Download Plasma Membrane Ghosts Form Differently When Produced from

Plant Cell Physiol. 40(1): 36-46 (1999)
JSPP © 1999
Plasma Membrane Ghosts Form Differently When Produced from
Microtubule-Free Tobacco BY-2 Cells
David A. Collings 1 , Tetsuhiro Asada and Hiroh Shibaoka
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka, 560 Japan
When lysed in an actin stabilizing buffer, protoplasts
made from tobacco BY-2 suspension culture cells formed
plasma membrane ghosts that retained both cortical actin
and microtubules. Distinct cytoskeletal arrays occurred:
the most common ghost array (type I) derived from
protoplasts in interphase and had random actin and microtubules, although the alignment of the actin was dependent, at least partially, on microtubule organization.
Type II ghosts were larger and more irregular in shape than
type I ghosts, and were characterized by a lack of microtubules and the presence of distinctive arrays of actin
bundles in concentric arcs. These ghosts derived from
protoplasts lacking cortical microtubules produced when
wall digestion occurred while the cells were in cell division, or from protoplasts isolated in the presence of 100
//M propyzamide. Because type II ghosts derived from
protoplasts of similar size to those that give rise to type I
ghosts, and because type II ghosts retained ordered actin
arrays while the parent protoplasts had random cortical
actin, type II ghosts apparently form differently to type I
ghosts. We speculate that instead of the protoplast being
sheared off to produce a round ghost, the plasma membrane tears and collapses onto the slide, ordering the actin
bundles in the process. One implication of this model
would be that cortical microtubules provide structural
support to the plasma membrane of the protoplast so that
only in their absence do the type II ghosts form.
walls (Mayumi and Shibaoka 1996, references therein;
Takesue and Shibaoka 1998). The cortical array undergoes rapid turnover (Hush et al. 1994, Hepler and Hush
1996) and its depolymerization with agents such as colchicine (Delay 1957, Green 1962, Hogetsu and Shibaoka 1978)
or oryzalin (Wasteneys 1992) transforms cell growth from
controlled elongation to isodiametric expansion by removing the controls on cellulose deposition. Cortical microtubules are retained after cell elongation ceases, although the
pattern of the microtubules changes away from transverse to longitudinal or oblique depending on the system,
and while ho longer controlling cell elongation, they still
function in the control of secondary wall deposition
(Giddings and Staehelin 1991). That these cortical microtubules closely associate with the plasma membrane has
been demonstrated by numerous microtubule ghosting
studies in which isolated patches of plasma membrane
retain cytoskeletal elements after the cortical cytoplasm has
been washed away (Marchant 1978, van der Valk et al.
1980, Kakimoto and Shibaoka 1986).
Cortical microtubules have at least one other functions within plants. Prior to the onset of mitosis, cortical
microtubules redistribute into a narrow strip, the preprophase band, which marks the ultimate division site
(Gunning 1982), possibly through the localized depolymerization of cortical microfilaments to leave a zone of actin
depletion (Cleary et al. 1992, Liu and Palevitz 1992). Several other functions for the cortical microtubules have
also been investigated, although not necessarily proved.
Animal and lower eukaryotic cells use microtubules as
tracks for vesicle motility, using microtubule motors of the
kinesin (Moore and Endow 1996) and dynein superfamilies
(Holzbaur and Vallee 1994) but to date, while these proteins may function in vesicle movement in pollen tubes,
there is no evidence for vesicle movement along the cortical microtubules of interphase higher plant cells (Asada
and Collings 1997). It was recently reported that microtubules in carrot protoplasts could regulate voltage-gated
calcium channels so that microtubule depolymerization increased the time that channels stayed open (Thion et al.
1996). It is also conceivable that the extensive cortical microtubule cytoskeleton might help stabilize or modulate the
plasma membrane, in a way analogous to the actin cytoskeleton in many lower eukaryotic and animal cells, but
there is no evidence to support this hypothesis. However, a
critical limitation exists for all the functions that cortical
Key words: Actin — Cytokinesis — Microtubules —
Mitosis — Plasma membrane ghost — Tobacco BY-2 cells.
The cortical microtubule cytoskeleton is important in
the control of plant morphogenesis, because microtubules control the direction of cell expansion by orienting
microfibril deposition in the cell wall (Green 1962, Giddings and Staehelin 1991). Cortical microtubules in elongating cells often align transversely giving transversely
aligned cellulose microfibril deposition (Shibaoka 1994,
Cyr and Palevitz 1995, Wymer and Lloyd 1996) but can
undergo cyclic reorientations that generate polylamellate
1
Corresponding author: David Collings, Department of Botany,
North Carolina State University, Raleigh, NC 27695-7612, U.S.A.
