Inhibition of Contractile Vacuole Function by ... - Universität zu Köln

Plant Cell Physiol. 46(1): 201–212 (2005)
doi:10.1093/pcp/pci014, available online at www.pcp.oupjournals.org
JSPP © 2005
Inhibition of Contractile Vacuole Function by Brefeldin A
Burkhard Becker 1 and Angela Hickisch
Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, D-50931 Köln, Germany
Brefeldin A (BFA) causes a block in the secretory system
of eukaryotic cells. In the scaly green flagellate Scherffelia
dubia, BFA also interfered with the function of the
contractile vacuoles (CVs). The CV is an osmoregulatory
organelle which periodically expels fluid from the cell in
many freshwater protists. Fusion of the CV membrane with
the plasma membrane is apparently blocked by BFA in S.
dubia. The two CVs of S. dubia swell and finally form large
central vacuoles (LCVs). BFA-induced formation of LCVs
depends on V-ATPase activity, and can be reversed by
hypertonic media, suggesting that water accumulation in
the LCVs is driven by osmosis. We suggest that the BFAinduced
formation of LCVs represents a prolonged diastole
phase. A normal diastole phase takes about 20 s and is difficult
to investigate. Therefore, BFA-induced formation of
LCVs in S. dubia represents a unique model system to
investigate the diastole phase of the CV cycle.
Keywords: Brefeldin A —Concanamycin A — Contractile
vacuole — Golgi — Osmoregulation — Scherffelia dubia —
V-ATPase.
Abbreviations: BFA, brefeldin A; Concan A, concanamycin A;
CV, contractile vacuole; EM, electron microscopy; LCV, large central
vacuole; LM, light microscopy; SR, scale reticulum.
Introduction
The contractile vacuole (CV) is a remarkable organelle
that is found in many protists and some freshwater sponges.
CVs are membrane-bounded vacuoles that slowly fill with fluid
(diastole) and periodically expel the fluid (systole) from the
cell and may serve primarily osmoregulatory functions (for
reviews see Patterson 1980, Hausmann and Patterson 1984,
Allen 2000, Allen and Naitoh 2002). Within the green algae,
CVs have been found in prasinophytes and many volvocalean
algae. CVs are absent in the vegetative cells of other green
algae but may appear in certain wall-less stages of their life
cycle (Hausmann and Patterson 1984). The morphology of
CVs in green algae has been described in many publications
investigating the ultrastructure of green flagellates or zoospores
(reviewed in Hausmann and Patterson 1984, Allen and Naitoh
2002). The number of CVs found in an organism is generally
constant and, therefore, is used as a character in systematics.
1 Corresponding author: E-mail, b.becker@uni-koeln.de; Fax, +49 221-4705181.
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;
Experimental evidence suggests that the CVs have an
osmoregulatory function (Patterson 1980, Allen 2000, Allen
and Naitoh 2002). However, the mechanisms of liquid accumulation
and expulsion are still not clear (Allen 2000, Allen and
Naitoh 2002). Vacuolar H + -ATPase was found to be associated
with the CV in Dictyostelium and Paramecium (Fok et al.
1993, Fok et al. 1995, Heuser et al. 1993), and it has been suggested
that V-ATPase catalyses the primary energy transduction
step for pumping water from the cytoplasm into the CV
(Nolta and Steck 1994). However, the existence of ATP-utilizing
water pumps cannot be ruled out completely (discussed in
Allen and Naitoh 2002).
In addition, vesicular transport is important for CV function,
as rab-like GTPases were localized to the CV of Dictyostelium
(Bush et al. 1994, Harris et al. 2001). In agreement with
this, electron microscopy (EM) has shown that coated pits are
associated with CV membranes in various organisms (Patterson
1980).
The CV in green algae consists of a fluid-filled membrane-bound
vacuole and associated coated-vesicles or vesicular
tubular membranes (Allen and Naitoh 2002, Hausmann and
Patterson 1984). In many algae, CVs repeatedly expel their
contents at discrete points of the cell surface. Some studies
have found membrane particle arrays associated with this
region of the plasma membrane (e.g. Weiss et al. 1977). The
structure and function of the CV of Chlamydomonas has been
investigated in some detail (e.g. Denning and Fulton 1989,
Domozych and Nimmons 1992, Gruber and Rosario 1979,
Luykx et al. 1997a, Luykx et al. 1997b, Robinson et al. 1998).
A total contraction cycle takes about 15 s in distilled water
(Luykx et al. 1997b). During the early stages of the diastole,
the CV of Chlamydomonas consisted of numerous vesicles of
about 70–120 nm diameter. These vesicles fused with each
other to form a large vacuole which grew by continued fusion
of small vesicles until the CV reached its final size of about
1.4 µm (Luykx et al. 1997b). H + -pumping pyrophosphatase and
V-ATPase have been suggested to be the primary energizing
source for water uptake in Chlamydomonas (Robinson et al.
1998). According to Robinson et al. (1998), the proton uptake
is followed by bicarbonate transport into the CV and osmotic
water uptake. However, direct proof for this mechanism has not
been presented yet.
The prasinophyte Scherffelia dubia is a green quadriflagellate
with a cell covering consisting of several different types
of scales. The scaly prasinophytes represent the ancestral stock
from which all other green algae and the land plants evolved

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(Nakayama et al. 1998, Chapman and Waters 2002). The flagella
are covered by three different types of non-mineralized
scales referred to as pentagonal, man (double, rod-shaped) and
hair scales based on their shapes as revealed by scanning electron
microscopy (SEM) and transmission electron microscopy
(TEM) (Melkonian and Preisig 1986, Becker et al. 1990). The
cell body is covered by a cell wall derived ontogenetically from
scales which coalesce extracellularly to form a cell wall called
a theca (McFadden et al. 1986, Melkonian and Preisig 1986).
