jf. Cell Sci. 31, 213-224 (1978)
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213
OSMOREGULATION IN THE ALGA
VACUOLARIA VIRESCENS. STRUCTURE OF
THE CONTRACTILE VACUOLE AND
THE NATURE OF ITS ASSOCIATION WITH
THE GOLGI APPARATUS
PETER HEYWOOD
Division of Biology and Medicine, Brown University, Providence,
Rhode Island 02912, U.S.A.
SUMMARY
The contractile vacuole of the chloromonadophycean alga Vacuolaria virescens is a permanent
structure that possesses a specialized membrane: subunits of this membrane have a diameter
of 21—24 nm and in places are arranged in a regular hexagonal pattern. The lateral walls of these
subunits form regularly spaced bristles or pegs which extend inwards from the trilaminar membrane for a distance of 13-15 nm. The contractile vacuole is situated immediately above an
extensive Golgi apparatus that covers most of the anterior surface of the nucleus. Vesicles of
Golgi origin give rise to subsidiary vacuoles which in turn empty into the contractile vacuole.
Golgi vesicles, subsidiary vacuoles and the contractile vacuole contain similar electron-dense
material. It is suggested that this material might be a highly hydrophilic substance which will
attract water from the cytoplasm into the Golgi vesicles, subsidiary vacuoles and contractile
vacuole from whence it is discharged from the cell. This method of osmoregulation is compared
to that occurring in other algae and protozoa.
INTRODUCTION
Many algae and protozoa possess contractile vacuoles (sometimes termed 'water
expulsion vesicles'). These osmoregulatory organelles expel a hypotonic solution from
the cell and so compensate for the influx of water from the surrounding medium due
to osmosis. Although contractile vacuoles are present in some marine organisms
(Dodge, 1973; Lloyd, 1928), they are usually absent from organisms inhabiting
marine or brackish water. For example, in the algal class Chloromonadophyceae
contractile vacuoles are present in the freshwater species (Fott, 1971; Mignot, 1967)
but are absent from the closely related marine representatives (Mignot, 1976; Toriumi
& Takano, 1973). A similar situation has been reported in laboratory cultures of
Chlamydomonas moeivusii (Guillard, i960). A mutant of this organism behaved like a
brackish water alga in that it required the osmotic pressure of the culture medium to
be higher than 1-5 atm. (1-013 x i o 2 kN m~2). Significantly, culture media of this
osmotic pressure caused the contractile vacuole of the wild strain to cease functioning
which led Guillard to the conclusion that ' the sole essential function of contractile
vacuoles in C. moewusii is elimination of water'. The physiological aspects of contractile vacuoles have received a good deal of attention and there is an extensive literature
214
P- Heywood
dealing with the effects on contractile vacuole function of such factors as the osmotic
pressure, pH, and temperature of the external medium (Ahmad & Couillard, 1974;
Czarska, 1964; Kitching, 1967; Organ, Bovee & Jahn, 1968; Rifkin, 1973). It has also
been demonstrated that adenosine triphosphate caused isolated vacuoles of Amoeba
proteus to contract (Prusch & Dunham, 1970) and accelerated the contraction cycle in
Paramecium multimicronucleatum (Organ et al. 1968).
The focus of this investigation is the freshwater chloromonadophycean alga
Vacuolaria virescens Cienkowsky. The earliest account of this organism (Cienkowsky,
1870) described the contractile vacuole, a conspicuous feature of the cell which is
presumably the basis for its generic name. Subsequently the contractile vacuole has
been examined with the light microscope (Dangeard, 1939; Poisson & Hollande,
1943; Spencer, 1971; Tschermak-Woess, 1954) and with the electron microscope
(Mignot, 1967; Schnepf & Koch, 19666). Schnepf & Koch (19666) described the
contractile vacuole of Vacuolaria virescens as a temporary structure which was formed
by fusion of Golgi-produced vesicles. They postulated that after discharging its
contents its membrane became part of the plasmalemma. The present report does not
support this idea: the evidence indicates that the contractile vacuole is a permanent
structure which possesses a specialized membrane similar to that occurring in some
other contractile vacuoles. The association of the contractile vacuole and the Golgi
apparatus is described and it is suggested that the function of the Golgi may be to
produce a hydrophilic substance which will attract water into the Golgi vesicles,
subsidiary vacuoles and contractile vacuole. In this manner water which has entered
the cell by osmosis will become sequestered into the contractile vacuole and can then
be discharged from the cell.
