Download Microtubule and F-actin dynamics at the division site in living

Journal of Cell Science 103, 977-988 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
977
Microtubule and F-actin dynamics at the division site in living Tradescantia
stamen hair cells
ANN L. CLEARY*, BRIAN E. S. GUNNING1, GEOFFREY O. WASTENEYS† and PETER K. HEPLER‡
Plant Cell Biology Group and 1Co-operative Research Centre for Plant Sciences, Research School of Biological Sciences, The Australian National
University, GPO Box 475, Canberra, ACT 2601, Australia
†Present address: Max-Plank-Institut für Zellbiologie, Rosenhof, D-6802, Ladenburg, Germany
‡Present address: Department of Botany, University of Massachusetts, Amherst, MA 01003, USA
*Author for correspondence
Summary
We have visualised F-actin and microtubules in living
Tradescantia virginiana stamen hair cells by confocal
laser scanning microscopy after microinjecting rhodamine-phalloidin or carboxyfluorescein-labelled brain
tubulin. We monitored these components of the
cytoskeleton as the cells prepared for division at preprophase and progressed through mitosis to cytokinesis.
Reorganisation of the interphase cortical cytoskeleton
results in preprophase bands of both F-actin and microtubules that coexist in the cell cortex, centred on the site
at which the future cell plate will fuse with the parent
cell wall. The preprophase band of microtubules is
formed from microtubules that polymerise and incorporate tubulin during prophase. The preprophase band of
actin may form either by reorganisation of pre-existing
filaments or by de novo polymerisation. Both cytoskeletal components disappear from the future division site
approximately five minutes prior to the breakdown of
the nuclear envelope. Cortical microtubules are undetectable throughout mitosis and cytokinesis, whereas cortical F-actin remains abundant, although it is notably
excluded from the division site. The phragmoplast, containing both F-actin and microtubules, expands towards
the cortical actin exclusion-zone through a region that
has no detectable microtubules or F-actin. The phragmoplast comes to rest in the predefined region of the
cortex that is devoid of F-actin. It is proposed that cortical F-actin may act as a “negative” template which
could position the phragmoplast and cell plate correctly.
This is the first in vivo documentation of F-actin dynamics at the division site in living plant cells.
Introduction
PPBs (MTs and F-actin) disappear at prophase (Staiger
and Lloyd, 1991), and throughout mitosis MTs are
restricted to the spindle while F-actin may remain in cortical and cytoplasmic networks. In vacuolate cells, F-actin
extends through phragmosomal strands of cytoplasm lying
in the plane of division, and is anchored at the division site
in the cell cortex to which it may guide the expanding
phragmoplast (Goosen-de Roo et al., 1984; Traas et al.,
1987; Goodbody and Lloyd, 1990). However, neither
phragmosomes nor cytoplasmic F-actin in its position are
seen in densely cytoplasmic cells (Staiger and Lloyd, 1991).
To date, all views of the cytoskeleton at the division site
have been obtained from fixed or detergent-extracted material. With the advent of microinjection techniques for introducing fluorescent markers into living, walled plant cells
(Zhang et al., 1990) it is now possible to make direct observations of cytoskeletal dynamics.
The aim of the present investigation has been to examine the distribution of both MTs and F-actin in living
Tradescantia stamen hair cells while they establish their
The division site is a predetermined region of the cell cortex
at which the cell plate will fuse at the end of cytokinesis
(Gunning, 1982). The expanding phragmoplast is guided to
the division site (Baskin and Cande, 1990), which provides
factors for the anchoring and maturation of the cell plate
(Mineyuki and Gunning, 1990). This site assumes its specialised properties during prophase (Gunning and Wick,
1985) and retains these throughout mitosis (Mineyuki and
Gunning, 1990). The most common, visible manifestation
of the division site in higher plants is the preprophase band
(PPB) of microtubules (MTs; Pickett-Heaps and Northcote,
1966), a collection of parallel equatorial MTs that forms
during the G2 phase of the cell cycle (Mineyuki et al., 1988;
Gunning and Sammut, 1990), and accurately predicts the
plane of division. Actin filaments colocalise with MTs at
the division site in some (Palevitz, 1987; McCurdy and
Gunning, 1990) but not all cell types (Seagull et al., 1987;
Cho and Wick, 1990).
Key words: cell cortex, division site, F-actin, microinjection,
microtubules, preprophase band, Tradescantia.
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A. L. Cleary and others
division site, using microinjection of specific cytoskeletal
probes into pre-mitotic cells, followed by confocal laser
scanning microscopy. The previous study of MTs during
mitosis in living Tradescantia stamen hair cells, while
detecting the PPB, failed to reveal the more sparse cortical
MT arrays (Zhang et al., 1990). We document, for the first
time, the often rapid redistribution of cytoskeletal elements
in the cortex of a living higher plant cell during division.
We confirm that both MTs and F-actin become oriented circumferentially during prophase and that both break down
immediately prior to nuclear envelope breakdown. However, of particular note, we find that F-actin remains in all
other regions of the cell cortex except the division site.
These studies thus demonstrate a unique cytoskeletal structural specialisation of the cell cortex that persists throughout division.