E-mail: [email protected]; fax: +1 919 515 3436.
36
Membrane ghosts from microtubule-free cells
microtubules could potentially support at the plasma
membrane, as the microtubules depolymerize during cell
division to form division specific arrays (Gunning 1982,
Baskin and Cande 1990).
We have previously demonstrated that membrane
ghosts produced from tobacco BY-2 suspension culture
protoplasts retain both cortical microtubules as well as an
extensive cortical actin cytoskeleton (Collings et al. 1998).
In this paper we show that while a large majority of ghosts
retained both actin and microtubules, a certain percentage of ghosts lacked microtubules and had distinctive actin
patterns. These ghosts derived from protoplasts that lacked
microtubules, either because they had not completed cell
division, or because of depolymerization induced by agents
such as propyzamide. Furthermore, the differences in actin
pattern suggest that there may be differences in the mechanisms of ghost formation, and thus that the cortical microtubules contribute to plasma membrane stability.
Materials and Methods
Plant material—Tobacco (Nicotiana tabacum L.) suspension
culture line BY-2 was subcultured weekly in modified LinsmaierSkoog medium (Akashi et al. 1988) and when necessary, synchronized by subculturing in aphidicolin (5/iM, 24 h) (Nagata et
al. 1981). Protoplasts were made from cells by wall digestion in
1% sumizyme (Shin-Nihon Kagaku Kogyo, Aichi, Japan), 0.1%
pectolyase Y23 (Seishin Pharmaceuticals, Tokyo, Japan), 0.5%
BSA and 0.3 M mannitol dissolved in modified LinsmaierSkoog-based buffer at pH 5.5 at 30°C for 60 to 70min, and the
resulting protoplasts pelleted, washed twice in wash buffer (10
mM PIPES pH 6.8, 100 mM KC1, 285 mM mannitol), and resuspended in wash buffer. Microtubule-free protoplasts were generated from whole cells that had been pretreated with propyzamide (10 or 100 /uM, 3h) (Akashi et al. 1988), with propyzamide
being included in the subsequent digest and wash buffers. Microtubule-stabilized protoplasts were made by incubating whole cell
prior to (30min) and during wall digestion with taxol (10/JM)
(Wako Pure Chemicals, Osaka, Japan).
To determine the mitotic index of samples, cells were fixed in
1% glutaraldehyde in either modified Linsmaier-Skoog buffer
(whole cells) or wash buffer (protoplasts), washed in PBS and then
stained with DAPI (1 fjg ml" 1 ). Protoplast diameters were measured from photographs of fixed protoplasts, and surface areas
calculated with the assumption that the protoplasts were spherical. The areas of plasma membrane ghosts were determined from
digitised photographic negatives using the imaging program NIH
image (NIH, Bethesda, MD, U.S.A.).
Immunolabeling of whole cells and protoplasts—Whole cells
were fixed and double-labeled for actin and microtubules.
Cells were attached to poly-L-lysine coated multiwell slides, and
pretreated with 400 fxM 3-maleimidobenzoyI-/v'-hydroxy-succinimide ester (MBS) in microtubule-stabilizing buffer (50 mM
PIPES, 5 mM EGTA, 2 mM MgSO4, pH 6.9 that contained 0.1%
triton X-100 and 200 ^M phenylmethylsulfonyl fluoride (PMSF)
(30min). Material was then fixed in stabilizing buffer containing
4.0% formaldehyde (30 min) and washed in phosphate buffered
saline (PBS; 2.68 mM KC1, 1.47 mM KH2PO4, 13.69 mM NaCl,
0.81 mM Na 2 HPO 4 , pH 7.4). Whole cells required brief wall
digestion (3 min) in the protoplast digest solution followed by
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PBS washes. Material was then blocked in incubation buffer ( 1 %
BSA, 0.05% tween-20 in PBS) (10 min), labeled with monoclonal anti-a-tubulin (Amersham, Amersham, England) (1/400 in
incubation buffer) (1 h), washed in PBS (30 min) and incubated in
secondary antibodies (FITC-labeled goat anti-mouse IgG, 1/200
in incubation buffer; Molecular Probes, Eugene, OR, U.S.A.) (1
h). After washing in PBS (30 min), cells were stained with DAPI
( l ^ g m l " ' , 30 s) and then labeled with rhodamine-phalloidin
(Molecular Probes) (0.08//M in PBS, 1 h). After a brief wash in
PBS, cells were mounted in 50% glycerol/PBS containing 0.1%
/7-phenylenediamine as an antifade agent.