Scales consist mainly of carbohydrates (Becker et al. 1991) and
are synthesized in the Golgi complex (Melkonian et al. 1991,
Becker et al. 1994). In vivo, the biogenesis of scales is
restricted to cell division. However, biogenesis of flagellar
scales can be induced by experimental amputation of the flagella.
Upon deflagellation, cells regenerate new flagella including
their scaly covering. Using this experimental system, the
anterograde transport of scales through the Golgi complex in S.
The contractile vacuole of S. dubia
Fig. 1 Structure of the contractile vacuole in S.
dubia as revealed by standard TEM. (A) Longitudinal
section through a flagellar regenerating
cell of S. dubia. (B) Higher magnification of the
CV region from a different cell. (C) Higher magnification
of the flagella groove of the cell shown
in (A), showing two growing flagella covered
with scales. (D–I) Early diastole phase: the scale
reticulum started to swell. The micrographs in
(D–G) and (H and I) represent consecutive sections
from the same cell, respectively. (K) Beginning
systole: a round vacuole discharged its
contents into the flagellar groove. Coated pits are
marked with arrows and pentagonal scales are
marked with arrowheads. bb, basal body; c, chloroplast;
cv, contractile vacuole; cw, cell wall; f,
flagellum; g, Golgi stack; m, mitochondrion; n,
nucleus; sr, scale reticulum. Bar = 0.5 µm.
dubia has been studied in detail (McFadden and Melkonian
1986a, McFadden et al. 1986, Becker et al. 1995, Perasso et al.
2000) and the results have contributed to the renaissance of the
cisternal progression model now called the cisternal maturation
model of intra-Golgi transport (Nebenführ 2003).
The fungal macrocyclic lactone brefeldin A (BFA) is a
commonly used inhibitor of protein secretion and Golgi function
in plant and mammalian cells (reviewed in Nebenführ et
al. 2002). When we treated S. dubia cells with BFA, we
observed that BFA interfered as expected with the structure and
function of the Golgi complex and, more surprisingly, with the
function of the CV. The two CVs swelled and finally formed
large central vacuoles (LCVs). We propose that BFA-induced
formation of LCVs in S. dubia represents a prolonged diastole
phase of the CV cycle. Therefore, BFA-dependent formation of
the large vacuole might offer a unique possibility to investigate
the diastole phase of a CV cycle in detail.

Fig. 2 BFA interferes with the function of the CV. Upon addition of
BFA to S. dubia cells, the two CVs swell (B, 5 min of BFA treatment)
and form a large central vacuole (LCV) at later stages (C, 10 min of
BFA treatment). (A) Control cells as seen in the light microscope. (D)
The rate of formation of LCVs depends on the temperature of the
medium.
Results
Structure of the contractile vacuole in Scherffelia dubia
When S. dubia cells were observed with the light microscope
(LM) in culture medium (5 mosM), two CVs were easily
detected near the anterior end of the cell on both sides of the
flagellar groove (see Fig. 2A for a light micrograph of a S.
dubia cell). The two CVs tended to alternate in their contractions,
with an average interval of 22.3 ± 3.5 s (n = 15, value
based on video recordings of single cells). However, in a few
cases, considerably shorter or longer contraction intervals were
observed (as short as 9 s and as long as 30 s), which sometimes
led to almost simultaneous contraction cycles. Each CV
The contractile vacuole of S. dubia 203
reached a maximum size of 1.4 ± 0.1 µm (n = 15) at the end of
diastole.
The ultrastructure of the CV was investigated in chemically
fixed cells (see Melkonian and Preisig 1986 for a detailed
description of the ultrastructure of S. dubia). At the end of diastole,
the CV consisted of a single vacuole with a smooth membrane
(Fig. 1A, B). At this stage of the CV cycle, we never
observed coated pits on the CV membrane. Sometimes pentagonal
scales (underlayer scales associated with the flagellar
membrane, see Fig. 1C, arrowheads) were found to be present
on the inner surface of the CV. The number of pentagonal
scales within the CV is low in interphase cells, when cells do
not synthesize flagellar scales (Table 1). In contrast, in CVs of
cells in which flagella biosynthesis (including biogenesis of
scales) has been induced by experimental amputation, we
observed a significant number of pentagonal scales (Table 1).
The two other scale types present on the flagella of S. dubia
were never observed within the CV.
In addition to the single vacuole representing the CV at
late diastole, a membranous reticulum containing numerous
coated pits (Fig. 1, arrows) is present in the CV region. In tangential
sections, the coated pits display honeycomb-like structures
typical for clathrin-coated pits (Melkonian and Preisig
1986). Coated pits often contain pentagonal scales (Fig. 1,
arrowheads). For this reason, the membrane reticulum was
called the scale reticulum (SR) by Melkonian and Preisig
(1986). At the beginning of the diastole, the SR swelled (Fig. 1
D–I; note the coated pits containing pentagonal scales indicated
by arrowheads) before a large round vacuole was formed (Fig.
