Research Article
2885
The SEC6 protein is required for contractile vacuole
function in Chlamydomonas reinhardtii
Karin Komsic-Buchmann, Lisa Marie Stephan and Burkhard Becker*
Botany, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 14 February 2012
Journal of Cell Science 125, 2885–2895
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.099184
Summary
Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in
Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion,
rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The
CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible
CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic
membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas
(CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the
Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains
overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction
interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the
mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and
systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and
water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an
indirect manner and flagellar length in Chlamydomonas.
Key words: Contractile vacuole, Flagella, Osmoregulation, SEC6, Exocyst, Chlamydomonas
Introduction
Contractile vacuoles (CVs) are osmoregulatory organelles found
in many unicellular freshwater protists without cell walls and
some sponges (Allen and Naitoh, 2002). CVs are membranebound cell compartments that periodically accumulate (diastole)
and expel (systole) water out of the cell, allowing cells to survive
under hypotonic conditions. Based on structure and behavior
about six basic types of CV have been described (Patterson,
1980). Despite this structural diversity the basic functions seem
to be conserved between different eukaryotes because the same
proteins and cellular processes have been found in Amoeba,
Dictyostelium, Paramecium, Trypanosoma and green algae [e.g.
V-ATPase (Becker and Hickisch, 2005; Fok et al., 2002; Heuser
et al., 1993; Montalvetti et al., 2004; Nishihara et al., 2008;
Robinson et al., 1998; Wassmer et al., 2005), aquaporin
(Montalvetti et al., 2004; Nishihara et al., 2008), vesicular
transport (Becker and Hickisch, 2005; Buchmann and Becker,
2009; Bush et al., 1994; Harris et al., 2001; Kissmehl et al., 2007;
Schilde et al., 2006; Stavrou and O’Halloran, 2006); see KomsicBuchmann and Becker for a summary of identified proteins and
cellular processes (Komsic-Buchmann and Becker, 2012)].
There are many accounts of the osmoregulatory role of CVs
(Allen, 2000; Allen and Naitoh, 2002), and it has been proposed
that water enters the CV by osmosis. V-ATPase and/or V-PPase
drive secondary active transport systems, allowing water to
follow passively through aquaporins. However, no acidification
of the CV (as expected for a proton-pump-mediated uptake
system) has ever been observed. Therefore, HCO32 has been
postulated to be the anion species continuously eliminated from
the cell through the CV (Robinson et al., 1998; Tominaga et al.,
1998). This would be similar to the situation for water transport
in animal epithelia (Hoffmann, 1986; Zeuthen, 1992), but
experimental evidence for a role of HCO32 in CVs has never
been presented. By contrast, experimental evidence points to the
involvement of phosphate in CV function in Trypanosoma and
Chlamydomonas (Rohloff et al., 2004; Ruiz et al., 2001) and K+
and Cl2 have been identified as the major osmolytes in the
cytosol and CV in Paramecium (Stock et al., 2002).
The structure and function of the CV in Chlamydomonas have
been investigated in some detail (Luykx et al., 1997a; Luykx
et al., 1997b; Robinson et al., 1998). At the end of diastole the
contractile vacuole of Chlamydomonas is spherical, expels the
liquid into the medium and the CV fragments into smaller
vacuoles (systolic phase; Fig. 1C). During diastole these smaller
vacuoles swell and fuse with each other to form again the
spherical vacuole at the end of a cycle (Luykx et al., 1997b)
(Fig. 1A). Several questions remain regarding the situation in
Chlamydomonas and more generally. (1) Exocytotic pore-like
structures were identified in ciliates (McKanna, 1973) but have
been very difficult to demonstrate in many green algae
(Buchmann and Becker, 2009; Luykx et al., 1997b). (2) How
the liquid leaves the cell in these systems is not clear, but
conspicuous intra-membrane particle arrays (up to 180 nm in
diameter) have been observed in the plasma membrane overlying
Journal of Cell Science 125 (12)
Journal of Cell Science
2886
Fig. 1. The contractile vacuole of Chlamydomonas reinhardtii CC3395. (A–C) The ultrastructure of the CVs in CC3395. The two CVs are located close to the
basal body (A). At the end of diastole (B) the CV membrane forms a contact zone with the plasma membrane marked by cytosolic electron dense material
between the membranes (arrows). In the systolic phase the CV fragments into smaller vesicles (C). The contact zone persists apparently until the end of systole
(arrow). The ‘bracelet’, a specialized plasma membrane region at the basis of the flagellum (F), is marked by an ellipse in A. M, mitochondrion; N, nucleus.