Immunolabeling plasma membrane ghosts—Protoplasts were
attached to poly-L-lysine-coated multiwell slides, and lysed in actin stabilization buffer (7 mM PIPES, 2 mM EGTA, 10 mM
MgCl2, 20 mM K + (as KC1/K0H to pH 6.4), 1.0% DMSO, 6 mM
DTT and 300 /uM PMSF) by a quick flick of the glass slide. The
resulting ghosts were washed in actin stabilization buffer (5 min),
fixed in actin stabilization buffer containing 4% formaldehyde
and 1% glutaraldehyde (30 min) and washed in PBS (30 min).
Ghosts was air dried (10 min), rehydrated in PBS, washed in cold
methanol (-20°C, 5 min), rehydrated in PBS and blocked in incubation buffer. Primary antibodies (monoclonal anti-actin, clone
C4 diluted 1/150, Lessard 1989, ICN, Costa Mesa, CA, U.S.A.;
polyclonal anti-soybean tubulin diluted 1/500, courtesy of Dr.
Koichi Mizuno, Osaka University) were applied concurrently in
incubation buffer (1 h). Ghosts were then washed in PBS (30 min)
and incubated in secondary antibodies (FITC-labeled goat antimouse IgG (Molecular Probes) diluted 1/200 and rhodaminelabeled goat anti-rabbit IgG (Organon Teknika, Durham, NC,
U.S.A.) diluted 1/500) applied concurrently in incubation buffer
(1 h). Slides were then washed in PBS (30 min) and mounted in
50% glycerol/PBS containing 0.1% p-phenylenediamine as an
antifade agent. Secondary antibody controls were negative and
there was no cross reactivity between either of the secondary
antibodies with the alternate primary antibodies.
Observation of samples—Whole cells were optically sectioned using an inverted Olympus microscope fitted with epifluorescence optics and equipped with a cooled CCD camera, with the
stacks deconvolved using the Delta Vision system (Applied Precision, Mercer Island, WA, U.S.A.). For observation of membrane
ghosts, a conventional Olympus microscope with epifluorescence
optics was used, with data recorded on ASA 400 T-max film
pushed to 1600 ASA. Negatives were digitised at 1350 pixels per
inch, and all output was linked to Adobe Photoshop.
Results
Three distinct types of cytoskeletal arrays on membrane ghosts—Using an immunofluorescent double labeling method, extensive actin and microtubule arrays can be
visualized on tobacco BY-2 suspension culture cell membrane ghosts (Collings et al. 1998). Based on both actin and
microtubule arrangements, these ghosts can be classified
into 3 distinct types (types I, II and III respectively). As it
became apparent that the variations between ghosts were
not due to inconsistencies in immunolabeling, but resulted
from variations in the protoplasts used to generate the
ghosts, we further characterized their type and possible
formation. Type I ghosts, which were the most common
ghost type, accounted for more than 90% of all ghosts.
These ghosts were generally round with both actin
38
Membrane ghosts from microtubule-free cells
Fig. 1-5 Three types of tobacco BY-2 membrane ghosts were characterized by their cytoskeletal organization, based on the simultaneous immunolabeling of actin and microtubules. Type I ghosts (1,2) were typically round and retained both actin (A) and microtubules (B), showing that both elements of the cytoskeleton attached directly to the plasma membrane. On control ghosts (1), actin and
microtubules were random, but on ghosts produced from cells pretreated with the microtubule-stabilizing agent taxol (10 ^M) for 3 h
prior to and during wall digestion, both microtubules and actin showed a high degree of alignment (2) implicating microtubules in the
organization of the cortical actin cytoskeleton. Type II ghosts (3, 4) were larger and more irregular in shape than type I ghosts retaining
actin (A), but generally lacking microtubules (C) when generated from either control (3) or taxol-treated protoplasts (4). Analysis of the
distribution of actin on type II ghosts (B) showed three domains. In the centre of these ghosts there was a round region, the size of a
normal type I ghost, where the actin was random (x). Within this area, many but not all type II ghosts had a region of reduced actin
(z) However, the distinguishing characteristic of type II ghosts was the presence of an irregularly shaped region (y) where concentric arcs
of actin focussed back onto the round area of random actin (x). A type III ghost had dense, random actin (A) but no microtubules (B),
although on a neighbouring type I ghost, both random actin and microtubules were retained (5).