Materials and methods
Cell culture
Tradescantia virginiana (L.) stamen hairs were dissected from
immature flower buds and immobilised in a thin film of 1%
Agarose VII (Sigma Chemical Co., St Louis, MO) containing
0.02% Triton X-100 (Sigma). Isolated hairs were cultured in a
medium containing 5 mM KCl, 0.1 mM CaCl2, and 5 mM Hepes,
pH 7.0. 1 mM probenecid (Sigma) was added to the culture
medium to retard sequestration of rhodamine-phalloidin into vacuoles.
Rhodamine-phalloidin
A 20 µl aliquot of stock (approx. 6.6 µM) rhodamine (Rh)-phalloidin (R-415, Molecular Probes Inc., Eugene, OR) was dried
down and resuspended in 5 µl of 100 mM KCl, sonicated for 10
min, and centrifuged (11,200 g) for 10 min at room temperature.
Tubulin
Sheep brain tubulin was extracted and tagged with 5-(and-6)-carboxyfluorescein succinimidyl ester as described by Wasteneys et
al. (unpublished). Pig brain tubulin, labelled with carboxyfluorescein, was kindly provided by Dr. Patricia Wadsworth, Department
of Biology, University of Massachusetts, Amherst, MA. Fluorescently labelled tubulin was diluted to 1-2 mg/ml in an injection
buffer (20 mM sodium glutamate, 0.5 mM MgSO4, 1 mM EGTA),
and centrifuged (11,200 g) for 10 min at 4°C.
Microinjection procedure
The procedure has been described in detail by Zhang et al. (1990).
Briefly, micropipettes constructed from borosilicate glass capillaries (World Precision Instruments Inc., Sarasota, FL) were backfilled with either Rh-phalloidin or carboxyfluorescein-labelled
tubulin and connected to a micrometer syringe via a water-filled
polyethylene tube. Microinjections were performed on a Zeiss
Axiovert 10 inverted microscope. Using a micromanipulator (Narishige Scientific Instrument Laboratory, Tokyo), the micropipette
was positioned against the side of the selected cell. Gentle tapping of the microscope resulted in the micropipette penetrating
shallowly into the cell. Hydraulic pressure was used to load the
cell with a small volume of solution, estimated to be about 1% of
cell volume (Zhang et al., 1990). The micropipette was removed
slowly (5-30 min) to allow the cell to form a callose plug around
the wound site.
Confocal laser scanning microscopy
Injected cells were imaged on a Biorad MRC-600 confocal laser
scanning system coupled to the inverted microscope on whose
stage the microinjections were performed. To minimise bleaching
of the fluorochromes and damage to the cells, the intensity of the
argon ion laser was reduced by introducing a 1% transmission
neutral density filter into the laser path. The overlap in the excitation spectra for fluorescein and rhodamine allowed us to use the
single channel blue excitation filter set for both fluorochromes. A
high numerical aperture (1.3 NA) Zeiss 40× oil-immersion lens
was used. Each image was compiled by averaging 10 frames using
the Kalman filter. High-resolution black and white monitor images
were photographed using Ilford Pan-F film (ASA 50). The state
of the cells was monitored throughout using differential interference contrast microscopy. In many cases confocal images of fluorescence were recorded along with transmission images in the
dual channel mode with a transmission detector.
Results
Microinjection of living cells
We describe here aspects of in vivo dynamics of cortical
MTs and F-actin in living Tradescantia stamen hair cells,
observed by confocal microscopy after microinjection of
either Rh-phalloidin or carboxyfluorescein-labelled tubulin.
An issue that needs to be addressed at the outset is whether
these experimental manipulations affect the course of cell
development. Two types of evidence indicate that the
images we have obtained depict cytoskeletal arrangements
similar to those in untreated cells. First, observations have
been made in cells in which microinjection had no adverse
effects on mitosis, which proceeded at the normal rate for
this material (e.g. Figs 8 and 13, see also Zhang et al.,
1990). Second, the veracity of sequential observations
obtained at various time intervals after microinjection has
been checked by comparing the appearance of all stages
attained post-injection, with the appearance of equivalent
stages in other cells observed immediately after microinjection. Thus a late telophase stage with a phragmoplast
looks the same in cells that have just been injected as in
cells injected one hour earlier (Figs 10A,B and 8O,P). This
comparison shows that microinjection of Rh-phalloidin
does not prevent a cell from rearranging existing actin filaments or forming a de novo array of F-actin.
Many cells that reach the stage of preprophase - late
prophase revert to interphase. This may arise from the
trauma of excision of the stamen hairs into culture medium,
since it occurs whether or not the cells are microinjected.
Consequently, it has proved difficult to obtain sequences of
cytoskeletal arrangements that occur before breakdown of
the nuclear envelope. Nevertheless, it has been possible to
compile complete developmental sequences from interphase to completion of cytokinesis using (a) images at
single time points (F-actin, 103 cells; MTs, 19 cells) placed
in order by reference to the state of the chromatin, the
appearance of the nuclear envelope, clear zone and mitotic
apparatus, all visible in differential interference contrast
views, and (b) from sequences (F-actin, 10 cells; MTs, 6
cells) that show changes preceding division, the entry into
mitosis and subsequent phases.