1A, B). During systole (lasting 1–2 s, based on video recordings),
the CV discharged its contents into the flagellar groove
(Fig. 1K). No small vacuoles similar to the situation in
Chlamydomonas (Luykx et al. 1997b), or a collapsed large vacuole
were found within the CV region (Fig. 1A, B). The only
other membranous organelle observed in the CV region was the
SR, indicating that the SR might be part of the CV complex of
Scherffelia. However, a continuity of the SR with the round
Table 1 Number of pentagonal scales observed in contractile vacuoles (interphase and flagellar regenerating cells) and large central
vacuoles (BFA-treated flagellar regenerating cells).
No. of CVs or
LCVs analysed
No. of pentagonal
scales observed
µm 2 membrane
analysed
No. of scales per µm 2 CV or
LCV membrane surface a
Control cells 14 2 3.18 0.6 ± 1.6
Flagellar regenerating cells 11 22 2.33 9.2 ± 6.5
5min BFA b 7 9 5.1 4.8 ± 3.0
10 min BFA b 11 25 6.9 3.6 ± 2.4
15 min BFA b 6 11 4.3 2.9 ± 2.0
20 min BFA b 6 8 4.5 1.5 ± 2.3
30 min BFA b 4 5 3.3 1.5 ± 1.1
a 2 2
The number of scales per µm of membrane surface was calculated for each CV. The average number of scales per µm of membrane surface and
the SD are given.
b
BFA was added 20 min after experimental deflagellation.

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vacuole at late diastole stages was never observed even when
serial sections were used to investigate a larger surface area of
the CV (data not shown).
Effect of BFA on the contractile vacuole
Upon addition of BFA to S. dubia cells, the two CVs
failed to expel liquid and swelled (Fig. 2B, 5 min of BFA treatment,
video 1 of the Supplementary material) which is accompanied
by an increase in total cell volume indicating the uptake
of water by the cell (see video 1 of the Supplementary material).
About 10 min after BFA addition, the cells lost their flagella.
Finally, >90% of the cells developed an apparent single
LCV (Fig. 2C, video 1 of the Supplementary material). Within
30 min, the LCV reached an average diameter of 6.7 ± 1.3 µm
(n = 20). A few cells (maximum 10% of the cell population)
remained motile and completely unaffected by BFA treatment
as judged by LM.
The formation of an LCV was completely reversible
(tested for up to 1 h of BFA treatment at 15°C, data not shown).
BFA-induced formation of an LCV was temperature dependent.
At 0°C (cells incubated with BFA on ice), no LCVs were
The contractile vacuole of S. dubia
Fig. 3 Electron microscopic analysis of the BFAinduced
formation of large central vacuoles (LCVs).
Flagellar regeneration was induced by experimental
amputation and the cells allowed to regenerate flagella
for 20 min before BFA was added. Cells were
treated for the indicated time with BFA and processed
for standard TEM. (A and B) After 5 min of
BFA treatment. (A) Longitudinal section through a
cell and (B) a cross-section of a cell: the CV was
enlarged, showed an irregular appearance and coated
pits (arrows) are associated with the enlarged CV.
(C) After 10 min of BFA treatment: a round vacuole
is formed. (D) After 15 min of BFA treatment. (E)
After 30 min of BFA treatment. bb, basal body; c,
chloroplast; cv, contractile vacuole; g, Golgi stack;
lcv, large central vacuole; n, nucleus; v, putative
polyphosphate-containing vacuole. Bar = 0.5 µm.
formed at all (Fig. 2D) and in cells incubated with BFA at 15°C
the formation of LCVs was significantly slower when compared
with cells treated with BFA at 25°C (Fig. 2D).
Ultrastructure of the CV in BFA-treated S. dubia cells
Cells were deflagellated and incubated for 20 min at 20°C
in culture medium before BFA was added. During the incubation
time (without BFA), the cells started to regenerate new
flagella and synthesized new flagellar scales (see Fig. 4A). At
20 min after deflagellation, all Golgi cisternae contain scales
except for the two to three cis-most cisternae (see below for
details). Cells were chemically fixed and processed for EM
prior to addition of BFA and 5, 10, 15, 20 and 30 min after adding
BFA. As a control, methanol alone was added 20 min after
deflagellation and cells were fixed for EM after 15 and 30 min
incubation time, respectively. No effect on the ultrastructure of
S. dubia cells was observed in the control samples (not shown).
Cells fixed at 5 min after addition of BFA contained
enlarged CVs of an irregular geometry (Fig. 3A, B). In contrast
to control cells, numerous coated pits are associated with
the CV of BFA-treated cells (Fig. 3C, arrows). At 10 min after

addition of BFA, the CVs were larger and had a more round
geometry (Fig. 3D). Only a few coated pits associated with the
CVs were observed. EM of later stages showed either a single
LCV (Fig. 3E, 15 min), which is in agreement with the LM
observations, or two LCVs (Fig. 3F, 30 min). Most probably,
cells always contained two LCVs which could not be resolved
by LM (see also below). In cells treated with BFA for >15 min,
only remnants of the SR were observed, indicating that the SR
was mainly adsorbed into the LCV. The number of pentagonal
scales per µm 2 of the LCV membrane decreased with BFA
The contractile vacuole of S. dubia 205
Table 2 Effect of BFA on the structure of the Golgi complex in S. dubia
Fig. 4 BFA affects the structure of the
Golgi complex in S. dubia. Flagellar regeneration
of S. dubia cells was induced by
experimental amputation and the cells
allowed to regenerate flagella for 20 min
before BFA was added. Cells were treated
for the indicated time with BFA and processed
for standard TEM. (A) Cells were
fixed 20 min after deflagellation. A longitudinal
section is shown. The Golgi stack is
filled with scales (arrowheads). At the rim
of the stack, numerous transition vesicles
can be seen (small arrows). All types of
scales are transported within the same vesicles
to the flagellar surface (arrow). (B)
Cells were treated with BFA for 5 min
before they were chemically fixed and processed
for EM. The number of Golgi cisternae
is reduced and only few transition
vesicles can be seen. The arrow points to a
vesicular tubular element which seems to
leave the stack. (C) After 10 min of BFA
treatment. The number of cisternae has
decreased further and all cisternae contain
scales (arrowheads). Numerous vesicles of
150 nm in diameter accumulate at the cisface
of the stack (arrows). bb, basal body; c,
chloroplast; g, Golgi stack; f, flagellum; lcv,
large central vacuole; m, mitochondrion; n,
nucleus; sr, scale reticulum. Bar = 1 µm.