(D) Frames from a light microscope time-lapse recording. Numbers indicate the time passed since the end of last diastole. The white arrow marks the CV, scale bar:
5 mm. (E) The growth of CC3395 in four different media (TAP/2, TAP, TAP-S and TAP-SS). The strain can grow in every medium tested. (F) The relationship
between the CV period, the CV volume and the efflux of each CV to the cell surface (n545). The bigger the cell surface is, the longer the CV period, the higher the CV
efflux and the larger the CV volume. (G) The mean values and the standard deviation of the data set in F, given as non-normalized and normalized to the cell surface.
The CV period shows higher variation in the normalized data set, whereas the normalized data set for the CV volume and the water efflux from a cell shows less
variation than the non-normalized data set. Numbers above the bars indicate the coefficient of variation for the different data sets.
the CV region (Weiss et al., 1977). These arrays apparently form
only during systole and are often matched by a similar array in
the CV membrane opposing the plasma membrane array (Weiss
et al., 1977). Both array are connected by cytosolic electron
dense material (Weiss et al., 1977) (Fig. 1A) and similar
cytosolic electron dense material has also been detected in
another green alga Mesostigma viride (Buchmann and Becker,
2009). (3) A role for cytoskeletal elements during the CV cycle
could only be demonstrated in Dictyostelium (Taft et al., 2008),
indicating that force generation during systole by cytoskeletal
elements does not play any role in most systems. Changes in
membrane structure have been implicated in water expulsion
during systole in Paramecium (Allen and Naitoh, 2002), but
whether this is a general mechanism remains to be seen. In
addition, our knowledge of how the CV cycle is controlled and
adapted to the need of the cell is at best fragmentary. Calcium,
protein kinases and cAMP have been implicated (Rohloff and
Docampo, 2008), but in no system is the CV really understood.
Chlamydomonas is a well-established protist model system
(Grossman et al., 2003). The genome of Chlamydomonas has
recently been sequenced (Merchant et al., 2007). Chlamydomonas
can be transformed using several methods (Coll, 2006; Grossman
et al., 2003). Silencing of genes using RNA interference (RNAi)
has been successfully introduced in Chlamydomonas and is
continuously improving (Schroda, 2006), and several proteins
have been expressed as GFP-tagged constructs (Fuhrmann et al.,
1999; Huang et al., 2007; Ruiz-Binder et al., 2002; Schoppmeier
et al., 2005), making it possible to observe the in vivo dynamics
of subcellular structures and/or proteins. For this reason we
have started a forward genetic approach to analyze CV function
Contractile vacuole of Chlamydomonas
in Chlamydomonas. Osmoregulatory mutants isolated after
insertional mutagenesis showed defects in CV structure and
function. We have analyzed a mutant in which membrane fusion
events related to CV function are apparently impaired. We show
that the deletion of the single Chlamydomonas SEC6 protein
accounts for the observed phenotype, indicating a role for
SEC6, and probably the exocyst complex, in CV function in
Chlamydomonas.
Results
Journal of Cell Science
Characterization of the contractile vacuole of
Chlamydomonas reinhardtii CC3395
We used Chlamydomonas reinhardtii strain CC3395 for the
mutant screen, which does not have a cell wall and is easily
transformed. We first characterized the CV of this strain using
light and electron microscopy (Fig. 1A–D). As in other
Chlamydomonas strains, the large round CV visible at late
diastole develops from small vacuoles (Fig. 1A) and forms close
contact zones with the plasma membrane at the end of diastole
(Fig. 1B), which apparently persist during systole (Fig. 1C).
CC3395 grows in media of different osmotic strengths (Fig. 1E,
see also Fig. 8A); note that TAP/2 contains only half of the mineral
nutrients of the other media. Cells had two CVs at the anterior end
in all media tested except TAP-SS (containing 120 mM sucrose,
increasing the total osmotic strength of the medium to 204 mosM).
In this medium less than 5% of the cells exhibited CVs that were
visible with a light microscope. CVs are only visible with the light
microscope in Chlamydomonas when it is in hypotonic medium;
therefore, this result indicates that the cytosolic osmolarity
of Chlamydomonas CC3395 is approximately 200 mosM.