The duration of F-actin time sequences is limited by
sequestration of Rh-phalloidin into vacuoles and by its loss
into neighbouring cells. Short sequential observations can
be obtained before the reagent disappears from the initially
MTs and F-actin at the division site
stained actin filaments. For longer time courses we found
that inclusion of the anion transport inhibitor probenecid in
the culture medium was advantageous. Probenecid at 10
mM completely inhibited sequestration of Rh-phalloidin,
but in addition cytoplasmic streaming ceased, mitosis
stopped at metaphase and cytokinesis was disrupted. The
extent of the side effects diminished with decreasing concentrations of probenecid, so that at 1 mM no inhibition of
cellular processes occurred. Sequestration of Rh-phalloidin
occurred in 1 mM probenecid, but at a much reduced rate
compared to cells in a probenecid-free medium. There was
no observable difference between fluorescence images
obtained from cells cultured in the presence or absence of
probenecid. For cortical microtubules, long-term observations were affected by the stability of the carboxyfluorescein-tubulin complex and by bleaching in the confocal
microscope, but there was no difficulty in obtaining
sequences that were long enough for the purposes of this
investigation.
Actin
The F-actin we observe ranges from brightly fluorescent
strands to much fainter, finer fluorescence that is not resolvable as separate elements. The degree of filament bundling
cannot be determined by fluorescence microscopy, and we
use the term “filament” in a generic, descriptive sense, not
implying single filaments at the molecular level.
In interphase cells, sparse cortical F-actin is oriented longitudinally (19 cells; Fig. 1). As cells approach prophase,
actin filaments become coaligned into oblique (4 cells; Fig.
2) and finally into predominantly transverse (5 cells; Fig.
3) arrays extending the length of the cells. In one cell, the
shift of cortical F-actin from longitudinal to transverse
occurred within 12 min (Fig. 5). It is not until after the
transverse alignment of F-actin is achieved that a change
in chromatin morphology is detected.
As chromatin condensation proceeds, transverse cortical
F-actin becomes concentrated in a wide band at the equator of the cell, in which position it predicts the future plane
of division (7 cells; Fig. 4). In two cells representative of
this stage of mitosis, PPBs of actin developed within 15
Figs 1-4. Rh-phalloidin labelling of F-actin in the cortical
cytoplasm of four premitotic cells. Scale bar, 10 µm.
Fig. 1. F-actin aligned predominantly longitudinal.
Fig. 2. Obliquely oriented, coaligned actin filaments.
Fig. 3. F-actin is aligned predominantly transversely along the
length of the cell.
Fig. 4. Preprophase. A wide band of transversely oriented actin
filaments girdle the mid region of the cell. Obliquely oriented
actin filaments diverge from the band.
Fig. 5. Two images taken at the same focal plane, as evidenced by
the spots (arrowheads), showing the realignment of cortical Factin from (A) predominantly longitudinal [0 min] to (B)
transverse along the length of the cell [12 min]. Scale bar, 10 µm.
Fig. 6. Formation of a transverse band of cortical F-actin. (A,B)
Initially, most F-actin is aligned approximately transversely on
both the upper (A) and lower (B) surfaces of the cell. Oblique and
randomly oriented actin filaments are visible also. (C,D) In the
same cell 15 min later there is a concentration of transverse Factin at the mid region (arrowheads) of both (C) upper and (D)
lower surfaces. Scale bar, 10 µm.
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(Fig. 6) and 17 min. Filaments at the edge of the actin PPB
diverge at oblique angles and traverse the cortical cytoplasm to the ends of the cells (Figs 4 and 6). In mid-late
prophase the width of the actin PPB decreases slightly (21
cells). Actin filaments remaining at the ends of the cells
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A. L. Cleary and others
Fig. 7. Formation of an actin exclusion-zone (arrowhead) in the cortex of a prophase cell. Scale bar, 10 µm. (A) F-actin is distributed
throughout the cell cortex [0 min]. (B) The density of F-actin diminishes predominantly in the equatorial region [7 min]. (C) An actin
exclusion-zone is formed [15 min]. (D) Mid plane of the cell at 15 min. The nuclear envelope has just broken down and diffuse
cytoplasmic fluorescence (arrows) is present amongst the chromosomes.
retain oblique or longitudinal orientations or may become
randomised.
The actin PPB disappears prior to the breakdown of the
nuclear envelope (Figs 7 and 8). Initially, F-actin at the
edge of the band remains, while filaments within the central region of the band stain less brightly and appear to be
fragmented (9 cells; Fig. 8A). Subsequently, all cortical
actin filaments disappear from the division site at the equator of the cell (11 cells; Figs 7 and 8C,D). In approximately
50% of cells at this stage of mitosis, sparse longitudinally
oriented actin filaments are present in the subcortical equatorial region. In three cells, clearing of the F-actin PPB took
approximately 15 min, reaching completion 4 or less minutes before nuclear envelope breakdown. Although F-actin
is abundant elsewhere in the cortex and cytoplasm (Figs
8D-J and 9B), it remains conspicuously absent from the
division site throughout mitosis (16 cells; Figs 8 and 9).
