Download Three-Dimensional Reconstruction of Tubular Structure of Vacuolar

Plant Cell Physiol. 44(10): 1045–1054 (2003)
JSPP © 2003
Three-Dimensional Reconstruction of Tubular Structure of Vacuolar
Membrane Throughout Mitosis in Living Tobacco Cells
Natsumaro Kutsuna 1, Fumi Kumagai 1, Masa H. Sato 2 and Seiichiro Hasezawa 1, 3
1
2
Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8562 Japan
Graduate School of Human and Environmental Studies, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan
;
Plant vacuoles are the largest of organelles, performing
various functions in cellular metabolism, morphogenesis
and cell division. Dynamic changes in vacuoles during mitosis were studied by monitoring tubular structure of vacuolar
membrane (TVM) in living transgenic tobacco BY-2 cells
stably expressing a GFP-AtVam3p fusion protein (BY-GV).
Comprehensive images of the complicated TVM configurations were obtained by reconstructing three-dimensional
(3-D) surface structures from sequential confocal sections,
using newly developed software, SSR (stereo-structure
reconstructor). Using the surface modeling technique, we
succeeded for the first time in clarifying the development
process of TVMs and the topological relationship between
TVMs and large vacuoles. TVMs, initially organized from
large vacuoles, elongated to encircle the spindle at metaphase. Subsequently, the TVMs invaded the equatorial
region from anaphase to telophase, and then they were
divided to the two daughter cells by the cell plate at cytokinesis. When the daughter nuclei were separating from the
cell plate, some TVMs enlarged to form large vacuoles near
the division site. Spatial analysis revealed that from anaphase until cytokinesis, TVMs connected the two large
vacuoles and functioned as a route for inter-vacuolar transport. Furthermore, the experiments using the inhibitor for
actin microfilaments indicated that the microfilaments were
indispensable for the development and the maintenance of
TVMs.
Keywords: 3-D reconstruction — AtVam3p — GFP — Mitosis
— Tobacco BY-2 cells — Vacuole.
Abbreviations: 3-D, three-dimensional; BA, bistheonellide A;
BY-GV, BY-2 cell line expressing GFP-AtVam3p fusion protein;
CLSM, confocal laser scanning microscopy; GFP, green fluorescent
protein; MF, microfilament; MT, microtubule; SSR, stereo-structure
reconstructor; TVM, tubular structure of vacuolar membrane; VM,
vacuolar membrane.
Introduction
Vacuoles constitute the largest organelles in plant cells,
and serve as multifunctional compartments (Wink 1993, Marty
3
1999). The large central vacuole occupies a considerable part
of the intracellular volume of most higher plant cells, and over
90% of the cell volume in mature tissues. The space-filling
properties and solutes of vacuoles therefore play an important
role in the growth and morphogenesis of higher plants. During
the rapid growth of higher plants, for example, increased vacuolar volumes can promote cell expansion without the need for
cell division. On the other hand, the large size of vacuoles
affects the dynamic and physical characteristics of plant cells,
and their structural behavior is thought to be important for cell
division and morphogenesis.
The morphological dynamics of vacuoles have been
studied by light microscopy, fluorescence microscopy and confocal laser scanning microscopy (CLSM), and a greater understanding of their structural dynamics has been facilitated by
labelling techniques using endogenous pigments (Palevitz and
O’Kane 1981, Palevitz et al. 1981), vital staining dyes (Hillmer
et al. 1989, Lazzaro and Thomson 1996, Emans et al. 2002)
and green fluorescent protein (GFP)-fusion proteins (Di
Sansebastiano et al. 1998, Cutler et al. 2000, Mitsuhashi et al.
2000, Hawes et al. 2001, Darnowski and Vodkin 2002, Saito et
al. 2002, Uemura et al. 2002). To date, however, little progress
has been made in characterizing the dynamic changes in vacuoles during the cell cycle, primarily because of the lack of
appropriate experimental systems in living plant cells.
In order to follow vacuolar dynamics during cell cycle
progression, we initially performed vital staining of the vacuolar membrane (VM, tonoplast) of tobacco BY-2 cells harboring developed vacuoles, by pulse-labelling with the styryl dye,
FM4-64 (Kutsuna and Hasezawa 2002). At interphase, the vacuoles were compartmentalized by cytoplasmic strands and, by
late G2 phase, the cytoplasmic strands, which extended from
the central nucleus, gathered to the mid-region of cell where
they formed the cytoplasmic plate that supports the mitotic
apparatus (phragmosome). Interestingly, the characteristic VM
structures were found to surround the mitotic apparatus at M
phase, and were recognized as small ellipses on each optical
section. However, by observing serial optical-sections, it
became evident that the VM structures were tubular in form,
and were thus designated as TVM (tubular structure of vacuolar membrane) (Kutsuna and Hasezawa 2002).
The use of FM4-64 for staining VMs is well suited for
confirming the existence of thin structures, such as TVM or
Corresponding author: E-mail, [email protected]; Fax, +81-4-7136-3706.