incubation time (Table 1), indicating that the membrane source
for LCV growth had a lower scale content than the CV and
‘diluted’ the number of scales per µm 2 of membrane in the
LCV. Similar results were obtained when interphase cells (no
flagellar regeneration) were treated with BFA.
Ultrastructure of the Golgi complex in BFA-treated cells
Beside affecting the structure of the CV, BFA also induced
changes in the structure of the Golgi complex of S. dubia. In
flagellar regenerating cells of S. dubia, the two Golgi stacks
Control cells 5 min BFA 10 min BFA
No. of stacks analysed 24 17 15
No. of cisternae a
14.3 ± 0.8 11.6 ± 1.1 8.3 ± 1.2
No. of trans-cisternae containing scales a 11.3 ± 0.7 10.5 ± 1.1 8.0 ± 1.3
No. of transition vesicles/stack a 10.6 ± 3.3 0.7 ± 1.1 0
Cells were deflagellated and allowed to regenerate for 20 min before BFA was added. Cells were fixed and
processed for EM prior to and 5 or 10 min after addition of BFA.
a Only clear cross-sections of Golgi stacks were analysed.

206
contained about 14 cisternae of about 1.5 µm diameter (Fig.
4A, Table 2). The cisternae were filled with flagellar scales
(arrowheads) except for the two or three cis-most cisternae
which are generally devoid of any scales (Fig. 4A). Numerous
transition vesicles (most probably COP1) can be see at the rim
of the stack (Fig. 4A, small arrows, Table 2). Secretory vesicles
containing all three types of scales transport the scales to
the cell surface (Fig. 4A, arrow). Upon addition of BFA, the
number of transition vesicles dropped within 5 min (Table 2,
Fig. 4B) and the number of cisternae decreased from 14 to
eight within the first 10 min (Table 2, Fig. 4B, C). At 10 min
after addition of BFA, all remaining cisternae including the cismost
cisternae contained scales (Table 2, Fig. 4C). At the cisface
of the stack, numerous small vacuoles (150 nm diameter,
arrows) accumulate. No further reduction of the number of cisternae
could be observed at longer BFA incubation times (e.g.
the Golgi stack in Fig. 3D contains seven cisternae after 15 min
of BFA treatment). However, after 15 min, the overall structure
of the cells was affected strongly by the growing LCV
(Fig. 3D, E). Therefore, it is difficult to rule out that the growing
LCVs inhibited a further BFA effect on the structure of the
Golgi complex. So far, we have not been able to find any conditions
which uncouple both BFA effects and would allow
investigation of the BFA effect on the Golgi structure without
affecting the CV, and vice versa.
V-ATPase activity is required for the formation of a LCV
V-ATPase function is required for CV function in many
organisms (summarized in Allen 2000, Allen and Naitoh
2002). To test whether V-ATPase activity is also required for
the formation of LCVs, cells were treated with BFA and/or the
V-ATPase-specific inhibitor concanamycin A (Concan A).
Concan A turned out to be very toxic for S. dubia, and cells
were dead within 15 min when treated with 1.5 µM (as judged
by LM). We were not able to observe any CV activity in cells
treated with Concan A. Due to the loss of CV activity, cells
The contractile vacuole of S. dubia
Fig. 5 TEM analysis of the effect of Concan
A on cells of S. dubia. Cells were treated
with Concan A and chemically fixed and
processed for electron microscopy. (A and
B) Longitudinal sections. (C) Cross-section.
Arrows point to numerous transition vesicles
seen at the periphery of the Golgi stack.
bb, basal body; c, chloroplast; g, Golgi stack;
f, flagellum; m, mitochondrion; n, nucleus;
sr, scale reticulum. Bar = 1 µm.
treated with Concan A first swelled and finally burst (not
shown), explaining the toxicity of Concan A observed. Cells
were viable in the presence of Concan A when 100 mM
sucrose was added to the medium (not shown). Under these
conditions, the cells remained motile although many cells
showed plasmolysis (see also below). EM confirmed the light
microscopic observations. In cells treated with Concan A for
10 min, not a single filled CV was detectable (Fig. 5A) and the
structure of the Golgi stacks was altered (Fig. 5). The number
of cisternae increased and the trans-cisternae showed a strong
Fig. 6 V-ATPase function is required for BFA-induced LCV formation.
Cells were treated with BFA or with BFA and Concan A for 5 or
10 min, fixed with osmium tetroxide and observed with a light microscope.
(A) After 5 min of BFA treatment. (B) After 5 min of BFA and
Concan A teatment. (C) After 10 min of BFA treatment. (D) After
10 min of BFA and Concan A treatment.