While some of the cortical actin filaments appear to be
stable (compare Figs 8K and 8O), new arrays of F-actin are
detected in late anaphase (see also Zhang et al., unpublished). Two populations of longitudinally oriented, fine
actin filaments are present in the interzone between the two
sets of chromosomes (11 cells; Figs 8L and 9D). The length
and separation of the two F-actin populations decrease, producing a phragmoplast-like structure between the re-forming nuclei (12 cells; Fig. 8N). The F-actin phragmoplast
expands towards the region of the cell cortex that is bereft
of actin filaments (Figs 8O,P and 10A,B). There is no
detectable cytoplasmic F-actin between the parent wall and
the leading edge of the phragmoplast (Figs 8L,N). The
intensity of F-actin fluorescence differs significantly
between the phragmoplast and the cortex. This suggests that
F-actin in the cortex is in thick bundles while the phragmoplast is a dense assemblage of thinner actin filaments.
This differential staining is not affected by probenecid.
Intense staining of the phragmoplast is observed only in
cells that have been killed by overloading with Rh-phalloidin. At the completion of cytokinesis, there is a sparse
population of actin filaments in the cell cortex and these
still avoid the original actin exclusion-zone (Fig. 8E). Cytoplasmic actin is concentrated on the side of the nuclei distal
to the new cell wall (Figs 8N,P and 10B).
Microtubules
Fluorescently labelled tubulin from both pig and sheep
sources incorporates into cortical MT arrays. Using confocal microscopy, these MTs can be resolved against the diffuse background fluorescence caused by unpolymerised
dimers. In interphase, MTs that have incorporated fluorescently labelled tubulin are sparse, and distributed randomly
in the cell cortex and cytoplasm (Fig. 11A,B). In prophase,
when chromatin condensation is apparent, the number of
Fig. 8. The distribution of F-actin throughout division imaged in a
single cell. Scale bar, 10 µm. Cell cortex (A,C,E,G,I,K,M,O). Mid
plane (B,D,F,H,J,L,M,P). Time elapsed from the first pair of
micrographs is given in [minutes]. (A,B) Prophase [0]. In the cell
cortex there is a dense array of actin filaments everywhere except
in the equatorial region (arrowhead) where actin filaments are
shorter and more sparsely distributed. Thick transverse bundles of
F-actin delimit the lower boundary of the forming exclusion-zone.
In the mid plane of the cell cytoplasmic F-actin extends through
the cytoplasm. (C,D) Nuclear envelope breakdown [3]. Within the
forming cortical actin exclusion zone (arrowhead) the number of
actin filaments is greatly diminished and only a few faint
transverse actin filaments remain at the boundary. Chromosomes
are faintly silhouetted against diffuse cytoplasmic fluorescence.
(E,F) Prometaphase [6]. The exclusion-zone is clearly evident in
the cell cortex and is maintained throughout the rest of mitosis
(arrowhead). (G,H) Metaphase [24]. (I,J) Mid anaphase [44].
(K,L) Late anaphase [50]. Two populations of longitudinally
oriented, fine actin filaments are labelled in the interzone between
the two sets of chromosomes. (M,N) Mid telophase [60]. A
phragmoplast composed of F-actin is present between the reforming nuclei (n). (O,P) Late telophase [67]. The F-actin
phragmoplast has expanded towards the cell periphery and now
occupies the cortical actin exclusion-zone.
MTs and F-actin at the division site
labelled cortical MTs increases and these amass in a wide
band in the midplane of the cell (Fig. 11C,D). Subsequently,
the majority of MTs become oriented transversely (Fig.
12A,B) and consolidate into a tight PPB (Figs 12C,D and
13A,B). In cells considered to be representative of their
stage of mitosis, the redistribution of cortical MTs to form
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an incipient PPB took 11 min (Fig. 11), and consolidation
of the band took 17 min (Fig. 12). There are no MTs in
the cortex outside of the mature PPB (Figs 12C and 13A),
and MTs are sparse or absent throughout the cytoplasm
(Figs 12D and 13B). Eventually, individual MTs can no
longer be resolved (Fig. 13C,D). The PPB of MTs decreases
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A. L. Cleary and others
Fig. 9. F-actin in a mitotic cell progressing from metaphase through to the completion of cytokinesis. Scale bar, 10 µm. Images are taken
at the surface (A,C,E) and mid plane (B,D,F) of the cell. Time elapsed from the first pair of micrographs is given in [minutes]. (A,B)
Metaphase [0]. There is a region in the cell cortex (arrowhead) bereft of all but a few longitudinally oriented actin filaments (arrow).
There are numerous cytoplasmic actin filaments surrounding the spindle. A few longitudinal actin filaments run parallel to the silhouetted
chromosomes (arrowheads). (C,D) Late anaphase [95]. In addition to thick cytoplasmic F-actin associated with the vacuoles (v) and the
side of the reforming nuclei (n) proximal to the old cell walls, finer, longitudinally oriented F-actin is now detected in the interzone
between the nuclei. (E,F) Two daughter cells [130]. Short cytoplasmic actin filaments are present around the nuclei (n). No actin is
present between the nuclei or in the region of the cortex corresponding to the exclusion-zone of the parent cell.
Fig. 10. Late telophase cell viewed 19 min after injection with Rh-phalloidin. Scale bar, 10 µm. (A) Actin phragmoplast is present in the
cortical actin exclusion zone. Short, randomly oriented actin filaments are present elsewhere in the cortex. (B) Profiles of the
phragmoplast are present at the cell periphery (arrowheads) and cytoplasmic F-actin is concentrated on the side of the nucleus away from
the new cell wall.
in intensity as fluorescence at the nuclear envelope
increases (Fig. 13A-F). The disappearance of the MT PPB
commenced approximately 10 min (1 cell) before nuclear
envelope breakdown, reaching completion ≤5 min beforehand (2 cells; Fig. 13E-H). Fluorescently labelled pig brain
tubulin incorporates into all mitotic MT arrays (Fig. 14AD). The MT phragmoplast expands to reach the parent cell
wall at the expected division site, in the absence of any
guiding cortical or cytoplasmic MTs (Fig. 14D).