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3-D structure of vacuoles in living BY-GV cells
cytoplasmic strands, but is unsuitable for time-sequential or
multi-focal observations due to fluorescence fading; thus making analysis of the complex structures and dynamics of TVMs
extremely difficult. In this study, therefore, we intended to clarify the structure and dynamics of TVM in order to elucidate the
functions of VM structures in plant cell division. We therefore
established a BY-2 cell line stably expressing a fusion protein
of GFP with AtVam3p/SYP22 (Kutsuna and Hasezawa 2002),
which belongs to the syntaxin family of Arabidopsis thaliana
proteins (Sato et al. 1997). Using BY-GV cells, we first followed the detailed changes in the vacuolar structures throughout mitosis. We subsequently developed software, termed SSR
(stereo-structure reconstructor), to construct three-dimensional
(3-D) images of the VM structures, especially TVMs, from
each series of optical sections generated by CLSM. From the
data obtained with this system, the structure and function of
TVMs during mitosis could be analyzed. Additionally, visible
effects of actin microfilament (MF) disruption on the structures of vacuoles were demonstrated stereologically. The doublelabeling experiments using GFP-AtVam3p and rhodaminephalloidin suggested that the MFs participated in the structural
adjustment of TVMs.
Results
Transgenic BY-2 cells for observing vacuolar structures
Tobacco BY-2 cells were transformed with Agrobacterium tumefaciens, harboring a 35S-GFP-AtVam3p construct,
essentially as described by An (1985), and the transformed cell
lines were designated BY-GV (BY-2 cells stably expressing
GFP-AtVam3p) (Kutsuna and Hasezawa 2002). From several
BY-GV cell lines we have chosen a line, designated BY-GV7,
for the following observations. The line displayed bright GFPfluorescence, and it could be maintained by 95-fold dilutions at
weekly intervals as in the case for the original BY-2 cells. In 2day-old BY-GV7 cells, 10–20% of cells were observed in the
M phase. Superficially, the shapes and sizes of the BY-2 cells
and BY-GV7 cells were indistinguishable. However, the BYGV7 cells emitted bright GFP-fluorescence that highlighted
the VM structures, especially TVM gross morphology. In BYGV7 cells, the VMs were more clearly labelled with GFPfluorescence than FM4-64 shown in Fig. 1 and 4 of Kutsuna
and Hasezawa (2002). Throughout the cell cycle, the VM
structures observed in BY-GV7, including TVMs, were very
similar to those observed in two other BY-GV lines whose
GFP-fluorescence was slightly lower than that of BY-GV7
(data not shown). The VM structures of BY-GV7 cells were
also similar to those of original BY-2 cells stained with FM464 (Fig. 1A), and in BY-GV7 cells the GFP-fluorescence perfectly overlapped with the FM4-64-fluorescence (Fig. 1C). The
vacuolar morphology could be observed by staining the
vacuolar lumen with Alexa 568 hydrazide (Fig. 1B) or 2¢,7¢bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Kutsuna and Hasezawa 2002), and the
Fig. 1 Vital-staining of vacuoles in tobacco BY-2 cells and BY-GV7
cells at telophase. (A, B) BY-2 cells, treated with FM4-64 (A) and
Alexa 568 hydrazide (B), were observed at telophase by CLSM.
TVMs were developed around the daughter nuclei. (C) A BY-GV7
cell, treated with FM4-64, was observed at telophase by CLSM. The
FM4-64 fluorescence of VMs (C-1) certainly overlapped with the fluorescence of GFP-AtVam3p (C-2). (D) A BY-GV7 cell, treated with
Alexa 568 hydrazide, was observed at telophase. The Alexa-fluorescence emitted from vacuolar lumen (D-1) was encircled by the GFPfluorescence (D-2). n, nucleus. Bars = 10 mm.
GFP-fluorescence encircled on the vacuoles was shown by
Alexa dye (Fig. 1D). These observations indicated that the fluorescence of GFP-AtVam3p certainly localized on the VMs in
BY-GV7 cells. Furthermore, based on the immunoblotting
analyses by the antibody against AtVam3p, the amount of the
GFP-AtVam3p fusion protein in BY-GV7 cells was not so high
(data now shown). From these results, we concluded that the
fused protein employed here was incorporated and located into
VMs and the vacuolar morphology, including TVMs, was not
artificially affected in BY-GV7 cells.
Vacuolar dynamics throughout mitosis
As described above, the BY-GV7 cells showed quite comparable growth rates to the original BY-2 cells, and they could
be highly synchronized by aphidicolin. More importantly, their
emitted GFP-fluorescence faithfully reflected the dynamic
changes in VM structures from G2 phase to G1 phase. As the
BY-GV7 observation system was superior, in terms of VM fluorescent brightness, slowness of fading and recovery from fading, to that using FM4-64 (Kutsuna and Hasezawa 2002), the
3-D structure of vacuoles in living BY-GV cells
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Fig. 2 Time-sequence observations of vacuolar structures throughout mitosis. In a living BY-GV7 cell, the dynamic changes in vacuolar structures were time-sequentially observed at the late G2 phase (0–40 min), prophase (60 min), metaphase (100 min), anaphase (160 min), telophase
(180 min) and early G1 phase (220 min). At the late G2 phase, thick cytoplasmic strands gathered at the central region of the cell. In a single optical section of the central region, several vacuolar compartments appeared to be isolated from the large vacuole by cytoplasmic strands (20 min,
arrowheads). They then changed their forms as TVMs at the G2/M interface (80 min, arrowheads). From anaphase to telophase, TVMs were also
observed in the equatorial region (180 min, arrowheads), and tended to align in the longitudinal direction. At cytokinesis (200 min), the TVMs
divided into the two daughter cells because of centrifugal expansion of the cell plate developing within the phragmoplast. After completion of
cytokinesis, some TVMs between the cell plate and daughter nuclei (200 min, arrowheads) developed into large vacuoles (220 min, arrowheads)
at the separating of the nuclei from the cell plate. n, nucleus. Bar = 10 mm.