Membrane ghosts from microtubule-free cells
39
Fig. 6-9 Type II ghosts derived from mitotic protoplasts, for while actin always showed typicalconcentric arcs, on the occasions when
microtubules were present, they were typical of dividing cells. (6) If cells were lysed with actin stabilization buffer containing fixative,
then some nuclei would be trapped on ghosts. Such a type II ghost showed typical concentric actin arrays (A) and microtubules in a
spindle (B). Preprophase bands of microtubules also occurred (7C, 8B), and were either accompanied by a clearly defined zone of actin
depletion (7A) or by an aligned preprophase band of actin in a field of random actin (8A). Note the 3 actin domains (x, y and z) on the
type II ghost (Fig. 7B), and that the ghosts adjacent to the type II ghost are typical type I ghosts which retained both actin and microtubules in random arrays (labeled I). (9) Phragmoplasts contained both actin (A) and microtubules (C), but only actin was
present elsewhere on such ghosts. On these ghosts, actin occurred in typical type II arrays (B), with the phragmoplast remnant occurring
in the zone of actin depletion (z), surrounded by a region of random actin (x) and concentric actin arcs (y).
40
Membrane ghosts from microtubule-free cells
(Fig. 1A) and microtubules in random arrays (Fig. IB), although there was some coalignment. Taxol pretreatments
gave highly ordered microtubule arrays, with cortical actin
aligned parallel to the microtubules (Fig. 2) suggesting the
potential for interactions between the cortical actin and
microtubules (Collings et al. 1998).
Type II ghosts accounted for fewer than 5% of ghosts,
and were generally larger and more irregular in shape than
the type I ghosts (Fig. 3, 4). In the absence of taxol
pretreatments, cortical actin occurred in concentric arcs
(Fig. 3A) but there were no microtubules (Fig. 3C). Pretreatment with taxol did not result in the modification of
the actin pattern on these ghosts (Fig. 4A), nor result in the
preservation of any microtubules (Fig. 4C). Type II ghosts
contained different actin domains. Most type II ghosts had
a round region, usually about the size of a typical type I
ghost, where actin was random (Fig. 3B, 4B; x) although
within this domain there was sometimes an area of reduced actin (Fig. 3B; z). Surrounding the round region was
the primary diagnostic feature for type II ghosts, a large
and irregularly shaped region where concentric arcs of actin (Fig. 3B, 4B; y) focussed back towards the central
round area.
Type III ghosts were rare, often being absent from
samples. Their cortical actin was dense and irregular
(Fig. 5A), but like type II ghosts, they lacked microtubules although neighbouring type I ghosts retained normal
microtubules (Fig. 5B). Because of their rarity, however,
type III ghosts were not studied further.
Type II ghosts derive from protoplasts trapped in
division—While the type I ghosts derived from interphase protoplasts, type II ghosts arose from protoplasts
retained in cell division. Evidence for this comes from numerous sources but the most compelling is that ghosts
would sometimes form with the nucleus still attached. This
was most easily observed in cells that were lysed in fixative.
In such cases, mitotic nuclei never associated with type I
ghosts, while type II ghost always associated with nuclei
undergoing division (Fig. 6).
In rare cases, microtubules in various mitotic and
cytokinetic arrays were found on type II ghosts where they
colocalized with specific actin structures. The occurrence of
these structures on ghosts demonstrated the fidelity of the
ghosting procedure. The most common of these forms
were preprophase bands of microtubules. Typically, a microtubule preprophase band (Fig. 7C) occured in the region
of depleted actin (Fig. 7B; z), while actin was found elsewhere on the ghost in typical type II configurations
(Fig. 7A), and adjacent ghosts contained both actin and
microtubules in characteristic type I arrays (Fig. 7A, B labeled I). Again, this demonstrated that the differences in
retention and pattern were not due to poor fixation or immunolabeling. On occasions, preprophase bands of microtubules were accompanied by aligned actin, although
elsewhere on the ghost actin was random (Fig. 8). Some
type II ghosts also retained portions of phragmoplasts,
complete with a central zone that excluded antibodies to
both actin (Fig.9A) and tubulin (Fig. 9C). As with most
preprophase bands, phragmoplast remnants occurred in
10
0
11
3
6
9
12
15
18
Time after aphidicolin removal (h)
0
3
6
9
12
15
Time after aphidicolin removal (h)
Fig. 10 Wall digestion prevented pre-mitotic cells from
dividing, but trapped mitotic and cytokinetic cells in division. A
BY-2 culture was synchronized for 24 h with aphidicolin (5 /^M),
and the mitotic index then determined at times after the block of
DNA synthesis was removed by washing the cells. A large peak in
cell division occurred at 10 h (-•-). Protoplasts made prior to the
onset of cell division from a small aliquot of the whole cell culture failed to start division (-•--). but protoplasts produced at the
peak in whole cell division remained in division after the whole
cells had ceased dividing (•*••). (xxxx; times when whole cells
aliquots were digested.)