Discussion
We have been able to visualise dynamic changes in the
organisation of the cortical cytoskeleton in living Trades cantia stamen hair cells, including F-actin arrangements
that provide the first indication of the establishment of division polarity, and direct evidence concerning the mode of
formation of the MT PPB.
Cytoskeletal probes
Fluorescently labelled tubulin is an excellent probe with
which to examine MT dynamics. It has been shown that
tubulins isolated from foreign sources including sheep brain
(present study; Wasteneys et al., unpublished), pig brain
(Zhang et al., 1990) and Paramecium (Vantard et al., 1990)
can copolymerise with plant tubulin, and that the resulting
MTs are competent to function in mitosis in living plant
cells.
Rh-phalloidin injected into living Haemanthus
endosperm cells (Schmit and Lambert, 1990) and animal
tissue-culture cells (Sanders and Wang, 1991) labels the
MTs and F-actin at the division site
Fig. 11. Early prophase cell injected with fluorescently labelled
tubulin and viewed at (A,B) 0 min and at (C,D) 11 min. Scale bar,
10 µm. (A) A sparse array of long, randomly oriented MTs
(arrowheads) is present in the cortex. (B) Mid plane of focus
showing cytoplasmic MTs (arrowheads) near the cortex and
nucleus. (C) MTs have increased in number and are organised into
a wide band at the equator of the cell. (D) At the mid plane of
focus, the MT band is seen as two bright areas in the cortex on
either side of the nucleus (arrowheads).
complete F-actin array present at the time of injection. Reinjecting the same cells after they have progressed through
mitosis reveals new populations of F-actin. It is proposed
that new F-actin is formed by de novo polymerisation. By
contrast, images of F-actin in Tradescantia stamen hair cells
that have been labelled and observed as they progress
through mitosis are similar to cells viewed immediately
after injection at all equivalent mitotic stages. Thus the
entire F-actin array, which at a given time may consist of
pre-existing and newly polymerised filaments (Zhang et al.,
submitted), can be visualised over considerable periods of
time. We have documented the reorientation and subsequent
disappearance of cortical actin filaments at the division site,
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Fig. 12. MTs in a prophase cell viewed at (A,B) 0 min and at
(C,D) 17 min. Scale bar, 10 µm. (A) In the cell cortex, there is a
wide band of transversely oriented MTs with individual MTs
diverging from the edges of the PPB (arrowheads). (B) Mid plane
of the cell. (C) MTs have concentrated into a tight PPB and
individual MTs are no longer visible. (D) Mid plane of the cell.
and also the formation and expansion of the actin phragmoplast toward the cortical actin exclusion-zone.
Our ability to observe complete F-actin arrays over
extended periods is reliant on the use of the anion exchange
inhibitor probenecid (Cole et al., 1991). If probenecid is not
added to the culture medium, the F-actin signal dissipates
so that little or no label remains 15-60 min after injection.
This occurs concomitantly with an increase in diffuse fluorescence in the vacuoles and staining of F-actin in adjacent cells. The kinetics of the interaction between phalloidin
and plant F-actin are unknown. However, a 50% loss of
phalloidin binding to animal F-actins has been recorded in
180 s, 1.2 h and 2.3 h, depending on the source of the actin
and the type of phalloidin derivative (Cano et al., 1992, and
references therein). It has been shown also that phalloidin
and actin monomers dissociate together from filaments
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A. L. Cleary and others
Fig. 13. Images of MTs at two focal planes in a single cell progressing from late prophase to breakdown of the nuclear envelope. Cell
cortex (A,C,E,G). Mid plane (B,D,F,H). Time from the first pair of images is given in [minutes]. Scale bar, 10 µm. (A,B) Mature PPB in
the cell cortex (arrowhead) and few cytoplasmic MTs [0]. (C,D) Mature PPB (arrowhead) in the cortex and an increase in tubulin
fluorescence associated with the nuclear envelope [5]. (E,F) The PPB is barely visible in the cortex (arrowhead) and fluorescence is
intense around the nucleus [11]. (G,H) The PPB has disappeared (arrowhead). The nuclear envelope has broken down as evidenced by the
tubulin fluorescence amongst the chromosomes [15].
(Cano et al., 1992). Thus, the loss of F-actin-specific fluorescence in Tradescantia could result either from the joint
dissociation of phalloidin and monomeric actin from
depolymerising filaments or from the dissociation of phalloidin from stable filaments. The addition of 1 mM
probenecid delays sequestration of Rh-phalloidin into vacuoles for 1-3 hours. Unbound Rh-phalloidin would be available to bind (or rebind) to either existing or newly formed
actin filaments.
The maximum concentration of Rh-phalloidin after injection into Tradescantia stamen hair cells was 0.27 µM
(assuming a 1% increase in cell volume, Zhang et al., 1990).
