the cytoskeleton have domi-
nated the literature but recent findings show that F-actin
plays an important part in spatial control (Traas et al.
1987; Schmit & Lambert, 1987; Kakimoto & Shibaoka,
1987; Lloyd & Traas, 1988). From these studies it
appears that F-actin could provide a cytoplasmic frame-
work that supports and organizes the cytoplasm during
cell division (for discussion, see also Lloyd, 1988). As
yet, the exact role of F-actin during the different stages of
division remains unclear and more information is needed
to complete existing models on cytoskeletal functioning.
Here, we have studied meiosis partly as an essential step
in pollen development, but even more importantly as a
typical example of cell division under strict geometrical
control. The PMCs are apparently unpolarized and the
two meiotic divisions involve the establishment of spindle
polarity and the subsequent determination of two div-
ision planes at right angles to each other. Meiosis
therefore provides an interesting tool for studying general
aspects of the coordination of cell division.
The formation of the spindle: the establishment of
polarity
In pre-meiotic interphase both microtubules and actin
filaments form random networks throughout the cyto-
plasm. During prophase both filamentous systems be-
come more dense around the nucleus (see also Van
Lammeren et al. 1985; Sheldon & Dickinson, 1986;
Hogan, 1987). At this stage the condensed chromosomes
move to the inner face of the nuclear envelope and it has
Eggplant cytoskeleton during meiosis 547
been suggested that microtubules might function in
chromosome pairing (Sheldon & Dickinson, 1986).
The PMCs do not show any obvious polarity until the
establishment of the spindle. This involves the concen-
tration of microtubule nucleation sites (MTNS) at the
two poles of the nucleus. This process is disturbed by
CIPC, which causes the splitting or replication of spindle
pole bodies of various structures. Similar results have
been reported for animal, algal, monocotyledonous and
dicotyledonous cells, suggesting that a phylogenetically
'universal' mechanism is affected (e.g. see Oliver et al.
1978; Clayton & Lloyd, 1984; Gunning & Hardham,
1982, for review). Our results do not support a role for F-
actin in determining spindle polarity: even in the pres-
ence of high concentrations of cytochalasin the micro-
tubular system retains a clear polarity. Interestingly,
similar results have been obtained with animal cells. For
instance, during early embryogenesis of Caenorhabditis
elegans centrosomal movements along the nuclear surface
are perturbed by anti-microtubule drugs but not by
cytochalasin D (Hyman & White, 1987; see also De
Brabander et al. 1986).
As soon as the MTNS are concentrated at the two poles
of the nucleus a spindle is formed that has pointed poles,
in contrast to the barrel-shaped spindles usually observed
in higher plant cells. We have observed that these poles
are always associated with the cell cortex and it is possible
that membrane-microtubule interactions are involved in
maintaining the position of the spindle.
RLP stains filaments and bundles within and around
the meiotic spindles of eggplant. This is not in agreement
with Sheldon & Hawes (1988), who could not localize F-
actin in association with the spindle microtubules and
concluded that both cytoskeletal systems act indepen-
dently during metaphase-telophase. Our results, how-
ever, are supported by a number of recent reports on
mitotic plant cells showing that actin does associate with
microtubules throughout division (Traas et al. 1987;
Schmit et al. 1985; Kakimoto & Shibaoka, 1987) and the
conflicting evidence could simply reflect differences in
stability of the mitotic actin system under different
preparative conditions.
It has been proposed that actin could function in
chromatid separation (Forer et al. 1979; Cande et al.
1977; Seagull et al. 1987). However, this is not firmly
established and it appears from in vitro experiments that
microtubule depolymerization is sufficient to explain
chromosome movement without the need for actin as a
force generator (e.g. see Koshland et al. 1988; Gorbsky et
al. 1988). Our results indicate a completely different role
for actin in mitosis: cytochalasins perturb spindle forma-
tion and therefore actin could be involved in spindle
formation or organization, i.e. in the reorganization of
the microtubular array during division. Likewise,
Kobayashi et al. (1987) have proposed that actin fila-
ments are actively involved in the re-alignment of micro-
tubules during differentiation of tracheary elements of
Zinnia. In these cells microtubules and actin filaments
are co-parallel. The microtubules switch their orientation
from longitudinal to transverse during re-differentiation,
but cytochalasin B inhibits microtubular re-orientation.
Previous reports have shown that cytochalasins do not
affect spindle formation or functioning in a number of
plant cells (e.g. see Schmit & Lambert, 1987; Lloyd &
Traas, 1988; for discussion, see also Lloyd, 1988). One
should realize, however, that part of the F-actin popu-
lation is resistant to cytochalasin treatments: in carrot
cells, for example, cytochalasin B and D are unable to
depolymerize spindle-associated actin (Lloyd & Traas,
1988). Therefore, the contrasting results obtained with
various cell types could be explained in terms of differ-
ences in sensitivity to cytochalasins.