Fig. 7 The CVs and the LCVs do not accumulate Neutral Red. Neutral
Red-stained living cells were viewed in the bright field light
microscope. Neither the CV (A) nor the LCV (B) are acidic enough for
a significant accumulation of Neutral Red. Numerous small vesicles
can be stained with Neutral Red (A and B), which are probably identical
to the putative polyphosphate-containing vacuoles surrounding the
nucleus (compare A and E). (A) Control cells. (B) Cells treated with
BFA for 20 min. (C) Electron micrograph of control cells processed
for standard TEM. N, nucleus; v, putative polyphosphate-containing
vacuole. Bar = 0.5 µm.
concave curvature (Fig. 5A). In many sections, the trans-cisternae
appeared as a multilamellated vesicular structure (Fig. 5A,
B). The formation of transition vesicles at the periphery of the
Golgi stacks was not affected (Fig. 5C).
When cells were treated with BFA and Concan A for 5 or
10 min, no LCV was formed (Fig. 6A–D). Cells lost their flagella
but the overall structure of the cells was not affected (Fig.
6B, D). We conclude that V-ATPase activity is required for
BFA-induced formation of LCVs, for CV function and for
maintenance of the structure of the Golgi complex in S. dubia.
The requirement for V-ATPase activity for CV function
and formation of LCVs led us to investigate whether CVs and
LCVs are acidic organelles. Control cells and BFA-treated cells
were incubated with Neutral Red and observed by LM. Neither
The contractile vacuole of S. dubia 207
the CV (Fig. 7A) nor the LCV (Fig. 7 B) was significantly
stained with Neutral Red. Instead, numerous small vacuoles
mainly with a perinuclear position accumulated Neutral Red
(Fig. 7A, B). The Neutral Red-containing vacuoles occupy the
same position in a cell as the putative polyphosphate-containing
vacuoles (compare Fig. 7A and C) described by Melkonian
and Preisig (1986).
Effect of the osmolarity of the medium on the formation of LCVs
To investigate the effect of the external medium on the
formation of LCVs, S. dubia cells were pre-incubated with a
sucrose (60–160 mM)-containing medium for 15 min (Fig.
8A). Many cells (67 ± 8%, n = 12) showed plasmolysis when
incubated with 100 mM sucrose, indicating that the osmolarity
of the cytosol is generally less than 100 mosM (Fig. 8, arrows).
Since this value is rather low, we determined the cytosolic
osmolarity directly. The method of Stoner and Dunham (1970)
as described by Stock et al. (2001) was used (see Materials and
Methods for details). Direct measurement of the cytosolic
osmolarity in a cell pellet gave a mean value of 93.1 ±
0.4 mOsmol (n = 3) which is in good agreement with the plasmolysis
data.
BFA was then added to the cells incubated for 15 min with
100 mM sucrose and the cells were incubated for another
30 min with BFA before fixation with osmium tetroxide. BFAinduced
formation of LCVs was inhibited by external sucrose
concentrations (Fig. 8B, C). An external sucrose concentration
of 100 mM inhibited the formation of LCV to 90% (Fig. 8B),
but many cells still displayed swollen CVs. At higher concentrations
of sucrose, most cells did not display swollen CVs
(Fig. 8C). Identical results were obtained when NaCl instead of
sucrose was used as osmoticum.
To test whether the LCVs are maintained under hyperosmotic
conditions, S. dubia cells were pre-incubated with BFA
for 30 min and then incubated with BFA/sucrose (100–
200 mM) for another 30 min before fixation with osmium
Fig. 8 BFA-induced formation of the LCV can be
inhibited (A–C) and reversed (D–E) by a hypertonic
medium. (A) Cells were treated with sucrose for 15 min.
Arrows indicate cells displaying plasmolysis. (B and C)
Cells were pre-incubated with sucrose (B, 100 mM; C,
160 mM) for 15 min and then with sucrose and BFA for
30 min. (D) Cells were incubated with BFA for 30 min.
(E and F) Cells were pre-incubated with BFA for 30 min
followed by a 30 min incubation with BFA/sucrose (E,
120 mM sucrose; F, 180 mM sucrose).

208
Fig. 9 BFA inhibits the CV in various prasinophyte algae. In T. cordiformis,
BFA also caused the formation of a LCV (A and B). In P.
tetrarhynchus, the cells swelled and finally burst after addition of
BFA, indicating that BFA also interfered with CV function in P. tetrarhynchus
(C and D). (E) BFA also interfered with CV function in M.
viride. Single frames of a time-lapse video showing the swelling of
two Mesostigma cells in the presence of BFA are shown. Numbers
refer to the time elapsed since addition of BFA.
tetroxide. After pre-incubation with BFA, the cells showed the
typical LCVs (Fig. 8D). When sucrose (120 mM) was added,
the LCV shrunk (compare Fig. 8D and E, video 2 of the Supplementary
material) and, at higher concentrations of sucrose,
cells displayed even smaller LCVs, two vacuoles or no vacuole
at all (Fig. 8F). In summary, the results presented indicate
that BFA-induced swelling of CVs can take place under hyperosmotic
conditions (Fig. 8B) and that LCVs shrink under
strong hyperosmotic conditions (Fig. 8D and E).