Preliminary data indicate that the cytosolic osmolarity varies
with growth conditions and status of the cells (unpublished own
observations), therefore only cells 4–6 days after subculturing (end
of log phase, Fig. 1E) were used in our analysis (see Materials and
Methods for details on cell culturing). In TAP medium the average
maximum diameter of the large round vacuole at the end of the
diastole was 1.7860.43 mm and the contraction interval
20.665.3 seconds (n545). The diastole lasted 19.465.0 seconds
and the systole 1.360.5 seconds (supplementary material Movie
1). From these results it can be calculated that in TAP medium (64
mosM) a Chlamydomonas CC3395 cell expels approximately
11.968.75 mm3/minute (approximately 2% of the total cell
volume per minute). Water uptake in a cell is directly
proportional to the cell surface area; we therefore performed a
linear correlation analysis between CV volume, CV period and
water efflux and the cell surface area of a cell. As is evident from
Fig. 1F, all three factors showed a good correlation to the cell
surface area [r250.6568 (CV period–cell surface area), r250.7907
(CV volume–cell surface area), r250.7872 (efflux–cell surface
area)]. Chlamydomonas cells considerably increase in size during
the cell cycle. Because the cells were not synchronized in our
cultures, we tried to normalize the CV data using the cell surface
areas determined for each cell from the videos used to characterize
the CVs. We then calculated the mean values and standard
deviations for the normalized and non-normalized data set
(Fig. 1G). As expected, the normalized data set showed less
variation than the non-normalized data set for the CV volume and
the water efflux from a cell (compare the coefficients of variation
indicated above the bars in Fig. 1G). By contrast, the standard
deviation obtained for the normalized data set was bigger for the
contraction interval of the CV, when compared with the non-
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normalized data set (Fig. 1G). These results indicate that cells use
mainly variation of the size of the CV to adapt to the increasing
water influx during cell growth, whereas the contraction period is
apparently regulated by a different factor.
Mutant screen
To isolate osmoregulatory insertional mutants we used the
mutant screen designed by Luykx et al. in combination with
insertional mutagenesis using the hygromycin B resistance
marker developed by Berthold et al. (see Materials and
Methods for details) (Luykx et al., 1997a; Berthold et al.,
2002). On TAP plates containing 0.06 M sucrose (TAP-S, 144
mosM) 2858 hygromycin-B-resistant clones were obtained, and
these were screened for failure to grow in TAP medium. Seven
mutant cell lines failed to grow at all in TAP medium. In
addition, 68 cell lines showed a different growth phenotype
(different growth rate, different color, etc.) than cells grown in
TAP medium containing 0.06 M sucrose, but were still able to
grow in TAP medium. Altogether we obtained a total of 75
potential osmoregulatory mutants (hereafter referred to as
Osmo1–Osmo75). We concentrated our work on seven cell
lines (Osmo12, 28, 32, 64, 66, 67, 75) showing a strong
phenotype (no growth in TAP medium, growth in TAP-S
medium).
Osmo64, 66, 67, 75 carry the same insertion of the
hygromycin B marker
Restriction enzyme site-directed amplification-PCR (RESDAPCR) (González-Ballester et al., 2005) was used to determine the
locus of insertion of the hygromycin B resistance marker for
Osmo12, 28, 32, 64, 66, 67 and 75. We obtained the 59 and 39
flanking sequences for Osmo64, 66, 67 and 75. All four isolated
strains contained exactly the same 33,641 base deletion
(Fig. 2A), indicating that the clones might have originated from
the same insertion event (possibly by cell division after the
insertion of the marker gene, during the recovery time after
transformation). By contrast, we were only able to determine the
39 insert flanking sequences for the other three mutants showing a
strong phenotype: Osmo28 (Fig. 2B), Osmo12 and 32 (Fig. 2C).
Primer walking indicated that also in these strains large deletions
(.9 kb) had occurred (Fig. 2B,C). Because the 59 insert flanking
sequences are identical for Osmo12 and Osmo32, these two
clones probably also originated from the same insertion event. At
present the deletion size in Osmo12, 28 and 32 is not known, so
all further work was carried out with Osmo75 as a representative
of Osmo64, 65, 67 and 75.
Characterization of Osmo75
We determined growth curves for Osmo75 in the same media
used for characterization of the parental strain and characterized
the mutant cell lines by video and electron microscopy (Fig. 3).
Fig. 3A shows the growth curves for Osmo75 in the different
media (also see Fig. 8A). The mutant was not able to grow in
media of low osmolarity. Assuming that the mutant cell lines
have a similar cytosolic osmolarity to that of the parental strain,
Osmo75 cells are able to grow under mild hypotonic conditions
(144 mosM) but fail to grow, or even die, under strong hypotonic
conditions (#64 mosM).