Metaphase I-interkinesis: the establishment of the
divisio)i planes
At telophase the daughter nuclei start to move to the two
opposite poles of the cell. They remain interconnected by
bundles of microtubules, which could function in the
migration by pushing the nuclei apart. During interkin-
esis microtubules still radiate out between the two nuclei.
The F-actin network that is still present throughout the
cytoplasm starts to concentrate in this zone and forms a
disk that divides the cytoplasm in two and indicates the
future division plane. This process closely resembles
early steps in phragmoplast formation in higher plant
cells and it has been proposed that the equatorial actin
functions in guiding the outgrowth of the new cell wall
(Schmit & Lambert, 1987; Lloyd & Traas, 1988).
However, PMCs of dicotyledons do not form a cross-wall
at this stage and therefore the equatorial actin system in
meiocytes must have a role other than helping to form a
dividing wall. Different observations suggest that a role
for F-actin may exist in the establishment of the future
division plane. (1) In a variety of plants, organelles
migrate to the equatorial region after the first meiotic
division, thus marking the future division site (Brown &
Lemmon, 1987, and references therein). The cytoplas-
mic disk of F-actin could obviously function in such a
process, re-distributing the cytoplasm in preparation for
cytokinesis. (2) The equatorial F-actin also extends to the
plasma membrane where the network seems to be more
dense. The disk of actin is therefore continuous across the
cell from one side to the other. As such it would
inevitably 'memorize' the division plane at the cortex. A
similar role has been proposed for the microtubular
preprophase band (PPB), which accurately marks the
future site of division at the cortex of polarized cells
(Lloyd, 1987; Traas, 1988, for reviews). At one time it
was thought that microtubules within the PPB were alone
responsible for marking the division site. Now it is known
that F-actin also occurs within that band (Palevitz, 1987;
Traas et al. 1987) as well as in the plane of the future
division site (Lloyd & Traas, 1988). It is important to
appreciate that there is no PPB of microtubules in
meiocytes to mark the division site. Perhaps the actual
plane of division in meiocytes is not initially fixed as it is
in somatic cells. However, it is clear that the actin
network alone is sufficient to form a raft that bi-lateralizes
the daughter nuclei and delineates the first division plane,
which is to be followed by another at right angles. The
absence of a PPB accentuates the involvement of F-actin
in these division processes. In cytokinesis in animal cells
548 jf. A. Traas et al.
F-actin is concentrated in the cortical division site (the
contractile ring) before and during furrowing. It has been
argued that astral arrays of microtubules radiating out to
the equatorial region determine the division planes (re-
view: Mabuchi, 1986). In plant meiocytes the micro-
tubules radiating out from the daughter nuclei to the
future division site could function in a similar way,
perhaps by transporting or guiding the actin network to
the equator.
The second meiotic division: spindle and phragmoplast
formation
During prophase II, microtubules extending freely into
the cytoplasm between the daughter nuclei start to
depolymerize, whereas those between the nuclear envel-
ope and the plasma membrane elongate. As in prophase
I, interactions with the membrane could help to stabilize
certain classes of microtubules, which then could partici-
pate in spindle formation/alignment.
After nuclear division, the microtubules again radiate
out from the nuclear envelope in telophase II. F-actin
invades the future division planes as it did after the first
division, but this time it remains there while the phrag-
moplast develops. Comparing the two meiotic divisions,
it seems that the concentration of actin in the division
planes has two distinct roles. First, the F-actin disk could
be involved in re-distributing the cytoplasm in prep-
aration for cytokinesis as was suggested above. Such a
process would be independent of the formation of a cross-
wall and constitutes a distinct step during meiosis.
During the second division, F-actin could initially func-
tion in the same way but, in remaining, would have the
additional role of guiding the outgrowth of the phragmo-
plast as proposed for other plant cells (Schmit & Lam-
bert, 1987; Lloyd & Traas, 1988). This suggestion is also
supported by the fact that cytochalasin treatments per-
turb the alignment of division planes and formation of the
phragmoplast. It is an interesting aspect of meiosis that
apparently the cell is able to uncouple these otherwise
closely linked steps, thus preventing phragmoplast for-
mation.
In summary, our results suggest that the microtubular
system primarily acts in the establishment of spindle and
cell polarity and that F-actin is involved in the establish-
ment and 'memorization' of the division planes and in the
organization of the cytoplasm during meiosis. Moreover,
reorganization of the cytoskeleton greatly depends on the
interaction between the two systems. These seem to be
general features not only of plant cell division but, as
discussed above, also of animal cell division.
It is tempting to describe processes like cell division
entirely in terms of microtubule dynamics. Yet, a number
of observations including our own also stress the import-
ance of microtubuleactin interactions in plant cell
division and it is likely that the cytoskeleton as a whole is
involved in the spatial control of cell division.
Meiosis offers an important tool for analysing the
coordination of cell division. Populations of meiocytes
can be obtained in which every cell is in division. Because
of their synchronous development, such cells will be
important in the next phase in which the molecular basis
of meiosis will be studied.
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