Fig. 11 There was a close correlation between the mitotic index
of a BY-2 culture and the percentage of type II ghosts. An
aphidicolin-synchronized culture began dividing about 6 h after
release, with the mitotic index peaking at 18% (-•- measured
prior to wall digestion). Protoplasts made from this culture
showed a similar response (--•-- measured after wall digestion), as
did the percentage of type II ghosts made from these protoplasts (••A--).
Membrane ghosts from microtubule-free cells
areas of reduced actin (Fig. 9B, z) while the actin elsewhere on the ghost showed typical type II patterns, with a
random round domain surrounded by concentric actin arcs
(Fig. 9A, B).
The effect of culture synchronization—Aphidicolin,
an inhibitor of DNA replication, synchronizes BY-2 cultures by blocking the cell cycle at S phase so that a peak in
cell division occurs about 10 h after aphidicolin removal
(Nagata et al. 1981). By generating protoplasts at various
times after aphidicolin removal, the correlation between
cell division and type II ghosts became clearer (Fig. 10, 11).
Protoplasts produced prior to the onset of division
failed to divide when cultured for 5 h, suggesting that the
cell wall is necessary for division (Fig. 10). However, a
sample of protoplasts made during cell division was retarded in the completion of division, as the mitotic index
remained high while that of the control sample returned to
lower levels, suggesting that the presence of the cell wall is
also necessary for the completion of division. Importantly
for this study, it was possible to generate protoplasts that
were in cell division and had depolymerized cortical microtubules. When protoplasts made at different times after
aphidicolin removal were made into ghosts, there was a
close correlation between the mitotic index of the initial cell
culture or resulting protoplasts, and the percentage of type
II ghosts (Fig. 11). In the synchronized culture, cell division started 6 h after washing and protoplasts made prior
to this did not form type II ghosts. Cell division peaked 9
h after washing with a mitotic index of 17 in whole cells
and 18 in protoplasts derived from these cells. This peak
corresponded to a peak in type II ghosts at 13% of all
ghosts. Subsequently, cell division and the number of type
II ghosts tapered off. A similar pattern also occurred after
subculturing, where the induced peak in cell division accompanied a similar peak in the number of type II ghosts
formed (data not shown).
Analysis of ghost areas—The peak of division in a
synchronized culture generated a mixture of interphase and
mitotic protoplasts which showed no significant differences in their diameters or surface areas (Table 1). However, type I ghosts generated from this protoplast mixture were on average only half the size of the type II ghosts,
41
and their average area of about 900 ^m 2 accounted for
roughly a quarter of the average surface area of protoplasts. If type II ghosts were to derive from the mitotic
protoplasts, as we argue, then their average area of 1,860
Urn1 would correspond to half the surface area of the
average mitotic protoplast. This suggests that either the
type II ghosts do not derive from mitotic protoplasts, or,
that there is some difference in the mechanism that forms
the type II ghosts.
Type II ghosts can be artificially induced by microtubule depolymerization—MicTotubules in tobacco BY-2
cells can be artificially depolymerized using propyzamide at
100fiM (Akashi et al. 1988). Microtubule depolymerization resulted in the formation of ghosts that mimicked type
II ghosts, having a similar distribution of actin and a significant increase in the ghost area. Cells were synchronized with 5 fiM aphidicolin (24 h), and then treated either
concurrently with aphidicolin and propyzamide (3 h; either
10 or 100 /uM) or with aphidicolin and taxol (30 min, 10
juM). Protoplasts were generated from these cells with
either propyzamide or taxol present, but without aphidicolin. Although the removal of aphidicolin released cells
from mitotic arrest, they were used within several hours
and confirmed to be non-mitotic so that the following
analysis is not confounded by the presence of true type II
ghosts.