MATERIALS AND METHODS
Vacuolaria virescens Cienkowsky was obtained from the Cambridge Culture Collection
(number LB 1195/1). It was grown on defined medium (Heywood, 1973) at 23 ± 1 °C and was
aerated with a gas mixture of 4 % COj in air. Illumination (1-94 x io 3 lux) was supplied by
Ecko daylight flourescent tubes either continuously or intermittently (16 h light, 8 h dark).
Living cells were examined by Nomarski differential interference microscopy using a Zeips
photomicroscope. The cells are extremely delicate and easily burst under the pressure of the
coverslip; to prevent this the coverslip was supported on small pieces of broken coverslip which
ensured a greater depth of liquid and so reduced the pressure. Cells were photographed with
an exposure of i/3Oth s at x 171 magnification on Plus-X film (125 ASA).
For electron microscopy cells harvested by gentle centrifugation were fixed for 1 h at 10 °C
in 2 % glutaraldehyde buffered to pH 6-5 with 007 M phosphate buffer. After several washes
in buffer they were postfixed in buffered 1 % osmium tetroxide; cells fixed in this manner are
shown in Figs. 2-4. Alternately, cells were fixed for 6 h at 0-2 °C in 1 % chrome-osmium fixative (Dalton, 1955); cells treated with this fixative are shown in Figs. 5-10. All cells were
subsequently dehydrated in a graded ethanol series and embedded in Epon. Sections cut with a
diamond knife were collected on copper grids coated with Formvar and carbon. Sections were
stained with aqueous uranyl acetate and lead citrate, and examined using a Philips 201 S electron microscope.
Osmoregulation in Vacuolaria
OBSERVATIONS
The contractile vacuole in Vacuolaria virescens may reach 8-10 /.cm in diameter and
is a conspicuous feature of the cell. It occurs at the anterior of the organism and
discharges close to the point of flagellar insertion. For a particular temperature and
Fig. 1. Two complete cycles of the contractile vacuole. These micrographs of a
stationary cell were photographed at 10-s intervals using Nomarski differential interference microscopy. The following stages can be distinguished: full (A, G, M), relatively
empty (c, D, H, 1, N), and filling (E, F, J-L, O, P). In B the arrow indicates the anterior
portion of a collapsed contractile vacuole which was photographed during the course
of emptying. The small dark structure to the upper right of the figure number in Fig. 1
is thought to represent a subsidiary vacuole. The sequence of changes occurring in this
region of the cell can be followed through successive frames; arrowheads in E, H, I, K
indicate instances where the subsidiary vacuole is probably emptying into the contractile vacuole. x 1370.
2i 6
P. Hey wood
growth medium the duration of the contractile vacuole cycle is relatively constant both
in individual cells and between members of a population. For example, in defined
medium (Heywood, 1973) at 23 °C the period was 49-62 s.
Fig. 1 follows the changes occurring in the contractile vacuole through 2 complete
cycles. In spite of the relatively long time between frames (10 s) the continuous
nature of the contractile vacuole cycle is apparent from this series, and therefore it
need hardly be pointed out that the designation of individual stages in this process is an
arbitrary one. However, it is useful to recognize the following stages: full (A, G, M),
relatively empty (c, D, H, I, N), and filling (E, F, J-L, O, P). One complete cycle of the
contractile vacuole shows the following sequence: it is full in A, and in B it was photographed during the course of emptying; in c and D the contractile vacuole is relatively
empty but its presence can be distinguished as an irregular area in the cell; in E and F
the contractile vacuole is filling, and in G it has attained its spherical full state. In one
instance, B, the contractile vacuole was photographed during the course of emptying.
These micrographs indicate that the contractile vacuole is a permanent structure
which is present throughout the contraction cycle even though it is less conspicuous
immediately after emptying. The empty contractile vacuole has an irregular outline
but as it fills it usually becomes somewhat elliptical (for example, frames j and o)
before it assumes the spherical shape which is its characteristic appearance during the
latter part of the filling period.