BFA effect on the contractile vacuole in other algae
To our knowledge, a BFA effect on the CV has never been
reported before. Therefore, we investigated whether BFA (1 µg
ml –1 ) had a similar effect on other prasinophyte algae. In the
closely related genus Tetraselmis cordiformis, the only known
freshwater Tetraselmis species, BFA also caused the formation
of a LCV (Fig. 9A, B). In Pyramimonas tetrarhynchus, the
cells swelled (Fig. 9C, D) and finally burst after addition of
BFA, indicating that BFA also interfered with CV function in
Pyramimonas. In contrast, no effect of BFA on the CV of
The contractile vacuole of S. dubia
Chlamydomonas reinhardtii was observed, which is in agreement
with earlier reports (Robinson 1993).
So far, all tested freshwater prasinophytes belong to the
chlorophyte lineage within the Viridiplantae. Therefore, we
also tested the effect of BFA on Mesostigma viride, the only
known scaly flagellate within the streptophyte lineage, which
includes the charophytes and land plants. M. viride cells were
incubated with BFA at 1 µg ml –1 and living cells observed by
LM. Within a few minutes, M. viride cells swelled and finally
burst when incubated with BFA (Fig. 9E). Thus it appears that
in all scaly green freshwater flagellates (prasinophytes), CV
function is sensitive to BFA.
Discussion
In this report, we analyse the function of the CV in S.
dubia using EM and inhibitor studies. We show that treatment
of S. dubia with BFA induced the formation of two large vacuoles
which appear as a single LCV in the light microscope, and
characterize the formation of LCVs regarding temperature
dependence, requirement for V-ATPase activity and effect of
hypertonic media.
Structure of the contractile vacuole
The EM data presented in this study indicate that in S.
dubia, the large round vacuole present at late diastole develops
from the SR. There are other arguments which are in favour of
an involvement of the SR in CV function. First, V-ATPase has
been localized by immunoelectron microscopy only to the SR
and to a lesser extent to the Golgi complex (Grunow et al.
1999). Remarkably, the round vacuole of the CV found at late
diastole phase was devoid of any labeling (Grunow et al.
1999). Several lines of evidence indicate that V-ATPase is
required for CV function in various systems including S. dubia.
Vacuolar H + -ATPase was found to be associated with the CV in
Dictyostelium and Paramecium (Fok et al. 1993, Fok et al.
1995, Heuser et al. 1993). The CVs of Dictyostelium (Temesvari
et al. 1996) and S. dubia cells (this study) were inhibited
by the V-ATPase inhibitor Concan A, and Concan A-treated
cells burst in hypotonic media (Temesvari et al. 1996, this
study). Treatment of Paramecium cells with concanamycin B,
another V-ATPase inhibitor, also strongly inhibited fluid output
from the CV (Fok et al. 1995).
Secondly, no SR or a similar organelle was found in
marine Tetraselmis species (Manton and Parke 1965,
Domozych et al. 1981, Domozych 1987, Marin et al. 1996),
which are closely related to Scherffelia (Nakayama et al. 1998),
and the overall ultrastructure of both genera is very similar
(Melkonian and Preisig 1986). Tetraselmis cordiformis is the
only known freshwater Tetraselmis species and also displayed
an LCV when the cells were treated with BFA. Unfortunately,
the ultrastructure of T. cordiformis is not well examined and the
published micrographs (Melkonian 1982) do not contain any

information regarding the presence or absence of a SR or a
similar structure.
Mechanism of water uptake into CVs and LCVs
Since LCVs are derived from CVs, the mechanism of
water uptake into both organelles is probably the same. Basically
two models are generally considered. Water could enter
the CVs and LCVs by osmosis, or ATP-energized water pumps
could move water across membranes against an osmotic gradient
(discussed in Allen and Naitoh 2002). The existence of
water pumps has been demonstrated (Zeuthen 1992). However,
an involvement of water pumps with water transport into
the CV has not been demonstrated yet. Water transport by
osmosis requires a higher osmolarity inside the CV than in the
cytosol. Unfortunately, the composition of the CV liquid is
not known. A long-held assumption is that the CV expels
water. However, there is little direct evidence for the composition
of the expelled liquid (reviewed in Allen 2000, Allen and
Naitoh 2002). For the CV of the amoeba Chaos chaos, Riddick
(1968) determined an osmolarity of 51 mOsmol (cytosol
117 mOsmol), and similar values have been found for Amoeba
proteus (Schmidt-Nielsen and Schrauger 1963). As pointed out
already by Heuser et al. (1993), water should not accumulate
under these conditions within the CV and therefore would
require water pumps. Recent estimates of the osmolarity (based
on Na + , K + Ca 2+ and Cl – activity measurements) of the CV and
cytosol of Paramecium showed that the osmolarity of the CV
was 1.5 times higher than that of the cytosol in Paramecium,
supporting the hypothesis that water uptake into the CV is
driven by osmosis in Paramecium (Stock et al. 2002a, Stock et
al. 2002b). In this study, we have shown that under strong
hyperosmotic conditions, LCVs shrunk, indicating that water
within LCVs followed osmotic gradients and supporting the
The contractile vacuole of S. dubia 209
Fig. 10 Cartoon showing the changes in the structure of the CV of S. dubia during a contraction cycle. The steps most likely inhibited by BFA
and Concan A are indicated. The lower panel shows the changes observed in the ultrastructure of a CV when cells were treated with BFA.
hypothesis that water uptake into CVs and LCVs was also
driven by osmosis in S. dubia, although this experiment cannot
exclude the presence of water pumps completely.