Video and electron microscopy was used to investigate
whether the observed growth defect is related to CV
malfunction. Video microscopy confirmed that indeed the CV
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Journal of Cell Science 125 (12)
Fig. 2. Insertion of the hygromycin B marker casette caused huge deletions. The corresponding areas of the genome of Chlamydomonas reinhardtii
(http://www.phytozome.net) are shown. The deletion, determined by RESDA-PCR (see Material and Methods) for these mutants, is indicated by the grey bar.
(A) In Osmo75 four genes are completely deleted (Cre20.g759800.t1.2–Cre20.g759950.t1.2) and two genes are truncated (Cre20.g759750.t1.2 and
Cre20.g760000.t1.2) owing to the insertion of the marker cassette in the genome. (B,C) In Osmo28 and Osmo12 and 32 only one flanking sequence of the marker
cassette could be determined. By primer walking a minimal deletion of 9 kb could be detected. Vertical arrows indicate the identified insertion site at the 39 end of
the marker cassette. Horizontal arrows indicate the positions of primers that failed to amplify the 59-flanking region of the marker cassette.
cycle was aberrant in Osmo75 cells (supplementary material
Movies 2–4). In all hypotonic media tested, all cells of the
parental strain have two CVs following a typical alternating CV
cycle with a large round vacuole at the end of the diastole
(Fig. 1D, Fig. 3B). By contrast, all Osmo75 cells showed CV
dysfunctions (changes in the number of CVs, the size and the
contraction interval of a CV) in TAP-S medium (Fig. 3B–L).
However, the CV phenotype was quite variable in a given
Osmo75 cell population. Many of the cells (61.3%) had multiple
smaller CVs in the region close to the basal bodies (Fig. 3D,E;
supplementary material Movie 3). Surprisingly 23.7% of the cells
did not have any CVs that were visible using light microscopy
(Fig. 3F; supplementary material Movie 4). In 8.0% of the
investigated cells one enlarged CV (Fig. 3G) was visible, and in
3.7% two enlarged CVs (Fig. 3C; supplementary material Movie
2) were visible. Finally, in 3.3% of the examined cells a mixed
morphotype was detected: one enlarged CV and multiple smaller
CVs (Fig. 3H).
Electron microscopy confirmed the light microscopy
observations (Fig. 3I,J). We could also detect the typical
contact zones formed by the CV membrane with the plasma
membrane during systole, although water expulsion was clearly
impaired in Osmo75 (see below; Fig. 4). Often contact zones in
Osmo75 appeared to contain less electron dense cystosolic
material between the plasma membrane and CV membrane than
in CC3395 cells (compare Fig. 1B with Fig. 3K,L; see also
Fig. 8H).
To investigate the behavior of individual CVs in the Osmo75
strain in more detail we selected videos of cells with two or one
enlarged CVs (Fig. 3C,G) and recorded the size of the CVs every
5 seconds in TAP-S medium (Fig. 4). For comparison, the size of
individual CVs in the parental strain was recorded also. CVs in
CC3395 showed an oscillating pattern. The diameter of a CV
increases during diastole and rapidly decreases during systole; at
the end of systole generally no CV is visible in the light
microscope (Fig. 4). However, it is noteworthy that in TAP-S,
CVs of CC3395 cells do not always completely empty (Fig. 4),
whereas in TAP medium the CV of CC3395 always completely
disappears (not shown). By contrast, CVs of Osmo75 cells show
irregular increases and decreases of the CV diameter or appear
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Journal of Cell Science
Contractile vacuole of Chlamydomonas
Fig. 3. The contractile vacuoles of the osmoregulatory mutant Osmo75. (A) The growth of Osmo75 in four different media (TAP/2, TAP, TAP-S and TAP-SS). The
strain grew in TAP-S and TAP-SS, but failed to grow, or even died, in TAP and TAP/2. (B–H) Cells of Osmo75 show variable CV morphologies. The graph (B) shows
the proportion of the various mutant and parental cells with the different CV morphologies (100 cells of each type were analyzed in triplicate). (C–H) Examples for the
different CV phenotypes. CVs are indicated by white asterisk. The arrowhead in F marks the cytoplasmic region normally displaying a CV. Scale bar: 5 mm.
(I–L) Electron micrographs of the CVs of Osmo75. (I) Four CVs are visible in one cell (multiple CVs per cell). Two of them show contact zones with the plasma
membrane (marked by arrows and are shown enlarged in K,L). (J) The mixed phenotype of Osmo75, one enlarged CV and multiple smaller CVs.
for some time constant (Fig. 4), but total discharges rarely
occurred.