system was considered suitable for time-sequential or multifocal observations. We therefore aimed to follow the detailed
changes in fluorescence during mitosis by CLSM, with special
emphasis on TVM dynamics around the mitotic apparatus
(Fig. 2). The observations were performed with one randomly
chosen cell at 20-min intervals. Thick cytoplasmic strands began
to gather near the central region of the cell at late G2 phase
(Fig. 2, 20 min, arrowheads). At this time, in optical sections,
some vacuolar compartments appeared sandwiched by the cytoplasmic strands and isolated from the large vacuole. From there,
the TVMs seemed to develop at the G2/M interface, to surround the mitotic apparatus at metaphase (Fig. 2, 80 min,
arrowheads), and then to invade the equatorial region from anaphase to telophase (Fig. 2, 180 min, arrowheads). The TVMs
tended to elongate along the longitudinal axis of cell extension, and were then divided by extensive growth of the cell
plate at cytokinesis (Fig. 2, 200 min, arrowheads). After cytokinesis, some of the TVMs that were located between the cell
plate and nucleus developed into the large vacuole at the time
when the nucleus was separating from the cell plate (Fig. 2,
220 min, arrowheads). These observations implied that the
TVMs developed from the vacuolar compartments around the
nucleus at late G2 phase, and were maintained until cytokinesis. However, we were unable to recognize each complete TVM
from single optical sections because the TVMs were winding
tubes running over a wide area around the mitotic apparatus.
3-D reconstruction of vacuolar structures
To understand the spatial configuration of TVMs and their
relationship with large vacuoles, we performed 3-D reconstructions of the vacuolar structures from a series of confocal
images taken along the z-axis by CLSM. Due to the lack of an
appropriate VM processing system, we developed a new software package, SSR (Kutsuna et al., in preparation), to perform
3-D modeling of a series of optical sections from the BY-GV7
cells. To overcome the complexity of vacuolar structures as
well as anisotropic resolution, which is typical of serial sections obtained by CLSM, we employed parallel contour reconstruction by which surfaces of 3-D objects are reconstructed by
connecting planar contours that represent cross-sections
through the objects (Fuchs et al. 1977). The obtained vacuolar
structures could be observed from any angle, and measurements of their volume and surface area could be obtained.
The 3-D vacuolar structures, which were reconstructed by
SSR at each stage of the cell cycle, are shown in Fig. 3. Each
image was constructed from serial optical sections obtained by
CLSM at 0.5–1.0 mm intervals. In the image at the late G2
phase, TVMs have not been developed yet, and the cytoplasmic strands run penetrating the large vacuole around central
region of the cell (Fig. 3A). Although some vacuolar compartments appeared to be isolated from the large vacuoles on the
single optical section (Fig. 2, 20 min), in fact these compartments connected with each other and with the large vacuole
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3-D structure of vacuoles in living BY-GV cells
Fig. 3 3-D structures of vacuoles. The images were constructed from a series of optical sections taken at 0.5–1.0 mm intervals. Vacuolar structures were classified and distinguished by color according to the area of each optical section (B, C, D’). Hence, the large vacuoles, the smaller
compartments and TVMs are presented in blue, sky-blue and green, respectively. (A) Vacuolar structures at late G2 phase. The large vacuole was
penetrated by thick cytoplasmic strands near the central region of the cell. (B) Vacuolar structures at prophase. The TVMs began to elongate from
the smaller compartment of large vacuole. At this time, the TVMs were short in length and few in number. (C) Vacuolar structures at metaphase.
The large vacuoles were divided by the cytoplasm, including the mitotic apparatus. All TVMs were attached to the large vacuoles (arrowheads)
and connected the two large vacuoles. (D, D’) Vacuolar structures at telophase. The division plane is represented by the orange line. TVMs
appeared to be cut off by the cell plate. m represents the position of the mitotic apparatus. Bars = 10 mm.
(Fig. 3A). In the image of vacuolar structures at prophase, the
cross-sections of vacuoles are divided into three classes by the
area size (Fig. 3B). The large sections, the middle ones and the
small ones are shown in blue, sky-blue and green, respectively.
This indicated that the TVMs, with small diameters and shown
in green, extended from the sky-blue compartments, which
were located at the periphery of the nucleus and in the midregion of the cell (Fig. 3B). The TVMs were few in number
and short in length at this stage. In the image obtained at metaphase, the large vacuole was divided in two by the cytoplasm
around the mitotic apparatus, proving that the TVMs elongated
around the mitotic apparatus and connected the two large vacuoles (Fig. 3C). This finding is the first demonstration of the
TVM dynamics using 3-D modeling. In the image at telophase, the cell plate was found to develop toward the edge of
the cell (Fig. 3D). Numerous TVMs shown in green were distributed near the division plane (Fig. 3D’). These TVMs were
therefore demonstrated to be cut off, when and where that part
of the cell plate was completed.
Lumenal connectivity of large vacuoles provided by TVMs
From the 3-D modeling using SSR we therefore obtained
a considerable amount of new information regarding TVMs,
and from these findings we focused our subsequent studies on
TVM cross-linking between two large vacuoles. In three optical sections, near the metaphase cell surface, obtained by
CLSM at 0.7 mm intervals, the large vacuole was bisected by
cytoplasm around the mitotic apparatus (Fig. 4). When the
TVM was traced from left to right, from the point where it was
connected to the large vacuole (Fig. 4, arrowheads), the TVM
appeared to connect both large vacuoles (Fig. 4). When the
vacuoles of metaphase BY-2 cells were stained with fluorescent dye, BCECF, large vacuoles with faint TVMs could be
observed (Fig. 5A, left). Photobleaching was performed with
strong excitation light only in the dashed area, after taking the
photograph. After a minute, BCECF fading was observed both
in the photobleached vacuole and in an additional vacuole (Fig.