We would expect the actual phalloidin concentration within
the cells to be significantly less than our estimate due to
the initial low solubility of Rh-phalloidin in the aqueous
injection buffer, and sequestering and loss to adjacent cells.
This low concentration has no adverse effect on cell mor-
phology, cytoplasmic streaming, or rate of mitosis (Zhang
et al., 1990), and is unlikely to stabilise existing filaments
(Dancker et al., 1975) or to promote actin polymerisation
(Wehland et al., 1977; Cano et al., 1992). Injection of a
low concentration of Rh-phalloidin to visualise F-actin in
living cells is a technique that has advantages over the use
of fixed cells (Lloyd, 1988; Baskin and Cande, 1990). While
the persistent cortical actin array was revealed in living
mitotic Tradescantia cells, neither it nor cytoplasmic actin
were detected following aldehyde fixation and staining with
Rh-phalloidin (Gunning and Wick, 1985). The fact that
cells remain alive after injection suggests that the introduction of artefacts is minimal. Nevertheless these results
need confirmation, for example through the imaging of fluorescently labelled plant actin that has been incorporated
into the endogenous pool. Parenthetically we note that fluorescent rabbit muscle actin injected into stamen hair cells
MTs and F-actin at the division site
985
mal cells occurs in 15 - 60 min (Goodbody and Lloyd,
1990).
The reorientation of cortical F-actin from longitudinal to
transverse occurs before there is any detectable chromatin
condensation and precedes the formation of an actin PPB
in the correct orientation. This phenomenon is similar to
the reorientation of cortical MTs observed in interphase
cells in which the expected plane of division is perpendicular or oblique relative to previous divisions (Cleary and
Hardham, 1989; Hush et al., 1990). In Tradescantia, the Factin reorients to match the future plane of division, prior
to either delineation of the division site or formation of
extensive arrays of labelled MTs. Cortical actin filaments
play an important, but as yet undefined, role in establishing cellular polarity in zygotes of fucoid algae (Kropf et
al., 1989). However, the results in Tradescantia are contrary to events in wounded pea roots, where cortical MTs
reorient prior to, and independently of, F-actin (Hush and
Overall, 1992).
Cortical MTs in young interphase Tradescantia stamen
hair cells visualised by both microinjection of labelled tubulin and ultrastructural examination (chemical and frozen
fixed cells; C. Busby and P.K. Hepler, unpublished observations), are not as prolific or well organised as those seen
by similar techniques in differentiating cells. Although the
MRC-500 model of the confocal microscope used by Zhang
et al. (1990) did not resolve the dispersed population of cortical MTs after microinjection, the present study has shown
that this component of the plant cytoskeleton can be seen
using similar preparations of labelled tubulin and the MRC600 confocal microscope (see also Wasteneys et al., unpublished). We believe that we are resolving individual MTs
in the cortex and cytoplasm, and associated with the nucleus
of interphase cells.
Fig. 14. Sheep brain tubulin incorporating into all components of
the mitotic spindle as a single cell progresses from prometaphase
to the end of cytokinesis. Time from the first pair of images is
given in [minutes]. Scale bar, 10 µm. (A) Prometaphase spindle
[0]. (B) Early anaphase spindle [9]. (C) Late anaphase spindle
[19]. (D) Phragmoplast [34].
(a) retards mitotic progression and (b) does not appear to
report dynamic activities of the endogenous F-actin system
(P.K. Hepler, unpublished observations).
Interphase arrays
We have visualised F-actin and MT arrays in the cortex of
interphase Tradescantia stamen hair cells. Actin filaments
are initially longitudinally oriented (present study; Tiwari
et al., 1984), but subsequently reorient to become transversely aligned along the length of the cell, an arrangement
detected also in extracted Tradescantia stamen hair cells
(Traas et al., 1987). Most changes in F-actin distribution in
Tradescantia stamen hair cells occur within 10 - 20 min, a
time course that is in accord with studies of actin dynamics in fixed plant cells; reinstatement of actin arrays in carrot
suspension culture cells after cytochalasin treatment takes
10 min (Traas et al., 1987), and reorientation of actin in
response to wounding of Tradescantia albovittata epider-
The division site
The division site in Tradescantia is identified by the appearance of transversely oriented bands of cortical MTs (Busby
and Gunning, 1980) and F-actin concomitant with the condensation of chromatin. Since its discovery by PickettHeaps and Northcote (1966), the PPB of MTs has been
shown to predict accurately the plane of division in many
higher plant cells (Gunning, 1982; Wick, 1991). One question which has been impossible to answer from studies of
fixed material is whether the MT PPB consists of new MTs
or pre-existing but rearranged MTs. The present study of
Tradescantia provides direct observations. It is apparent
from the number and distribution of MTs at interphase and
prophase, that new MTs must have been polymerised in
order to form the PPB. This is a rapid process, with the
PPB in one Tradescantia stamen hair cell appearing within
11 min. This provides support for the hypothesis that the
band is formed from newly polymerised MTs (Staiger and
Lloyd, 1991; Gunning, 1992). A potential site for MT
nucleation is at the nuclear envelope (Wick and Duniec,
1983; Tiwari et al., 1984; Clayton et al., 1985; Gunning,
1992; Chevrier et al., 1992). However, in Tradescantia
stamen hair cells there is no detectable increase in MT density at the surface of the nucleus during PPB formation.