Taken together the observed osmoregulatory phenotype
indicates that in Osmo75 membrane fusion events during the
CV cycle are impaired. The multiple small vacuoles might be
caused by inefficient homotypic vacuolar fusion during diastole.
The enlarged CVs are possibly caused by failure to terminate
systole and achieve water expulsion.
Finally we noted that Osmo75 cells had a distinct flagellar
length phenotype. Flagella of Osmo75 cells were much longer
(9.6361.55 mm) than those of the parental CC3395 strain
(6.9761.05 mm; Fig. 5).
Protein targeting to the CV is not impaired in Osmo75
To test whether protein targeting to the CV is impaired in
Osmo75 we tried to develop a GFP marker system for CV in
Chlamydomonas. Aquaporins have been implicated in CV
function in several other organisms (Montalvetti et al., 2004;
Nishihara et al., 2008). The genome of Chlamydomonas
reinhardtii encodes only two putative aquaporins (Anderberg
et al., 2011) CreMIP1 (Cre12.g549300; www.phytozome.net)
and CreMIP2 (Cre17.g711250). RT-PCR showed that CreMIP1
but not CreMIP2 is expressed in vegetative cells (data not
shown). We reasoned that CreMIP1–GFP might be a useful
marker to investigate whether protein targeting to the CV is
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Journal of Cell Science
Fig. 4. Osmo75 fails to expel liquid from the cells
efficiently. The diameter of individual CVs was determined
every 5 seconds and used to calculate the surface area of
individual CVs in the parental strain CC3395 and Osmo75.
Each line represents a different CV. Whereas CVs in the
parental strain show a reiterating pattern, CVs of Osmo75
cells show a completely irregular behavior.
impaired in the Osmo75 cell line. In addition, expression of
CreMIP1–GFP might confirm that, as in other systems,
aquaporins are localized to the CV. The full-length cDNA of
CreMIP1 was cloned into the GFP expression vector pJR38
(Neupert et al., 2009). Osmo75 and the UVM4 strain (which was
specifically developed for GFP expression in Chlamydomonas)
(Neupert et al., 2009) were transformed with a linearized
ScaI–XbaI fragment of pJR38-MIP1-GFP, coding for CreMIP1–
GFP and the APHVIII protein (paromomycin resistance).
Paromomycin-resistant clones were selected and screened for
GFP expression. Fig. 6 shows the results of this experiment. Nontransformed cells showed some background fluorescence in the
GFP channel (Fig. 6A). However, as is evident from Fig. 6B–E,
CreMIP1–GFP clearly localized to the CV in the UVM4
(Fig. 6B,C) and Osmo75 (Fig. 6D,E) strains, indicating that
protein targeting of the CreMIP1-GFP construct to the CV is not
impaired in Osmo75. In addition, using CreMIP1-GFP in the
UVM4 genetic background we always observed that the CV
membrane and plasma membrane apparently did not intermingle
with each other during the CV cycle (Fig. 6C).
Rescue of Osmo75
Based on the available genome sequence (www.phytozome.net),
the 33,641 base deletion in Osmo75 affects six gene models. Four
putative proteins are deleted and two additional putative proteins
are truncated. Table 1 summarizes the available information on
the six gene models. Gene model Au9.Cre20.g759900.t1 encodes
the only putative SEC6 protein in Chlamydomonas. SEC6
proteins have been shown to be part of the exocyst complex
Fig. 5. Flagellar length of Chlamydomonas reinhardtii CC3395, the
mutant Osmo75 and three rescued strains (Osmo75-SEC6GFP).
Significant differences from CC3395 are indicated by asterisks (*P#0.05,
***P#0.001). Significant differences between the rescued strains and
Osmo75 are indicated by hashes (###P#0.001); n537, 37, 29, 33 and 32,
respectively, left to right.
(Bröcker et al., 2010). The exocyst complex belongs to the group
of multi-subunit tethering factors required for efficient membrane
fusion events and is involved in polarized secretion in many
eukaryotic systems. Membrane fusion events occur during the
diastole and at the beginning of systole during a CV cycle.
Therefore the deletion of a protein similar to SEC6, possibly
affecting exocyst function, seems a probable molecular cause for
the observed phenotype of Osmo75. For this reason we
concentrated our work on this protein, referred to as CreSEC6.