5A, right). This not only suggested that both large vacuoles,
which were bisected by the cytoplasmic plate, were topologi-
3-D structure of vacuoles in living BY-GV cells
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Fig. 4 Connections between the large vacuoles and TVMs at metaphase of a BY-GV7 cell. Three optical sections were obtained by scanning
from the cell surface at 0.7 mm intervals. The large central vacuole was divided into two by the cytoplasm, including the mitotic apparatus, from
where the TVMs were derived (arrowheads). The TVMs were observed to connect the two large vacuoles through three serial sections. l, large
vacuole. Bar = 10 mm.
cally connected by TVMs, but also that the vacuolar contents
were always able to diffuse between the large vacuoles through
the TVMs.
Role of actin MFs on the morphology of TVMs
To clarify the mechanism for development and maintenance of tubular structures characteristic of TVM, effects of
cytoskeleton inhibitors on the morphology of vacuoles were
observed using the BY-GV7 cells. We found that development
of TVMs were inhibited by the treatment with 1 mM bistheonellide A (BA), a dimeric macrolide, that was recently
reported to inhibit polymerization of G-actin (Saito et al. 1998,
Hoshino et al. 2003). At the late G2 phase, BA treatment caused
the formation of several spherical vacuoles around the nuclei
(Fig. 6A-1, arrowheads). The 3-D image showed that the newly
formed spherical vacuoles were not connected to the large vacuoles via VMs, and connectivity between two large vacuoles
was disrupted (Fig. 6A-2). Moreover, the disruption of MFs
inhibited the development and maintenance of TVMs at the
metaphase (Fig. 6B). Instead of TVMs, the spherical vacuoles
were observed (Fig. 6B-1). Treatments with 100 mM cytochalasin B and D, other actin inhibitors, caused similar changes in
the structure of vacuoles (data not shown).
The disappearance of TVMs, by the destruction of MFs,
might cause the loss of lumenal connection between the two
large vacuoles. Thus, the vacuolar lumen of BY-2 cells was
vital-stained with BCECF, and then their MFs were destroyed
with BA. Subsequently, the photobleaching was performed on
Fig. 5 Connectivity of the lumen of two large
vacuoles at metaphase. The vacuolar lumens of
metaphase BY-2 cells were stained with BCECF,
and the cells then observed by fluorescence microscopy. Photobleaching was performed by the irradiation with the strong excitatory light only to the
dashed area. (A) The large central vacuole of the
central cell was divided into two sections (prebleach). In addition to the large vacuoles, TVMs
were also identified by faint fluorescence of
BCECF (arrowheads). However, after photobleaching, the fading of the green fluorescence was
not only limited to the left vacuole but also
observed in the right vacuole (post-bleach). (B) A
BA-treated cell. Instead of TVMs, some spherical
vacuoles were observed around mitotic apparatus
(pre-bleach). The fading, observed in the left large
vacuole, did not spread to the right vacuole (postbleach). l, large vacuole. Bars = 10 mm.
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3-D structure of vacuoles in living BY-GV cells
Fig. 6 Effect of BA on the organization of TVMs. BY-GV7 cells were treated with 1 mM BA which have immediate effect on actin-depolymerization. After 30 min of the treatment, structures of vacuoles were observed by the GFP fluorescence. The single optical sections (A-1, B-1) and
the corresponding 3-D vacuolar structures (A-2, B-2) of living BY-GV7 cells were shown. Connections between the large vacuoles were lost and
several spherical vacuoles were formed around the nuclei at the late G2 phase (A-1, arrowheads). In contrast to untreated cells, TVMs were not
developed and the spherical vacuoles (B-1, arrowheads) were observed at the metaphase (B). n, nucleus. Bars = 10 mm.
the metaphase cells, which were untreated or treated with BA
(Fig. 5). In contrast to the untreated cell (Fig. 5A, right), the
BCECF fading did not spread to another large vacuole in the
BA-treated cell (Fig. 5B, right). As expected from the 3-D
image (Fig. 6), the disruption of MFs actually caused the separation of vacuoles and the lumenal disconnection between the
two large vacuoles, suggesting that the MFs may be necessary
for the maintenance of TVMs as the lumenal connection.
These experiments with the inhibitor indicated that the
MFs were involved in the organization of TVMs. To gain
insight into the spatial relationship of them, BY-GV7 cells were
stained with rhodamine-phalloidin to visualize the MFs and
were observed by CLSM (Fig. 7). Some TVMs seemed to be
surrounded by or colocalized with the MFs during mitosis (Fig.
7A, B). On the other hand, when pretreated with BA for
30 min, such MFs were rarely observed (Fig. 7C), as described
in Hoshino et al. (2003).
Discussion
In this study, we have established a cell line BY-GV7 that
stably expresses a GFP-AtVam3p fusion protein, and examined the dynamics and 3-D structures of plant vacuoles during
mitosis. GFP fluorescence in the VM of BY-GV7 cells was
more intensive and durable than by staining with the fluorescent dye, FM4-64. In the latter case, the fluorescence gradually
darkened, and some particulate artifacts appeared in the vacuoles within several days (Kutsuna and Hasezawa 2002). In con-
Fig. 7 Double labelling of VMs and actin MFs in mitotic cells. To visualize VMs and MFs simultaneously, BY-GV7 cells were stained with
rhodamine-phalloidin under condition that did not disturb VM structures. The green GFP fluorescence of VMs and the red rhodamine fluorescence of MFs were observed by CLSM. (A) A metaphase cell. A part of TVMs was surrounded in MFs (arrowhead). (B) A telophase cell. Some
TVMs were colocalized with MFs around the cell plate (arrowhead). (C) A telophase cell pretreated with BA. Small spherical vacuoles were
observed around the daughter nuclei. MFs were destroyed by BA treatment. n, nucleus. Bars = 10 mm.