Alternatively, PPB MTs could be nucleated in the cell
cortex (Hepler and Palevitz, 1974; Gunning et al., 1978).
986
A. L. Cleary and others
The available evidence does not support the “bunching up”
of pre-existing MTs as the mechanism of PPB formation
(Pickett-Heaps, 1969; Doonan et al., 1987; Lloyd and Traas,
1988; Flanders et al., 1990), although this remains a feasible proposition for the subsequent narrowing of the band.
The PPB is not instated as a coaligned group of MTs; rather,
transverse order is achieved as the band narrows to the midplane of the cell. The narrowing of the band and loss of
resolution of individual MTs is consistent with observations
of fixed material (Wick and Duniec, 1983; Gunning and
Sammut, 1990; Gunning, 1992).
Actin occurs at the division site (Kakimoto and Shibaoka,
1987; Palevitz, 1987; Traas et al., 1987; McCurdy and Gunning, 1990; Mineyuki and Palevitz, 1990; Cleary et al.,
1992), although its presence is not universal (Clayton and
Lloyd, 1985; Seagull et al., 1987; Cho and Wick, 1990).
Formation of the F-actin PPB in Tradescantia stamen hair
cells is achieved by retaining transverse F-actin in the midplane of the cell while the recently-formed transverse alignment is lost at the ends of the cells. Narrowing of actin
bands, as seen in Tradescantia, occurs also in cells of
Allium epidermis (Palevitz, 1987) and wounded Trades cantia epidermis (Goodbody and Lloyd, 1990), but not
wheat root tips (McCurdy and Gunning, 1990). At its most
developed, the PPB of F-actin in Tradescantia is wider than
the corresponding MT PPB, and individual F-actin bundles
are resolvable throughout.
Cytoskeletal inhibitors have been used to elucidate the
roles of MTs and F-actin in defining the division site. The
initial formation of the actin band has been shown to be
dependent on MTs (Katsuta et al., 1990; McCurdy and Gunning, 1990; Mineyuki and Palevitz, 1990). Conversely, formation of the MT PPB is independent of actin (Cho and
Wick, 1990; Katsuta et al., 1990; Mineyuki and Palevitz,
1990; Hush and Overall, 1992), although narrowing of the
MT band relies on the integrity of the F-actin system (Palevitz, 1987; Mineyuki and Palevitz, 1990; Mineyuki et al.,
1991a). Examination of sites of MT polymerisation and the
relative roles of MTs and F-actin in the formation of the
division site in Tradescantia should be possible using
microinjected cells.
Disappearance of PPBs
It is well established that the PPB of MTs disappears in
unperturbed cells in late prophase (Gunning, 1982; Wick
and Duniec, 1984; Murata and Wada, 1992) or early
metaphase at the latest (Traas et al., 1987). In most cells,
cortical actin also disappears before the breakdown of the
nuclear envelope, irrespective of whether the actin was
organised into PPBs (Palevitz, 1987; Seagull et al., 1987)
or networks (Clayton and Lloyd, 1985; McCurdy and Gunning, 1990). Cells that retain cortical actin networks
throughout mitosis include carrot (Traas et al., 1987; Lloyd
and Traas, 1988) and tobacco (Kakimoto and Shibaoka,
1987) suspension culture cells, and Haemanthus endosperm
cells (Schmit and Lambert, 1987). However, there is no
cytoskeletal delineation of the division site in these cells
during mitosis.
In Tradescantia, we have determined that both F-actin
and MTs disappear from the division site approximately 5
min (or less) prior to nuclear envelope breakdown. The
decrease in length of actin filaments and the loss of fluorescence at the PPB site suggests that F-actin disassembles.
We do know that the appearance of the exclusion-zone is
not due to lack of Rh-phalloidin, as it is seen in freshly
microinjected cells. We cannot, however, discount the possibility that F-actin may be selectively bound by factors,
such as accessory proteins, that prevent Rh-phalloidin from
staining it (Cano et al., 1992). Subject to this caveat, what
distinguishes Tradescantia stamen hair cells from other cell
types examined is that F-actin is not at the division site but
is retained elsewhere in the cell cortex. The general disruption, beginning in late prophase, of the previously highly
ordered system of cortical F-actin, correlates with the cessation of cytoplasmic streaming in these cells (Ota, 1961).
In wheat root tip cells also, there is evidence that F-actin
is absent from mature cortical PPB sites at mid-late
prophase (Fig. 4C-F of McCurdy and Gunning, 1990);
thereafter cortical F-actin was not detected by antibody
localisation in the fixed cells. In mitotic Haemanthus
endosperm cells, which have no predefined plane of division and lack PPBs of MTs (De Mey et al., 1982), cortical
actin filaments in the midplane of the cells reorient to lie
parallel to the spindle axis while remaining as a network
(interphase configuration) at the spindle poles (Schmit and
Lambert, 1987, 1990). Schmit and Lambert (1987) suggest
that the realignment is brought about by mitotic stresses but
they do not exclude the possibility that the changes result
from clearing of transverse actin filaments in the midzone.
The cell cycle kinase p34cdc2 has been detected in all
eukaryote cells studied to date (Lamb et al., 1990; and references therein), including plant cells (John et al., 1989).