Ghosts made from control protoplasts retained both
random actin and microtubules (see Fig. 1) and were on
average about 17% of the surface area of the protoplasts
from which they were made (Table 2). Ghosts from taxoltreated protoplasts had ordered actin and microtubule arrays (see Fig. 2) and were not significantly different in size
from the controls. When cells were treated with propyzamide (10 fjM; 3 h), microtubules were present in whole
protoplasts and were found on all ghosts derived from
these cells, with actin retained in normal type I-like arrays
(data not shown). When compared to the surface area of
the cells, these ghosts were not significantly larger than
controls (Table 2). However, 100^M propyzamide treatments which depolymerize microtubules in whole cells
(Akashi et al. 1988) and hence, presumeably, in protoplasts gave ghosts almost twice the size of control ghosts or
Table 1 Type II ghosts are larger than type I ghosts
Protoplast or ghost type
Protoplasts (n=10)
Protoplast
Protoplast
diameter
surface area
Cum) (± SEM) Cum2) ( ± SEM)
Ghosts ( n = l l )
Interphase protoplasts/type I ghosts
33.9±1.3
3,650±290
890± 80
24
Mitotic protoplasts/type II ghosts
33.1 ±1.1
3,480±250
l,860±210
53
42
Membrane ghosts from microtubule-free cells
Table 2 Ghosts produced with propyzamide mimic type II ghosts
Whole cell pretreatment
Protoplasts (n>100)
Protoplast
Diameter
surface area
(jum) (±SEM)
Cum2) (±SEM)
Ghosts (n=44)
Ghost area
Ghost area
{% of protoplast
Gum2) (±SEM)
surface area)
Control protoplasts
50.0 ± 1.1
7,240 ±480
1,200 ±90
17
Taxol(lO^M)
47.6±0.7
6,470±260
l,260±110
19
Propyzamide (10/JM)
50.4±1.0
8,230±370
l,470±130
18
Propyzamide (100fiM)
47.7±0.6
7,240 ±190
2,320±190
32
ghosts produced from propyzamide treatments that did not
affect microtubules (Table 2). The majority of the ghosts
formed from 100 ^M propyzamide treatments retained no
microtubules (Fig. 12C) and showed actin in patterns suggestive of type II ghosts (Fig. 12A), being random in a
round region at the centre of the ghost (Fig. 12B; x) and
having concentric actin arcs in an irregularly-shaped region
outside of this (y). Areas of depleted actin (similar to
Fig. 3B, 7B, 9B; z) were not clearly seen. However, some
ghosts from cells treated with 100 //M propyzamide
retained partially-disrupted microtubule arrays, and in
these cases the ghosts were regular in shape and had actin
in type I arrays (Fig. 13). Thus, microtubule depolymerization with propyzamide had similar effects on ghosts as
did the microtubule depolymerization that occurs during
cell division.
Is the area of reduced actin equivalent to actin depletion zones found in other dividing cells?—The location on
type II ghosts of preprophase bands and phragmoplast
remnants within the domains of reduced actin suggested
that this region might be equivalent to the zones of actin
depletion zones described in other mitotic systems by
Cleary et al. (1992) and Liu and Palevitz (1992). These
zones form at the site of the preprophase band after the
Fig. 12-13 The microtubule antagonist propyzamide (10 ^M; 3 h) modifies ghost formation. (12) The majority of ghosts were irregularly-shaped and similar in size to type II ghosts. These ghosts had actin (A) in typical type II ghosts patterns; (B: x, central domain;
y-irregular domain with concentric actin arcs), but retained no microtubules (C). (13) However, some propyzamide-treated ghosts
retained microtubules that were partially disrupted (B), and in these cases, the ghosts were more regular in shape and retained actin in
random, type I arrays (A).
Membrane ghosts from microtubule-free cells
43
Fig. 14-16 A zone of actin depletion was not observed in the division plane of either whole cells or protoplasts. Cells were immunolabelled for tubulin and stained with rhodamine-phalloidin for actin, optically sectioned at 0.5 ^m intervals and deconvolved, with
the images shown being projections of 10 planes. (14) During preprophase, microtubules form a band in the cortex which marks the site
of subsequent wall fusion and division of the daughter cell (D), with some microtubules also occurring in cytoplasmic strands projecting
away from the nucleus (B). Actin was present throughout the cytoplasm (A) and cortex, where there was no evidence for actin depletion
the division plane (C). (15) During cytokinesis, the phragmoplast contains both actin (A) and microtubules (B). Cortical actin is retained
throughout division, and under the growth conditions used, there was never any indication of a zone of actin depletion in the cortex
to mark the division site (arrows). (16) A rhodamine-phalloidin labeled protoplast (fixed as with whole cells with the inclusion of 200
mM mannitol, but not immunolabeled) showed no indication of actin depletion in the cortex (A). The protoplast was in anaphase, with
the division plane marked by arrows and spindle axis at an angle out of the page. (B) DAPI-staining.