3-D structure of vacuoles in living BY-GV cells
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Fig. 8 Schematic representation of vacuolar structures at mitosis in BY-2 cells. (A) The large central
vacuole is compartmentalized around the nucleus by
cytoplasmic strands at the G2 phase. Some of the
cytoplasmic strands accumulate at the central region
of the cell. (B) Some vacuolar compartments convert to TVMs at the G2/M interface. When the large
vacuole is divided into two sections, some TVMs
connect both vacuoles at metaphase. (C) After chromosomal segregation, TVMs invade into the equatorial region at telophase. The TVMs begin to be cut
off by the cell plate that develops within the phragmoplast. (D) When cytokinesis is complete, all the
TVMs are divided into the two daughter cells.
Thereafter, some TVMs between the cell plate and
daughter nuclei develop into large vacuoles at the
early G1 phase. n, nucleus. red, VM; sky blue, vacuole; green, MT.
trast, the BY-GV7 cells showed stable GFP fluorescence due to
the constitutive expression of the GFP-AtVam3p fusion protein
by the CaMV 35S promoter. From the observations of BY-GV7
cells double-labelled with GFP-AtVam3p and FM4-64 (Fig.
1C) or GFP-AtVam3p and Alexa hydrazide (Fig. 1D), the fluorescence of GFP-AtVam3p seemed to be located in the whole
VMs and not to be located in the limited part of VMs. In this
context, it has been previously demonstrated that the GFPAtVam3p was localized to the VMs and could be used as a fluorescent probe for the VM structures in transgenic A. thaliana
(Uemura et al. 2002). Specific features of the BY-GV7 cells,
notably fluorescence intensity, durability and sharpness, enabled us to perform not only studies on vacuolar morphology in
living cells but also time-sequential and multi-focal observations. In addition, the shapes and sizes of the BY-GV7 cells
were indistinguishable from the original BY-2 cells, their
growth rates were essentially similar, and the cells could be
synchronized by the same method described in Nagata and
Kumagai (1999) (data not shown). Therefore, at the peak of
mitotic index, a large number of cells at various mitotic and
premitotic stages could be easily observed at the same time.
The structure of VMs in living plant cells are dynamically
modified within a brief time-span, and their configurations are
sometimes very complicated and intricate. Spherical structures, designated as ‘bulbs’, have been reported within the
lumen of vacuoles in rapidly expanding cells of A. thaliana
(Saito et al. 2002), and cylindrical and sheet-like structures that
invaginate into the vacuolar lumen have been observed in several tissues of transgenic A. thaliana expressing a GFPAtVam3p (Uemura et al. 2002). These structures were reported
to exhibit considerable motility. Time-lapse observations and
acquisition of 3-D images were indispensable for comprehending these structures. During mitosis, TVMs also displayed
dynamic changes in their conformations, with entangled and
complicated structures (Fig. 2), and 3-D image processing
served as a powerful tool for understanding such structures. Of
the numerous approaches to 3-D image processing, we selected
parallel contour reconstruction, which performs surface reconstructions, and then developed specific computer software,
SSR, as tool for reconstructing 3-D structures and analyzing
the series of CLSM images. By employing the contour-based
modeling system, we have succeeded in reconstructing and visualizing 3-D vacuolar structures containing TVMs (Fig. 3, 6).
Compared to the single optical sections, the 3-D images reconstructed by SSR provided more comprehensive views of the
morphological changes induced by the MF disruption (Fig. 6).
In addition, the newly developed SSR software could be used
to quantify 3-D structures, such as of the surface area and volume of both vacuoles and whole cells (data not shown). It is
notable that the SSR software can be widely applied to various
biological objects, especially for membranous structures even
in large plant cells, in which the CLSM images are prone to
anisotropic resolution and to greater topological differences
between serial sections of whole cells. In addition, by combining SSR with a real-time observation system using CLSM, we
think that we will be able to monitor the dynamic images of 3D structures over a long time-period. In such cases, SSR may
be suitable for investigations of plant cell morphology.
Our results on the dynamics of TVMs are summarized in
Fig. 8. At the late G2 phase, the cytoplasmic strands converge
on the central region of the cell, and compartmentalize the
large central vacuole around the nucleus (Fig. 8A). The TVMs
then elongate from some of these vacuolar compartments at the
G2/M interface. Although the large vacuole is segregated into
two parts by the cytoplasm and mitotic apparatus, they are connected to each other by the TVMs at metaphase (Fig. 8B).
Some TVMs invade the equatorial area after chromosomal segregation at anaphase. When the cell plate is formed, the TVM
bridges between the large vacuoles are cut off by the developing cell plate (Fig. 8C). After cytokinesis, some TVMs between
the cell plate and daughter nuclei develop into large vacuoles at
the early G1 phase (Fig. 8D). With respect to TVM behavior,
the vacuolar-tubular continuum in the secretory trichomes of
chickpea were reported to be continuous between the stalk cells
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3-D structure of vacuoles in living BY-GV cells
and to function in the rapid delivery of solute from the trichome base to the secretory head cells (Lazzaro and Thomson
1996). In BY-GV7 cells, the TVMs connected the two large
vacuoles and appeared to be cut by the cell plate during cell
plate formation. It is possible that this TVM bridge is the precursor of the continuum observed in stalk cells of chickpea.