Microinjection of p34cdc2 alters cytoskeletal (MT and Factin) organisation in living mammalian cells (Lamb et al.,
1990), and its putative detection in the PPB of higher plants
(Mineyuki et al., 1991b) suggests a role in the regulation
of the PPB. Recent experimental evidence has shown that
cytoplasmic factors are involved in the disappearance of the
MT PPB (Murata and Wada, 1992), and that cortical MT
depolymerising factors are ATP-dependent (Sonobe, 1990).
It has also been proposed that MTs may be differentially
regulated by Ca2+/calmodulin complexes activated by
localised ion fluxes (Cyr, 1991).
Function of the division site
The function of the cortical division site is to (i) provide
precise spatial guidance for the expanding phragmoplast
(Gunning, 1982) and (ii) provide receptive sites and maturation factors for the cell plate (Hepler and Palevitz, 1974,
Mineyuki and Gunning, 1990). The question remains as to
how these functions are executed in plant cells.
In vacuolate cells, the phragmosome, a network of transvacuolar strands that extend in a plane between the
nucleus/spindle and the cortex, accurately predicts the plane
of division (Sinnott and Bloch, 1941). Phragmosomes have
been recorded in cells with and without PPBs (Gunning and
Wick, 1985). MTs (Bakhuizen et al., 1985; Flanders et al.,
1990; Katsuta et al., 1990) and F-actin (Goosen-de Roo et
al., 1984; Kakimoto and Shibaoka, 1987; Seagull et al.,
1987; Traas et al., 1987; Lloyd and Traas, 1988; Goodbody
and Lloyd, 1990) have been identified within the phragmosome, and both are necessary for phragmosome forma-
MTs and F-actin at the division site
tion (Venverloo and Libbenga, 1987). Phragmosomes are
not usually visible in Tradescantia stamen hair cells, but
they can appear during division (Gunning, 1982) and after
centrifugation (Ota, 1961). Staiger and Lloyd (1991)
suggest that the distribution of cytoplasmic MTs and F-actin
would define the “phragmosomal” cytoplasm in densely
cytoplasmic cells and that these elements may be destroyed
by aldehyde fixation. Neither our study of living Trades cantia stamen hair cells nor a previous study of aldehydefixed cells (Gunning and Wick, 1985) revealed phragmosomal F-actin. Thus, the proposal that phragmosomal actin
guides the expanding phragmoplast to the division site
(Lloyd and Traas, 1988) does not seem applicable to
Tradescantia stamen hair cells.
While there is no apparent cytoskeletal basis for longdistance guidance of the phragmoplast in Tradescantia
stamen hair cells, the persistent cortical array of F-actin
could be involved in the final positioning of the cell plate.
Ota (1961) showed that if the spindle in a mitotic Trades cantia stamen hair cell was displaced by centrifugation, the
growing cell plate expanded to the lateral walls but did not
fuse until it and the nuclei migrated back to the expected
division site. Ota (1961) described this phenomenon as the
cell plate “sliding” along the parent wall. One may envisage a mechanism whereby cortical actin provides a guide
along which a displaced cytokinetic apparatus moves, via
interactions between the phragmoplast F-actin, MTs or
associated proteins and the cortical F-actin. The cell plate
could become immobilised in the region of the cell cortex
in which F-actin is absent (or obscured). Similarly, in
Triticum seedlings that are continuously centrifuged, most
divisions that produce guard mother cells have cell plates
that are able to locate the predefined division site (Galatis
et al., 1984). By contrast, in the highly asymmetrical divisions that produce subsidiary cells, often only part of the
cell plate fuses at the site of the PPB. Actin, as we have
described in Tradescantia, could be involved in positioning
of these cell plates, but may fail when the nuclei are held
in irregular positions. We note that actin exclusion-zones
have not been visualised in the cortex of fixed or extracted
stomatal cells (Cho and Wick, 1990). While we have considered cells that have been centrifuged, the mechanism
could apply also to unperturbed cells where reorientation of
late anaphase-telophase spindles is necessary for correct
alignment of the cell plate. Previous studies of Tradescantia
stamen hair cells (Gunning and Wick, 1985; Mineyuki and
Gunning, 1990) and guard mother cells (Palevitz and Hepler,
1974; Cho and Wick, 1990), have shown that placement of
the cell plate is sensitive to cytochalasin B.
Mineyuki and Gunning (1990) have discussed the accumulation of cell plate maturation factors at the division site
in Tradescantia stamen hair cells. Our results do not add to
their observations, except to suggest that F-actin, as well as
MTs, may be involved in imparting specialisation to the
division site. MTs (Seagull and Heath, 1980) and F-actin
(Jung and Wernicke, 1991) may function in controlling the
distribution of membrane components such as cellulose-synthesising complexes. Nevertheless, in Tradescantia, there is
no evidence that specialisation of the division site involves
the localised deposition of wall thickenings (Packard and
Stack, 1976; Galatis et al., 1982; Sawidis et al., 1991).
987
We thank Dr. Patricia Wadsworth, Department of Biology, University of Massachusetts for supplying us with fluorescently
labelled pig brain tubulin. P.K.H. acknowledges the support of a
Fulbright Grant from the Australian-American Educational Foundation and a United States Department of Agriculture Grant (9137304-6832). G.O.W. is supported by a Queen Elizabeth II
Fellowship from the Australian Research Council.
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(Received 6 July 1992 - Accepted, in revised form, 24 August 1992)