RT-PCR revealed that CreSEC6 is expressed in the parental
strain (Fig. 7, lanes 1–4) under all tested osmotic conditions,
whereas we failed to amplify the same PCR fragment from the
Osmo75 strain (Fig. 7, lane 5). Based on the Augustus gene
model in Phytozome (www.phytozome.net) we expected the fulllength cDNA to be 2019 bases (672 aa) long excluding both
UTRs. However, the isolated full-length cDNA was 2439 bases
(812 aa) long. Comparison of the full-length cDNA with the
transcript and protein sequence in Phytozome indicates that the
predicted protein sequence in Phytozome misses one exon. This
exon is also present in the published Arabidopsis sequence. Blast
analysis of the full-length cDNA sequence showed that CreSEC6
is 31% identical (47% similar) to the SEC6 from Arabidopsis
thaliana.
To confirm that CreSEC6 is indeed responsible for the
observed phenotype of Osmo75 we tried to rescue the mutant
with a CreSEC6–GFP fusion protein again using pJR38 as the
expression vector. On TAP plates 312 clones resistant to
hygromycin B and paromomycin were obtained. Osmo75 cells
do not grow in TAP medium, so this already indicated a
successful rescue. For all clones investigated no defects in CV
function could be observed by light microscopy of living cells,
and the long flagellar phenotype was nearly completely rescued
when analyzed in three randomly selected rescue cell lines
(Fig. 5).
To characterize the rescued strains in more detail, ten strains
were randomly selected (Osmo75-A5, A9, C11, D10, E3, F6, F9,
G6, H5 and H7). RT-PCR analysis showed that the CreSEC6GFP construct is expressed in all ten strains (Fig. 7, lanes 6–15)
and at a higher level than the endogenous SEC6 in CC3395. Thus
all rescue cell lines are SEC6 overexpressors. Although all ten
rescue strains grew at all osmotic conditions tested, some strains
(e.g. A5 and D10) did not grow as well on strong hypotonic
media and isotonic media, possibly because of different insertion
sites of the CreSEC6-GFP construct in the various strains.
Detailed light microscope analyses of the CV cycle revealed
interesting differences between the ten rescued Osmo75 strains
investigated and the parental strain. Whereas the contraction
interval was not significantly altered in all ten rescue strains; the
ratio of CV:cell surface area and water efflux:cell surface area
Journal of Cell Science
Contractile vacuole of Chlamydomonas
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Fig. 6. Expression of CreMIP1-GFP in
the UVM4 and Osmo75 background.
(A) Untransformed UVM4 cell.
(B,C) UVM4 cell transformed with
CreMIP1-GFP. (D,E) Osmo75 cell
transformed with CreMIP1-GFP.
(B,D) Overview of the whole cell.
(C,E) Time-lapse images of the CV region
of the cells shown in B and D, respectively.
Numbers indicate the time (in seconds).
Exposure time for individual frames was
2.2 seconds. PH, phase contrast; Ex 460500, excitation wave length. Scale bars:
5 mm.
increased significantly (CV volume:cell surface area, P#0.001
for seven of the ten rescue strains; water efflux:cell surface area,
P#0.001 for all ten rescue strains). Electron microscopy
confirmed that the CV structure was completely restored
(Fig. 8E–I). The ultrastructure of the CV in the rescued strains
examined (G6, Fig. 8E–I; C11 not shown) was indistinguishable
from that of the parental strain.
Discussion
The molecular mechanisms of CV function are still poorly
understood. Over the last year several proteins have been
implicated in CV function in several systems [for a recent
summary, see Komsic-Buchmann and Becker (KomsicBuchmann and Becker, 2012)]. Generally, proton pumps,
SNAREs, Rab proteins and calcium signaling have been shown
to be important for CV function. The current models suggest that
water uptake into the CV is by osmosis, energized by proton
pumps, and that aquaporins facilitate this process. Although our
knowledge about water uptake into the CV has greatly increased
in recent years, the mechanism of water expulsion has not been so
well studied in most systems. To increase our knowledge on CV
function in green algae and in general we choose a forward
genetic approach using Chlamydomonas as a model system and
investigated the cellular localization of a Chlamydomonas
aquaporin.
Early genomic analyses indicated only a single aquaporin in
the genome of Chlamydomonas. A recent detailed analysis of
algal MIPs indicated the presence of at least a second isoform
(Anderberg et al., 2011). However, RT-PCR indicated that
CreMIP2 is not expressed in C. reinhardtii, whereas CreMIP1
could be easily detected. Using a CreMIP-GFP construct we
could clearly show that MIP1 is localized to the CV. In vivo
observations of the CV indicated the CV membrane to be a stable
compartment with no intermixing with the plasma membrane.