Conversely, if TVMs accidentally escape being cut off at cytokinesis, the TVMs may penetrate into the new cell wall as in
the case of the vacuolar-tubular continuum.
The viscosity of the lumen of lytic vacuoles is considerably lower than that of the cytoplasm, a property that appears
valuable for bulk transport. In fungal hyphae, motile tubular
vacuoles have been implicated in longitudinal transport of
nitrogen and phosphorus compounds (Ashford 1998). In our
photobleaching experiment (Fig. 5A), we demonstrated that
lumens of the two large vacuoles were connected by TVMs,
and that lumenal solutes could probably diffuse freely. Compared to the result of BA treated cells in Fig. 5B, the BCECF
fading in Fig. 5A was not due to the cellular damage caused by
the excitation light of photobleaching. It is uncertain how solutes are mixed within the lumen and whether any mechanism
of positive stirring exists. The time-course pattern of BCECF
fading could be explained by passive diffusion. The connectivity of vacuolar lumens through the TVMs also suggests that
specific conditions, for example osmotic pressure, pH, membrane potential, and electrolyte concentrations, were maintained at equal levels in the two large vacuoles.
It is noteworthy that TVMs were found connected to the
large vacuole throughout mitosis and cytokinesis, suggesting
that they may function as branches of the large vacuole. Some
TVMs invaded the equatorial region between anaphase and telophase (Fig. 2, 180 min), and subsequently co-localized with
the developing phragmoplast within which the cell plate was
forming (Kutsuna and Hasezawa 2002). For cell plate formation, large amounts of membranous components appear to be
consumed by vesicle transport of the cell plate materials. In the
contrast, the surface area and volume of the cell plates were
decreased dramatically during their maturation (Otegui et al.
2001). Thus, the removal of excess membranes from the cell
plates should occur accompanied by the maturation of cell
plates. The appearance of multivesicular bodies and small vacuoles, adjacent to cell plates, has also been reported (Samuels
et al. 1995, Otegui et al. 2001). These lytic compartments
seemed to be involved in the recycling of membrane lipids as
the downstream organelles of the cell plates. In our observations (Fig. 1, 2, 3D), some TVMs were located near the developing cell plates. Therefore, these TVMs may have the possible
role in the recruitment of the membrane components. In addition, it is possible that TVMs fulfill other roles, for example in
the protection or positioning of the mitotic apparatus and in
preservation of VMs for vacuolar enlargement at the G1 phase.
We previously demonstrated that microtubules (MTs)
were indispensable to the organization of TVMs that encircle
the chromosomal region (Kutsuna and Hasezawa 2002). Never-
theless, disruption of MTs did not affect the tubular structure of
TVMs themselves (Kutsuna and Hasezawa 2002). In this study,
we found that development and maintenance of TVMs were
directly or indirectly dependent on the actin MFs from inhibitor experiments (Fig. 5B, 6). The exact functions and mechanisms of MFs in the TVM development are hitherto unknown.
In higher plant cells, MFs are involved in organization of the
preprophase band (Eleftheriou and Palevitz 1992), phragmoplast (Zhang et al. 1993) and radial network of MTs (Miyake et
al. 1997). Because the treatments for staining MFs by the fluorescent probes tend to deform VM structures, few studies demonstrating spatial relationships between MFs and VMs have
been reported. Consequently, little is known about relationship
between the vacuoles and MFs in higher plants. The dynamics
of vacuoles in Allium stomatal cells require the actin MFs
(Palevitz and O’Kane 1981). In epidermal cells of transgenic A.
thaliana expressing a GFP-AtVam3p, cylindrical and sheet-like
structures invaginated into the vacuolar lumen were observed
(Uemura et al. 2002). While their morphology was not disrupted, the movement of them was blocked with the MF depolymerizing drug, cytochalasin D (Uemura et al. 2002). These
reports also suggest the existence of MF-dependent machinery
for deformation of vacuoles. In this article, the localizations of
TVMs and MFs were shown at metaphase (Fig. 7A) and at telophase (Fig. 7B). The MFs were partially colocalized with the
TVMs. These results implied that the VM dynamics was
directly regulated through colocalization or contact with MFs.
In summary, using our newly developed SSR software as a
tool for modeling, analyzing and viewing 3-D biological image
data, we have been able to demonstrate the following: (1) BYGV7 cell line is suitable for time-sequence observations of
VMs; (2) SSR can clarify complicated cellular structures such
as TVMs; (3) each TVM is a tubular structure and is derived
from the periphery of the large vacuole; (4) TVMs actually
connect two large vacuoles, and act as a pathway for the transport of soluble vacuolar contents during mitosis; and (5) development and maintenance of TVMs depend on MFs. Our future
studies will focus on analyzing in detail the structure and function of plant vacuoles, especially in TVMs, using BY-GV7
cells and our SSR software.
Materials and Methods
Plant material
At weekly intervals, suspension cultures of a tobacco BY-2 cell
line, derived from seedlings of Nicotiana tabacum L. cv. Bright Yellow
2, were diluted 95-fold with a modified Linsmaier and Skoog (LS)
medium (Linsmaier and Skoog 1965), as described (Nagata et al.
1992). The cell suspension was agitated on a rotary shaker at 130 rpm
at 27°C in the dark.