Taken together, the results obtained support the following
model for CV function in Scherffelia (Fig. 10). At early diastole,
the SR swells due to osmotic uptake of water energized by
V-ATPase. At late diastole, the remaining SR is disconnected
from the developing round vacuole typical of the CV at late
diastole, before the CV discharges its content during systole
into the flagellar groove. Whether the vacuolar membrane is
incorporated into the plasma membrane and recycled by endocytosis
or collapses and is disconnected from the plasma membrane
cannot be decided based on the current knowledge.
However, so far, collapsed CV membranes have not been
reported in Scherffelia (Perasso et al. 2000, Melkonian and Preisig
1986, this study) and, therefore, the latter does not seem
likely.
The structure of the CV in S. dubia is in marked contrast
to the only other well characterized CV within the green algae:
C. reinhardtii. In C. reinhardtii, the large round vacuole of late
diastole develops from numerous smaller vacuoles (Luykx et
al. 1997b). Similar vacuoles have never been found in S. dubia
(Melkonian and Preisig 1986, Perasso et al. 2000, this study).
Another important difference between Chlamydomonas and
Scherffelia is that CV function in Chlamydomonas was not
inhibited by BFA (Robinson 1993, this study). These differences
are not really surprising since green algae most probably
evolved in a marine environment and invaded the freshwater
habitat several times independently.
Molecular basis of the BFA effect
Currently, the molecular basis for the BFA-induced formation
of LCVs is not known. It is well established that in mam-

210
malian cells, BFA targets a subset of sec7-type GTP exchange
factors (GEFs) that catalyse the activation of small GTPases
called ARFs (Jackson and Casanova 2000). ARF1 is the best
studied member of this protein family. Upon binding of GTP,
ARF1 is responsible for the formation of COP1-coated vesicles
at the Golgi complex as well as the formation of clathrin/
AP1-coated vesicles at the trans-Golgi network (TGN) (Scales
et al. 2000, Spang 2002). The molecular targets of BFA have
been found in the Arabidopsis genome (Cox et al. 2004) and it
is generally assumed that the primary BFA effect is similar in
plants and in animals (for a recent review see Nebenführ et al.
2002). This study revealed that the initial effect of BFA on the
structure of the Golgi complex is also similar in S. dubia. The
Golgi stacks lose the peripheral transition vesicles and the
number of cisternae decreases as in land plants. Maturation of
cisternae during the fist minutes seemed to continue, as 10 min
after addition of BFA all cisternae contained scales, whereas in
control cells the first two to three cis-most cisternae were generally
devoid of scales. In S. dubia, we observed a second
effect of BFA. The CVs swelled and failed to discharge their
contents into the medium. In this study, we have presented evidence
that the CV in Scherffelia develops from the SR. Since
we could not observe a connection between the SR and the
round vacuole at a late diastole phase, we propose that during
the CV cycle, the developing round vacuole and the SR are disconnected
(Fig. 10), a process which is analogous to late steps
in vesicle budding and which might be regulated by an ARF
protein. BFA might inhibit this process which then leads to the
observed swelling of the vacuole (Fig. 10).
Beside the inhibition of the retrograde transport from the
Golgi to the endoplasmic reticulum, BFA induced in land
plants the formation of a Golgi-derived ‘conglomerate of
tubules and vesicles’ termed the BFA compartment (Satiat-
Jeunemaitre et al. 1996) which also included endocytotic compartments
(reviewed in Nebenführ et al. 2002), and inhibited
endocytosis of a styryl dye in BY-2 cells (Emans et al. 2002),
indicating that also in land plants BFA acts on post-Golgi compartments.
It is currently not known whether the BFA effect on
the endocytotic pathway has the same molecular basis as the
Golgi effect. In this context, it is interesting that we recently
have found in our expressed sequence tag (EST) sequencing
project of the prasinophyte M. viride three different cDNAs for
a class 1 ARF protein in interphase cells (A. Simon and B.
Becker, unpublished). The CV function in Mesostigma is also
inhibited by BFA (this study). Therefore, it seems possible, that
a specific ARF might exist which is involved in regulating the
CV function in freshwater prasinophyte algae.
Whether the SR has to be disconnected as a prerequisite
for exocytosis during the CV cycle, or whether BFA also interfered
directly with exocytosis of the CV is currently an open
question. Another possibility is that COP1- and/or clathrin/
AP1-mediated vesicular transport is required for proper function
of the CV. In this case, the observed effect of BFA on the
function of the CV would be an indirect one. However, the lat-
The contractile vacuole of S. dubia
ter model cannot easily explain the observed consumption of
the SR by the growing LCVs. Additional work will be required
to resolve this issue.
Several different species of green algae have been treated
with BFA, but a BFA effect on the CV has, to our knowledge,
never been reported before (e.g. Dairman et al. 1995, Salomon
and Meindl 1996, Haller and Fabry 1998, Noguchi et al. 1998,
Domozych 1999, Noguchi and Watanabe 1999, Callow et al.
2001). All of the tested species are either marine (Enteromorpha),
do not possess a CV (Micrasterias, Closterium,
Scenedesmus) or belong to the volvocalean algae (which
includes Chlamydomonas), which explains why a BFA effect
on the CV has not been reported yet. Therefore, a brief survey
of the BFA effect on the CV in green flagellates was undertaken.