In low-power electron micrographs of Vacuolaria virescens the arrangement of the
contractile vacuole and other organelles becomes more apparent (Fig. 2). The cell
lacks a cell wall and is bounded by a thin cell membrane. The outer layer of the cell
contains chloroplasts which are held in position by endoplasmic reticulum or narrow
strands of cytoplasm. There is a large nucleus surrounded by a layer of cytoplasm
containing mitochondria and endoplasmic reticulum. An extensive Golgi apparatus
occurs between the anterior surface of the nucleus and the contractile vacuole. This
region of the cell is seen in greater detail in Figs. 3-5, in which 3 categories of membranebound structures with electron-dense contents can be recognized; in ascending order
of size and in increasing distance from the surface of the nucleus these are Golgi
vesicles, subsidiary vacuoles and the contractile vacuole.
Earlier light-microscope descriptions of Vacuolaria virescens noted the presence of a
'supranuclear cap' extending over the anterior surface of the nucleus and producing
vesicles which ultimately formed the contractile vacuole (Poisson & Hollande, 1943).
Electron micrographs of this region indicate that the supranuclear cap is an extensive
Golgi apparatus (Figs. 3-5; see also Mignot, 1967; Schnepf & Koch, 19666). During
the course of examining many electron micrographs (including serial sections) through
this region of the cell I have been unable to detect any other structure which might be
responsible for channelling water into the contractile vacuole; for example, there is
nothing comparable to the system of fluid segregation tubules which surrounds the
contractile vacuole in some protozoa (McKanna, 1974, 1976). It is therefore possible
to confirm earlier observations (Schnepf & Koch, 19666) that the Golgi apparatus is
responsible for water secretion in this organism.
Figs. 3-5 illustrate differences between the images obtained with the 2 types of
Osmoregulation in Vacuolaria
217
Fig. 2. Longitudinal section through a cell of Vacuolaria virescens showing the cell
membrane, chloroplasts, nucleus (n), nucleolus {mi), Golgi apparatus (j>) and contractile vacuole (c). To aid in orientation the labelling is placed in the same position
in both this figure and in the higher-magnification view of the anterior region of this
cell (Fig. 3). X5160.
2l8
P. Heywood
Osmoregulation in Vacuolaria
219
fixation employed in this investigation. Both gave similar results for most cell components but the chrome-osmium fixative resulted in a more intensive staining of cell
structures; in particular, it is apparent that the contractile vacuole, subsidiary vacuoles
and Golgi vesicles contain electron-dense material (Figs. 3, 4). This material is not
preserved after fixation in glutaraldehyde followed by postfixation in osmium tetroxide
(Fig. 5). However, in cells fixed in this manner the contractile vacuole membrane
shows a regular substructure (Figs. 5-10), which is not preserved in the absence of
glutaraldehyde (Figs. 3, 4). The only remaining difference between the 2 types of
fixation is that the Golgi vesicles and subsidiary vacuoles are somewhat smaller after
fixation in chrome-osmium than after fixation in glutaraldehyde and postfixation in
osmium tetroxide. These observations obviously raise questions about fixation
artifacts, in particular, whether the contractile vacuole membrane really possesses a
regular substructure, and whether the Golgi vesicles, subsidiary vacuoles and contractile vacuole contain some material other than water. In thefirstinstance it is unlikely
that faulty fixation would produce a regular specialization of the membrane; it is
more probable that this appearance corresponds to a structure found in living cells
which is not preserved byfixationin chrome-osmium. This conclusion is strengthened
by the fact that similar types of modified membranes, sometimes termed 'coated
membranes' or 'bristle coat structures', are known to occur in other situations involving transport, particularly transport of water (Aaronson & Behrens, 1974; Franke,
Kartenbeck & Spring, 1976; Hoffman, 1976; McKanna, 1974, 1976; Morre", Mollenhauer & Bracker, 1971). Similarly, it is unlikely that faulty fixation would cause
electron-dense material to be present in the Golgi vesicles, subsidiary vacuoles and
contractile vacuole unless there had been widespread cytoplasmic damage, in which
case this material could have leaked into these regions of the cell. There is no sign of
such damage nor is this material present in other regions of the cell. Furthermore,
similar material occurs in the Golgi apparatus of Glaucocystis where this organelle is
also involved in water secretion. Light-microscope observations on living cells of
Vacuolaria virescens surrounded by India ink indicated that in some instances the
water eliminated by the contractile contained mucus (Tschermak-Woess, 1954). This
presumably corresponds to the material preserved in the contractile vacuole after
treatment with the chrome-osmium fixative. The presence of electron-dense material
in the Golgi vesicles, subsidiary vacuoles and contractile vacuole suggests a mechanism
by which water molecules can enter the osmoregulatory system and be bound there
Fig. 3. Higher magnification of the contractile vacuole region of the cell in Fig. 2. The
micrograph includes an oblique section through the base of theflagellarroot(/r) which
makes contact with the anterior surface of the nucleus (n). Electron-dense material is
present in vesicles produced by the Golgi apparatus (g), and in the subsidiary vacuole
(s) and the contractile vacuole (c). The arrowhead indicates a region where the subsidiary vacuole was about to empty into the contractile vacuole. x 14850.