Transformation and establishment of the BY-2 cell line expressing the
GFP-AtVam3p fusion protein
Agrobacterium tumefaciens strain, C58C1, was transformed with
the GFP(S65T)-AtVam3p construct. A 4-ml aliquot of 3-day-old BY-2
3-D structure of vacuoles in living BY-GV cells
cells was inoculated with 100 ml of an overnight culture of the transformed A. tumefaciens as described (An 1985). After a 2-day incubation at 27°C, the cells were washed four times in 5 ml LS medium, and
then plated onto solid LS medium containing 250 mg liter–1 kanamycin
and 500 mg liter–1 carbenicillin. Calluses that appeared after 10 d were
transferred onto new plates and cultured independently until they
reached a diameter of about 1 cm, at which time they were transferred
to 20 ml liquid LS medium in 100-ml Erlenmeyer flasks and agitated
on a rotary shaker at 130 rpm at 27°C in the dark. After 1 month, a cell
line suitable for VM structural observations was selected by identifying GFP-fluorescent cells by fluorescence microscopy. The selected
cell line, designated BY-GV7 (BY-2 cell line expressing GFPAtVam3p fusion protein No.7), could be maintained by 95-fold dilutions at weekly intervals, and be synchronized similarly to the original
BY-2 cells.
Staining of vacuoles and vacuolar membranes
To stain the inside of vacuoles, 2¢,7¢-bis-(2-carboxyethyl)-5-(and6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM, Molecular
Probes Inc., Eugene, OR, U.S.A.) and Alexa 568 hydrazide (Molecular Probes) were used. BCECF-AM was added to the cell suspension at
the final concentration of 10 mM. The cells were incubated for 1 h at
27°C, washed with fresh culture medium, and then incubated for an
additional 6 h at 27°C. Alexa 568 hydrazide was used essentially
according to Emans et al. (2002). To visualize the VM in BY-2 cells,
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64, Molecular Probes) was used
(Kutsuna and Hasezawa 2002). Cells between 10 and 20 h after labelling were used to observe VMs.
Staining of actin MFs
For simultaneous observations of MFs and VMs, BY-GV7 cells
were suspended in a solution containing 50 mM PIPES (pH 6.8),
1 mM MgSO4, 5 mM EGTA, 1% glycerol, 3% sucrose, 0.03% saponin
(ICN Biomedicals Inc., Aurora, OH, U.S.A.), 250 mM m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS; Pierce Chemical Co., Rockford, IL, U.S.A.) and 0.3 mM rhodamin-phalloidin
(Molecular Probes). The cells were incubated for 5 min at room temperature, and then observed.
Time-sequential and multi-focal observations
For time-sequence observations, 2-day-old BY-GV7 cells were
transferred into Æ 35 mm Petri dishes with Æ 14 mm coverslip windows at the bottom (Matsunami Glass Ind., Ltd., Osaka, Japan). The
dishes were placed onto the inverted platform of a fluorescence microscope (IX, Olympus, Olympus Co. Ltd., Tokyo, Japan), equipped with
a confocal laser scanning head and control systems (CLSM GB-200;
Olympus). This observation system, which allows observation of VM
dynamics in BY-GV7 cells with minimal damage, was found to be
ideal for our studies. The 2-D images were processed digitally using
Photoshop software (Adobe Systems Inc., San Jose, CA, U.S.A.).
Reconstruction and computer visualization of 3-D structures of
vacuoles
Reconstruction and visualization of the 3-D structures of vacuoles were performed using SSR, an originally developed software for
3-D structure reconstruction (Kutsuna et al., in preparation), running
under SerioWare GNU/Linux on an Athlon XP 2200+ personal computer (Advanced Micro Devices Inc., Sunnyvale, CA, U.S.A.)
equipped with 2 gigabytes of random access memory. Serial optical
sections taken at 0.5–1.0 mm intervals were displayed and analyzed
with SSR. Once the 3-D surface data was reconstructed, vacuolar models were displayed either by surface rendering or by volume render-
1053
ing, using the Open Visualization Data Explorer (International Business Machines Corp., White Plains, NY, U.S.A.).
Photobleaching of BCECF within vacuoles
The BY-2 cells were stained with BCECF as described above,
and monitored with a fluorescence microscope (BX, Olympus)
equipped with a CCD camera system (DP70, Olympus). Following
observation, and confirmation that BCECF fluorescence was limited to
the vacuolar lumens, photobleaching was performed by irradiating
only a small area of the cell with strong excitation light using an iris.
Inhibitor treatment
The BY-GV7 and BY-2 cells were treated with 1 mM bistheonellide A (BA, Wako Pure Chemical Ind., Osaka, Japan) or 100 mM cytochalasin B and D (Sigma, St. Louis, MO, U.S.A.) for disruption of
MFs as described previously (Hoshino et al. 2003) and were then
observed by the fluorescence microscopy and the CLSM.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas (No. 10219201 and 15031209) from the
Ministry of Education, Culture, Sports, Science and Technology, Japan,
and the Nissan Science Foundation (S. H).
References
An, G. (1985) High efficiency transformation of cultured tobacco cells. Plant
Physiol. 79: 568–570.
Ashford, A.E. (1998) Dynamic pleiomorphic vacuole systems: are they endosomes and transport compartments in fungal hyphae? Adv. Bot. Res. 28: 119–
159.
Cutler, S.R., Ehrhardt, D.W., Griffitts, J.S. and Somerville, C.R. (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in
cells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. USA 97: 3718–
3723.
Darnowski, D.W. and Vodkin, L.O. (2002) A soybean lectin-GFP fusion labels
the vacuoles in developing Arabidopsis thaliana embryos. Plant Cell Rep. 20:
1033–1038.
Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S. and Neuhaus, J.-M. (1998)
Specific accumulation of GFP in a non-acidic vacuolar compartment via a Cterminal propeptide-mediated sorting pathway. Plant J. 15: 449–457.