All tested scaly green flagellates showed an effect of
BFA on the CV. However, only S. dubia and T. cordiformis
developed LCVs, whereas in P. tetrarhynchus and M. viride, no
LCV formation was observed. BFA interfered with CV function
in both species, and the cells swelled and finally burst in
hypotonic media. In contrast, no BFA effect on the CV of
Chlamydomonas was observed (Robinson 1993, Haller and
Fabry 1998, this study). Inhibition of CV function by BFA
seems to be common within prasinophytes which represents the
ancestral stock from which all other green algae and the land
plants evolved (Nakayama et al. 1998, Chapman and Waters
2002), but not all prasinophyte develop an LCV. In addition,
the absence of any BFA effect in Chlamydomonas and other
volvocalean algae (Dairman et al. 1995) indicates that
advanced chlorophyte algae might have lost the BFA sensitivity
of the CV.
To summarize, cells of S. dubia treated with BFA fail to
terminate the early diastole phase of the CV cycle. The cells
continue to accumulate ions in the CV, which leads to additional
water uptake and swelling of the CV. This basically represents
a prolonged diastole phase. A normal diastole phase
takes about 20 s and is therefore difficult to investigate. Therefore,
we conclude that BFA-induced formation of LCVs in S.
dubia will represent a unique model system to investigate the
diastole phase of the CV cycle.
Materials and Methods
Strains and culture conditions
Scherffelia dubia Pascher emend. Melkonian et Preisig (Melkonian
and Preisig 1986) strain CCAC019 was obtained from the Culture
Collection of Algae at the University of Cologne and cultured as
previously described (Grunow et al. 1993). Mesostigma viride Lauterborn,
Tetraselmis cordiformis (Carter) Stein and Pyramimonas tetrarhynchus
Schmarda were kindly provided by Dr. M. Melkonian
(Botanical Institute, University of Cologne) and cultured in 100 ml
Erlenmeyer flasks containing 50 ml of a modified Waris solution
(McFadden and Melkonian 1986b) at 15°C and a 14/10 light/dark
cycle. Chlamydomonas reinhardtii strain CC-3395 was kindly provided
by Dr. K.-F. Lechtreck (Botanical Institute, University of
Cologne) and cultured in TAP medium in a 14/10 light/dark cycle at
25°C.

Inhibitor treatments
A stock solution (1 mg ml –1 ) of BFA (ICN) was prepared in
methanol. Cells were incubated with BFA at a concentration of 1 µg
ml –1 of BFA, the lowest concentration inhibiting growth in S. dubia.
Concan A was a gift of Dr. G. Thiel, Technical University of Darmstadt,
Germany and was used at a concentration of 1.5 µM (stock solution
15 mM in MeOH). Inhibitor treatments were performed at 15°C if
not indicated otherwise.
Light microscopy
For direct LM observations, 5 µl of a cell suspension was used
per slide (24×50 mm coverslip). Such preparations immobilize most
cells and last at least 15 min without any significant change in CV
activity. Observations were made either with a Zeiss IM 35 microscope
equipped with a ×63 oil immersion objective in the brightfield
or differential interference contrast mode, or with a Nikon Eclipse 800
microscope ×60 or ×100 oil immersion objectives in the brightfield or
differential interference contrast mode. The Nikon microscope is
equipped with a Spot CCD camera (Diagnostic Instruments, MI,
U.S.A.) and micrographs taken were analysed with the Metamorph
software (version 4.5, Universal Imaging Corporation, PA, U.S.A.).
Electron microscopy
Samples for EM were taken from interphase cells and during
flagellar regeneration before BFA treatment and at various time points
after adding BFA, and fixed with glutaraldehyde and osmium tetroxide
as described previously (Perasso et al. 2000). Further processing of
the samples was standard (McFadden and Melkonian 1986b). EM
micrographs were taken with a Phillips CM10 electron microscope
(Eindhoven, The Netherlands).
Quantitative analysis of the number of pentagonal scales per CV/LCV
membrane surface
The number of pentagonal scales and the diameter of the CV/
LCV was determined on micrographs. The perimeter of the circular
profile of the CV/LCV was calculated and the membrane area of the
CV/LCV on a given section was estimated using the known thickness
of the section (70 nm). In cases where an elliptical profile for the CV/
LCV was encountered, the major and minor radii were measured and
the perimeter calculated using the approximation formula of Ramnujan
for the perimeter of an ellipse.
Determination of the cytosolic osmolarity
The method of Stoner and Dunham (1970) as described by Stock
et al. (2001) was used. Cells were washed twice with WEES-S (a salt
solution containing only the mineral components of the culture
medium). Cells were pelleted and the osmolarity of the pellet was
determined using a freezing point osmometer (Osmomat 030,
Gonotec, Berlin). The value was corrected for the volume containing
WEES-S in the extracellular space within the pellet which was determined
as described by Stock et al. (2001) except that blue dextran
(Amersham) was used instead of Congo red. Briefly, cells were pelleted
and the pellet volume was measured (15–50 µl). The cells were
resuspended in 1 ml of a blue dextran solution (0.5% in WEES-S) and
pelleted again. The supernatant was carefully removed and the cells
resuspended in a known volume of WEES-S. Cells were pelleted and
the concentration of blue dextran in the supernatant was determined
with a spectrophotometer. The volume of extracellular space within the
pellet can be calculated from the dilution of dextran blue (Stock et al.
2001).
The contractile vacuole of S. dubia 211
Supplementary material
Supplementary material mentioned in the article is available to
online subscribers at the journal website www.pcp.oupjournals.org.
Acknowledgments
We thank Lars Vierkotten and B. Wustman for help with electron
microscopy, and M. Melkonian for helpful discussions. This study
was supported by the Deutsche Forschungsgemeinschaft.
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(Received March 29, 2004; Accepted November 4, 2004)