These results suggest that similar to the CV in other systems, the
membrane of the Chlamydomonas CV contains an aquaporin
(Montalvetti et al., 2004; Nishihara et al., 2008). In addition, as in
many other systems the CV membrane and the plasma membrane
do not intermingle during the CV cycle (Patterson, 1981; Zanchi
et al., 2010), suggesting that potential membrane fusion events
follow the kiss-and-run mechanism.
For the mutant screen we selected Chlamydomonas CC3395,
which has no cell wall. Analyses of the CV cycle in CC3395
showed that, overall, the situation is very similar to strain 137c
(average diameter at end of systole, contraction interval),
indicating that the cell wall has only a minor effect on water
Table 1. (Putative) proteins of Chlamydomonas affected in the Osmo75 strain
Name of model
Cre20.g759750.t1
Cre20.g759800.t1
Cre20.g759850.t1
Cre20.g759900.t1
Cre20.g759950.t1
Cre20.g760000.t1
Annotation
Phytoene dehydrogenase
SpoU rRNA methylase family
CGI-12 protein related
Exocyst complex component Sec6
Protein of unknown function (DUF789)
None
Deletion/Truncation
139 b truncation of the 39 end of the 39UTR
Deletion
Deletion
Deletion
Deletion
1089 base truncation of the 59 end
Expression
+
+
+
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Journal of Cell Science 125 (12)
Journal of Cell Science
Fig. 7. Expression of SEC6 in various
Chlamydomonas strains. RT-PCR was performed using
the parental strain in four different media of different
osmotic strengths, and Osmo75 (upper panel) and 10
rescue strains of Osmo75 (two lower panels). The genes
targeted by the primers used are indicated at the top. Lane
numbers refer to the different templates used and are
explained on the right. The centrin gene was used as
loading control. Expected length: SEC6, 126 bp; SEC6GFP, 222 bp; centrin, 94 bp.
uptake in Chlamydomonas. However, our results indicate that the
cytosolic osmolarity of the cells is slightly higher in CC3395 than
in 137c.
Insertional mutants were generated and screened for CV
dysfunction. Four of the obtained mutants (Osmo64, 65, 67 and
75) show the same 33,641 base deletion, indicating that the clones
might have originated from the same insertion event (possibly by
cell division after the insertion of the marker gene during the
recovery time). Such large deletions are not uncommon in
Chlamydomonas after transformation (Gonzalez-Ballester et al.,
2011) and have been proposed to depend on the type of marker
(large size, full plasmid) and the transformation method used
(Gonzalez-Ballester et al., 2011). However, as we obtained large
deletions using a small linear DNA fragment, most probably the
transformation method is more important in this respect.
The 33,641 base deletion in Osmo75 includes the only SEC6
protein, encoded in the Chlamydomonas genome. Characterization
of the phenotype indicated that membrane fusion events during
diastole (homotypic vacuolar fusion) and systole (exocytosis) do
not operate efficiently in the CV in Osmo75, leading to hypotonic
sensitivity of the cells. In addition, cells had long flagella. Rescue
of the Osmo75 phenotype with a CreSEC6-GFP construct
confirmed that indeed the deletion of CreSEC6 is responsible for
the observed defect in CV function and flagellar length in Osmo75.
The SEC6 protein is part of the exocyst complex, which belongs to
the multi-subunit tethering factors (MTCs) (Bröcker et al., 2010).
MTCs are ancient facilitators of membrane fusion events, and
current knowledge indicates that every membrane fusion event
requires its own tethering factor (Koumandou et al., 2007). The
exocyst complex has been shown to be required for efficient
exocytosis in various systems (Bröcker et al., 2010; Zhang et al.,
2010). In this respect the observed phenotype in Osmo75 is
surprising in two aspects. First, exocytosis is inefficient (leading to
enlarged CVs) and homotypic vacuolar fusion (leading to many
smaller CVs) does not take place efficiently. Work in the yeast
system indicates that homotypic vacuolar fusion is mediated by the
HOPS complex (Bröcker et al., 2010). The HOPS complex in yeast
consists of six different subunits (Bröcker et al., 2010); two
subunits have so far not been found in the Chlamydomonas
genome (Koumandou et al., 2007). Given the observed phenotype
in Osmo75 it is tempting to speculate that in Chlamydomonas
SEC6 is also involved in HOPS complex-mediated homotypic
vacuolar fusion. Second, the SEC6 deletion mutant in
Chlamydomonas is viable, whereas SEC6 is essential for growth
in yeast (Potenza et al., 1992), and Arabidopsis T-DNA insertion
lines that disrupt SEC6 expression fail to produce homozygous
progenies (Hála et al., 2008). The latter is caused by defects in
pollen germination and growth, indicating a major role in polar
secretion in plants. However, in Chlamydomonas polar secretion
seems not completely impaired, as the cells are able to form longer
flagella. This is in striking contrast to the requirement of the
exocyst in ciliogenesis in animals (Das and Guo 2011; Zuo et al.,
2009). Exocyst localizes to the base of primary cilia in MDCK
epithelial cells (Rogers et al., 2004) and deletion of SEC10
abolishes ciliogenesis (Zuo et al., 2009), whereas overexpression
of SEC10 led to elongated primary cilia. By contrast, deletion of
SEC6 in Chlamydomonas caused elongated flagella, whereas
overexpression of SEC6–GFP did not change the flagellar length.