Eleftheriou, E.P. and Palevitz, B.A. (1992) The effect of cytochalasin D on preprophase band organization in root tip cells of Allium. J. Cell Sci. 103: 989–
998.
Emans, N., Zimmermann, S. and Fischer, R. (2002) Uptake of a fluorescent
marker in plant cells is sensitive to brefeldin A and wortmannin. Plant Cell
14: 71–86.
Fuchs, H., Kedem, M. and Uselton, S.P. (1977) Optimal surface reconstruction
from planar contours. Comm. ACM 20: 693–702.
Hawes, C., Saint-Jore, C.M., Brandizzi, F., Zheng, H., Andreeva, A.V. and
Boevink, P. (2001) Cytoplasmic illuminations: in planta targeting of fluorescent proteins to cellular organelles. Protoplasma 215: 77–88.
Hillmer, S., Quader, H., Robert-Nicoud, M. and Robinson, D.G. (1989) Lucifer
yellow uptake in cells and protoplasts of Daucas carota visualized by laser
scanning microscopy. J. Exp. Bot. 40: 417–423.
Hoshino, H., Yoneda, A., Kumagai, F. and Hasezawa, S. (2003) Examining the
roles of actin depleted zone (ADZ) and preprophase band (PPB) in determining the division site of higher plant cells using a BY-2 cell line expressing
GFP-tubulin (BY-GT). Protoplasma in press.
Kutsuna, N. and Hasezawa, S. (2002) Dynamic organization of vacuolar and
microtubule structures during cell cycle progression in synchronized tobacco
BY-2 cells. Plant Cell Physiol. 43: 965–973.
Lazzaro, M.D. and Thomson, W.W. (1996) The vacuolar-tubular continuum in
living trichomes of chickpea (Cicer arietinum) provides a rapid means of solute delivery from base to tip. Protoplasma 193: 181–190.
1054
3-D structure of vacuoles in living BY-GV cells
Linsmaier, E.M. and Skoog, F. (1965) Organic growth factor requirements of
tobacco tissue cultures. Physiol. Plant. 18: 100–127.
Marty, F. (1999) Plant vacuoles. Plant Cell 11: 587–599.
Mitsuhashi, N., Shimada, T., Mano, S., Nishimura, M. and Hara-Nishimura, I.
(2000) Characterization of organelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells. Plant Cell Physiol. 41: 993–1001.
Miyake, T., Hasezawa, S. and Nagata, T. (1997) Role of cytoskeletal components in the migration of nuclei during the cell cycle transition from G1 phase
to S phase of tobacco BY-2 cells. J. Plant Physiol. 150: 528–536.
Nagata, T. and Kumagai, F. (1999) Plant cell biology through the window of the
highly synchronized tobacco BY-2 cell line. Meth. Cell Sci. 21: 123–127.
Nagata, T., Nemoto, Y. and Hasezawa, S. (1992) Tobacco BY-2 cell line as the
“HeLa” cell in the cell biology of higher plants. Int. Rev. Cytol. 132: 1–30.
Otegui, M.S., Mastronarde, D.N., Kang, B.-H., Bednarek, S.Y. and Staehelin,
L.A. (2001) Three-dimensional analysis of syncytial-type cell plates during
endosperm cellularization visualized by high resolution electron tomography.
Plant Cell 13: 2033–2051.
Palevitz, B.A. and O’Kane, D.J. (1981) Epifluorescence and video analysis of
vacuole motility and development in stomatal cells of Allium. Science 214:
443–445.
Palevitz, B.A., O’Kane, D.J., Kobres, R.E. and Raikhel, N.V. (1981) The vacuole system in stomatal cells of Allium. Vacuole movements and changes in
morphology in differentiating cells as revealed by epifluorescence, video and
electron microscopy. Protoplasma 109: 23–55.
Saito, C., Ueda, T., Abe, H., Wada, Y., Kuroiwa, T., Hisada, A., Furuya, M. and
Nakano, A. (2002) A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis. Plant J. 29: 245–255.
Saito, S., Watabe, S., Ozaki, H., Kobayashi, M., Suzuki, T., Kobayashi, H.,
Fusetani, N. and Karaki, H. (1998) Actin-depolymerizing effect of dimeric
macrolides, bistheonellide A and swinhilide A. J. Biochem. 123: 571–578.
Sato, M.H., Nakamura, N., Ohsumi, Y., Kouchi, H., Kondo, M., HaraNishimura, I., Nishimura, M. and Wada, Y. (1997) The AtVAM3 encodes a
syntaxin-related molecule implicated in the vacuolar assembly in Arabidopsis thaliana. J. Biol. Chem. 272: 24530–24535.
Samuels, A.L., Giddings, Jr., T.H. and Staehelin L.A. (1995) Cytokinesis in
tobacco BY-2 and root tip cells: a new model of cell plate formation in higher
plants. J. Cell Biol. 130: 1345–1357.
Uemura, T., Yoshimura, S.H., Takeyasu, K. and Sato, M.H. (2002) Vacuolar
membrane dynamics revealed by GFP-AtVam3p fusion protein. Gene. Cell.
7: 743–753.
Wink, M. (1993) The plant vacuole: a multifunctional compartment. J. Exp. Bot.
Suppl. 44: 231–246.
Zhang, D., Wardsworth, P. and Hepler P.K. (1993) Dynamics of microfilaments
are similar, but distinct from microtubules during cytokinesis in living, dividing plant cells. Cell Motil. Cytoskel. 24: 151–155.
(Received April 18, 2003; Accepted July 30, 2003)