Fig. 4. The proximal face of the Golgi apparatus Cg) is situated close to the surface of
the nucleus (n). Golgi vesicles show an increasing gradation in size towards the distal
face. Electron-dense material is present in the Golgi vesicles, subsidiary vacuoles (s)
and the contractile vacuole (c). Large numbers of membrane profiles and small
vesicles occur in this region of the cell, x 24000.
P. Heytoood
Osmoregulation in Vacuolaria
for sufficient time to allow for their expulsion from the cell. The latter is an important
consideration since in the absence of any restraint water molecules contained within
vacuoles have a strong tendency to cross the limiting membrane and to re-enter the
surrounding cytoplasm (McKanna, 1976). It is postulated that the Golgi apparatus is
producing a strongly hydrophilic substance which will attract water molecules into
10
Figs. 7-10. Sections through contractile vacuole membrane. Arrowheads in Figs.
8-10 indicate the arrangement of bristles on the cytoplasmic surface of the membrane.
Figs. 7, 8 contain surface views of these membrane subunits. x 100 000.
Fig. 5. Longitudinal section through the nucleus (JI), Golgi apparatus (g), subsidiary
vacuoles (J), and contractile vacuole (c). Theflagellum(/) and portions of the flagellar
root (/>) are also present. The contractile vacuole which was relatively empty at the
time of fixation is seen in oblique section with the result that 2 compartments appear to
be present. The intervening region of contractile vacuole membrane is present in surface view and is shown in greater detail in Fig. 6. x 16 500.
Fig. 6. Higher magnification of the region delimited by asterisks in Fig. 5. The subunit structure of the membrane can be seen in this view of the surface. An arrangement of 4 subunits (arrows) around a central subunit is present. The hexagonal nature
of this packing arrangement was demonstrated by photographic rotational enhancement, x 150000.
222
P. Heytvood
Golgi vesicles, subsidiary vacuoles and contractile vacuole and maintain them there
until they are eliminated from the cell. It is not known whether the subsidiary vacuoles
are permanent structures into which the Golgi vesicles empty their contents and which,
in turn, discharge into the contractile vacuole, or whether they are transient structures
formed by fusion of the Golgi vesicles, and which will disappear after they have
emptied into the contractile vacuole. In both instances there will be considerable
recycling of membranes and membrane components: the number of small vesicles and
membrane profiles in this region (Figs. 3-5) indicate that such a process is occurring.
At present there is little information about the mechanism of contractile vacuole
discharge. For example, it is not known whether this structure empties to the exterior
at a specific site. The cause of the contraction is uncertain: close examination of the
cytoplasm around the vacuole has failed to reveal any structures (for example,
microfilaments) which might be implicated in this process. However, in the course of
these investigations new information was gathered on the structure of the contractile
vacuole membrane. Perpendicular sections indicate that regions of the trilaminar
membrane possess regularly spaced bristles or pegs which extend from the membrane
into the cytoplasm (Figs. 8-10). These bristles extend inwards from the membrane
for a distance of 13-15 nm and are spaced at a distance of 21-24 nm. In surface views
(Figs. 6—8) the contractile vacuole membrane is observed to consist of regular subunits.