microtubules and actin in the band depolymerize at
prophase, and thus mark the site of eventual cell plate fusion with the parent cell wall. In contrast to ghosts, where
the microtubule preprophase band often lies in an area of
reduced actin (Fig. 7), the whole tobacco BY-2 suspension culture cells used for this study did not exhibit actin
depletion zones in the plane of cell division during either
preprophase of cytokinesis. During preprophase, microtubules concentrated at the periphery of the cell in the
preprophase band (Fig. 14B, D) while actin was present in
the cytoplasm (Fig. 14A) and in the cortex where there was
no evidence for a zone of actin depletion (Fig. 14C). There
was no evidence for any region of depleted actin. During
cytokinesis, microtubules were present primarily in the
phragmoplast (Fig. 15B) while actin occurred in the
phragmoplast, throughout the cytoplasm and at the site of
cell plate fusion so that no zone of actin depletion was
present (Fig. 15A). Actin was also present throughout the
cortex of a protoplast fixed while in mitosis, again with no
suggestion of a zone of actin depletion (Fig. 16).
Discussion
Type I ghosts from interphase protoplasts—Both cortical actin and microtubules associate with the plasma
membrane ghosts made from interphase plant cells, including Zinnia mesophyll (Kobayashi 1996) and tobacco
BY-2 suspension cells (Collings et al. 1998). We propose
that ghosts form from interphase cells as follows
(Fig. 17A). Even if the microtubules in the original cells are
transverse, microtubules in protoplasts become randomized during cell wall digestion (Hasezawa et al. 1988).
Cortical actin in protoplasts is random because its alignment depends, at least in part, on the alignment of the
microtubules (Collings et al. 1998). A spherical protoplast attached to a poly-L-lysine-coated slide flattens somewhat, so that a round piece of the plasma membrane forms
the contact site between the slide and the protoplast. On
addition of the hypotonic lysis buffer and mechanical disruption, the protoplast shears off leaving the round contact
site, and random microtubules (not shown) and actin. Our
estimate of 15 to 25% of the surface area being found in
ghosts (Table 1, 2) agrees with the estimate of van der Valk
et al. (1980) of about 20%, and is consistent with a spherical cell being attached to a flat surface.
Type II ghosts from microtubule-free protoplasts—
Three lines of evidence demonstrate that type II ghosts
form from microtubule-free protoplasts. First, there is a
direct correlation between the number of dividing cells and
the percentage of cells that show type II patterns. Second, preprophase bands and phragmoplast remnants were
occasionally found well preserved on ghosts, and in all
such cases, these ghosts showed type II actin patterns.
However, the best evidence that type II ghosts derive from
Membrane ghosts from microtubule-free cells
44
17A
microtubules
actin
ghost actin
arrays
Fig. 17 Proposed mechanisms for type I ghost formation in the presence of cortical microtubules (A) and type II ghost formation in
the absence of microtubules (B). Microtubules are shown in the left-hand panel, while actin is shown in the remaining images. (A) Type
I ghosts form from cells that have random cortical microtubules and cortical actin when the protoplast shears off. This leaves a ghost
with random microtubules (not shown) and actin (shown). (B) We propose that type II ghosts form from cells free of cortical microtubules that have random cortical actin when the plasma membrane tears and collapses onto the slide. Areas initially in contact with the
slide retain random actin (x), whereas on the irregularly-shaped areas of membrane that have collapsed onto the slide, cortical actin is
artefactually aligned into concentric arcs (y).