Photographic rotational enhancement of selected areas (for example, the subunits
indicated by arrows in Fig. 6) indicates that the subunits are arranged in a hexagonal
pattern. The diameter of the subunit corresponds to the 21-24 nm distance between
adjacent bristles in sections through the membrane, thus indicating that the bristles
represent sections through the lateral walls of the membrane subunits.
DISCUSSION
The timing of the contractile vacuole cycle (49-62 s at 23 °C) is in agreement with
that observed by earlier investigators, for example, 36-42 s at 18-20 °C (Spencer,
1971), 80 s at 18 °C (Poisson & Hollande, 1943), and 48 s at an unspecified temperature
(Schnepf & Koch, 19666). The latter authors concluded that the contractile vacuole is
a temporary structure formed by fusion of vesicles produced by the Golgi apparatus.
They suggested that after discharging its aqueous contents the contractile vacuole
membrane became part of the plasmalemma. This is a reasonable speculation since a
flow of membrane material from the Golgi apparatus to the cell surface is known to
occur in other instances (Dauwalder, Whaley & Kephart, 1972; Morre' et al. 1971).
However, the distinctive structure of the contractile vacuole membrane indicates that a
similar mechanism is not applicable to Vacuolarta virescens since this type of membrane
specialization is not present in the membranes of the Golgi vesicles, subsidiary
vacuoles or in the plasmalemma. Moreover, light microscopy using Normarski
differential interference microscopy indicates that the contractile vacuole is a permanent organelle.
The regularly spaced bristles or pegs extending from the contractile vacuole
membrane into the cytoplasm resemble 'bristle coat structures'. This type of 'coated
Osmoregulation in Vacuolaria
223
membrane' is known to occur in other situations involving transport of water and
electrolytes (McKanna, 1974, 1976). It is possible that this specialization may confer
additional strength on the membrane to enable it to withstand the pressure changes
occurring during the contractile vacuole cycle. Alternatively, the bristle elements
may have an active role in effecting the emptying of the contractile vacuole.
McKanna (1976) has termed the 'coated membrane' a 'permeability modulating
membrane coat' and has described the occurrence of this coat in the fluid segregation
organelles of some protozoa. In these organisms the Golgi apparatus is not associated
with the contractile vacuole. Instead the contractile vacuole is surrounded by a network of fluid segregation tubules which are confluent with the contractile vacuole.
McKanna (1974, 1976) has discussed how this type of arrangement can account for
the entry of water molecules into the osmoregulatory system and for their remaining
within this system until their discharge from the cell. In contrast, the contractile
vacuole system of Vacuolaria virescens possesses an extensive Golgi apparatus in place
of the network of fluid segregation tubules. The presence of electron-dense material
within the Golgi vesicles, subsidiary vacuoles and contractile vacuole suggests an
alternate method for fluid segregation. If the Golgi produces a highly hydrophilic
substance this will attract water from the cytoplasm into the Golgi vesicles, subsidiary
vacuoles and contractile vacuole and will retain water in these compartments until its
discharge from the cell. It is known that some extracellular material has a high affinity
for water, for example, the acid mucopolysaccharide hyaluronic acid can bind 68-520
ml of water per g (Fitton Jackson, 1964). This observation led to the suggestion that
'similar materials while still in the Golgi cisternae or vesicles could influence intracellular hydration' (Dauwalder et al. 1972). In order to substantiate this model for
secretion in Vacuolaria virescens it will be necessary to isolate the substance produced
by the Golgi apparatus (for example, from the culture medium in which cells were
grown) and determine its physical characteristics. In the absence of this type of
information there is evidence from, comparative studies that a similar mechanism may
be employed by other algae. For example, the Golgi apparatus of Glaucocystis which is
also involved in water secretion produces vesicles containing electron-dense material;
the possibility that this organelle may secrete mucilage together with the water has
been recognized (Schnepf & Koch, 1966 a). Furthermore, the observation by Dodge
(1973) that the contractile vacuole frequently occurs between the Golgi apparatus and
the plasmalemma suggests that this mechanism of water secretion may be widespread
in the algae.
I am most grateful to Ms Amy Davidoff for her careful and skilled assistance in preparing
the illustrations and in typing the manuscript. This investigation was supported by a Biomedical Research Support Grant from Brown University.
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