mitotic protoplasts is that when ghosts were found with
attached nuclei, only those ghosts with mitotic nuclei
showed actin patterns consistent with type II ghosts. Interestingly, this study would suggest that protoplasts can
form from cells already undergoing division, but that
digestion prior to division prevents division from occurring. Therefore, a cell wall, or some factor within the wall,
seems necessary for cell division. (Cell divisions in these
mitotic protoplasts are different to those cell divisions that
occur in protoplasted cells one to several days after wall
digestion, and after the subsequent recovery of some cell
wall material (Fowke and Gamborg 1980). )
Do type II ghosts form differently from type I
ghosts?—We propose a model for the formation of type II
ghosts that is different to the formation of type I ghosts
(Fig. 17B). We reason that microtubule-free protoplasts,
which have random cortical actin, form a round contact
site when they bind to slides, similar to microtubulecontaining protoplasts. On lysis, the contact site forms the
round central area of a type II ghost (Fig. 17B; labeled x)
which is similar to a type I ghost. Because membrane-associated actin is random in microtubule-free protoplasts,
the actin in the central, round region of type II ghosts is
also random. We suggest that protoplast lysis involves the
plasma membrane tearing and folding down onto the slide,
rather than the protoplasts shearing off, with this additional membrane becoming the irregular lobes characteristic of type II ghosts. While this model remains speculative,
it can explain two unusual features of type II ghosts:
—Type II ghosts have concentric arcs of actin (Fig. 17B;
labeled y), yet form from protoplasts with a random cortical actin cytoskeleton. However these arcs form, they are
most probably artefacts generated by the lysis procedure.
The collapse of extra membrane onto the slide is a mechanism by which the artefactual alignment of actin might
occur.
—This extra membrane also explains how type II ghosts
can be significantly larger than type I ghosts even though
mitotic or propyzamide-treated protoplasts are of similar
sizes to protoplasts that have cortical microtubules. This
also explains how type II ghosts can correspond to 50% of
the surface area of the parent protoplast.
However, this model does not necessarily explain why
type II ghosts should have an area of reduced actin
(Fig. 3B, 7B, 9B; z) at their centre. One possibility is
that this corresponds to the zone of actin depletion that
marks the plane of cell division in cells growing in whole
plants (Cleary et al. 1992, Liu and Palevitz 1992). That
phragmoplast remnants on ghosts always occurred in the
areas of reduced actin would support this contention.
Nevertheless, we found no evidence for such a zone in
either whole cells or protoplasts of BY-2 cells grown under the experimental conditions used, suggesting that another unknown mechanism causes the areas of depleted
reduced actin on ghosts.
Implications of type II ghosts formation—The model"
of ghost formation that we propose (Fig. 17B) implies that
fundamental changes occur to the plasma membrane as a
Membrane ghosts from microtubule-free cells
result of microtubule depolymerization, notably, that in
the absence of microtubules, the plasma membrane becomes more susceptible to disruption so that it can collapse
onto the slide during lysis. Thus, microtubules may play a
role in the structural integrity of the protoplast. There is
little in the literature, however, to suggest that microtubules play such a role in either protoplasts or whole plants.
Nevertheless, a possibly related phenomenon was found by
Abe and Takeda (1986, 1989) in experiments analysing
the effects of cytoskeletal antagonists on protoplast electrofusion. While actin disruption did not affect the rate of
protoplasts fusion, it promoted the spherulation of fused
protoplasts, but microtubule disruption reduced the rate of
fusion, resulting in more cells that lysed, and also reduced the rate of spherulation. These data might be interpreted to mean that the cortical actin cytoskeleton is partially responsible for the shape of the protoplast, and that
by depolymerizing the cortical microtubules, the protoplasts become less stable. Similarly, Nagata (1989) found
that transformation rates increased in BY-2 protoplasts
produced from cell cultures synchronized in mitosis. While
this effect was attributed to the breakdown of the nuclear
membrane, were the cortical microtubules to be responsible for plasma membrane stability, then their depolymerization during cell division might increase the rate at which
DNA could enter the cell on electroporation.
Do microtubules provide structural support to the
plant plasma membrane?—The experimental data presented
in this paper suggest that the cortical microtubule cytoskeleton can provide structural support to the plasma
membrane of protoplasts. However, these data do not
necessarily imply that such support occurs in whole cells,
where the cell wall normally provides significant support to
the plasma membrane. Nevertheless, in certain, physiological-relevant situations such as plasmolysis, where the
membrane no longer is in contact with the wall, it is conceivable that microtubules might be important for stability.
Future research might therefore, directly test our proposed
model of ghosts formation by direct observations of
protoplast lysis, and determine what roles microtubules
play in membrane stabilization in protoplasts using biophysical tests of membrane strength and flexibility.
The authors wish to thank Dr. Koichi Mizuno for gifts of
antibodies. DAC was the recipient of a Foreign Researchers Fellowship from the Japanese Society for the Promotion of Science,
and acknowledges the advice and support of Dr Nina Allen (North
Carolina State University) .
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(Received May 26, 1998; Accepted October 26, 1998)