The reason for the difference in behavior between these two
systems is currently not clear.
To our knowledge this is the first report of an involvement of
SEC6 (and probably the exocyst complex) in CV function.
Recently, Zanchi et al. reported that a secA mutant in
Dictyostelium discoideum developed a large vacuole, which
was shown to be derived from the CV (Zanchi et al., 2010). SecA
is the Dictyostelium homologue of the yeast SEC1 and the
mammalian Munc18 proteins (SM proteins), which are involved
in vesicle docking during exocytosis and have been shown to
interact with the exocyst complex, pointing to a role of the
exocyst complex in CV function in Dictyostelium. However, in
contrast to the SEC6 deletion in Osmo75, the SecA mutation in
Dictyostelium leads only to an enlarged CV and not to a multiple
CV phenotype, supporting the idea that the phenotype of Osmo75
indicates a dysfunction of two different cellular processes
(homotypic vacuolar fusion and exocytosis).
Interestingly, in a recent study Morgera et al. showed that
SEC6 regulates exocytosis by interaction with SEC1 (Morgera
et al., 2012). SEC1 [the yeast plasma membrane SM protein
(plasma membrane sec1/Munc18-like proteins)] binds to the tSNARE SEC9, inhibiting the formation of the SNARE complex
required for exocystosis. SEC6 releases SEC1 from SEC9, thus
allowing exocytosis to proceed (Morgera et al., 2012). Given the
function of the exocyst complex in other systems and these new
findings, it seems plausible that exocyst in Chlamydomonas is
required for the formation of the close contact zones between the
plasma membrane and CV membrane and polar secretion in
2893
Journal of Cell Science
Contractile vacuole of Chlamydomonas
Fig. 8. Characterization of the rescued strains Osmo75-SEC6GFP. (A) The growth of CC3395, Osmo75 and ten randomly selected rescued strains Osmo75SEC6GFP (A5 to H7, as listed) on agar plates with different osmotic strengths ranging from strong hypotonic (32 mosM, TAP/2) to isotonic (204 mosM, TAP-SS).
(B) Comparison of the CV period of CC3395 (n545) with the CV periods of the ten rescued strains Osmo75-SEC6GFP A5 to H7 (n520). Only E3 and F6 have
significantly different CV periods from that of CC3395 (*P#0.05). (C) Comparison of the CV volume relative to the cell surface area of CC3395 (n545) and the
rescued strains Osmo75-SEC6GFP A5 to H7 (n520). Only two rescued strains, C11 and D10, have similar CV volumes to CC3395, the others all differ
significantly (*P#0.05, ***P#0.001). (D) The CV efflux relative to the CV surface area of CC3395 (n545) and the rescued strains Osmo75-SEC6GFP A5 to H7
(n520). The efflux of all rescued strain is significantly higher than the efflux of CC3395 (***P#0.001). (E–I) Electron micrographs of one rescued strain,
Osmo75-SEC6GFP-G6. At the end of diastole the CV forms contact zones with the plasma membrane (E, arrows, and enlarged in H). Two CVs are visible in the
cell shown in F, the left CV at mid diastole and the right CV in early diastole. The contact zones seem to persist until the end of the systolic phase (G arrows, and
enlarged in I).
flagellar biogenesis. SEC6 might be required for water expulsion
to proceed efficiently in the CV cycle by releasing a similar block
as the SEC1 block observed in yeast.
Materials and Methods
Cell cultures
The following strains were used in this study: Chlamydomonas CC 3395 (arg7-8
cwd mt1) (Shimogawara et al., 1998) and UVM4 (cwd mt+ arg7) (Neupert et al.,
2009). Cells were cultured in TAP medium (Gorman and Levine, 1965), the
medium for CC3395 and all derived mutant cell lines were supplemented with
additional arginine. To achieve different osmolarities the medium was either
diluted with aqua dest. (TKA X-CAD, Thermo Electron LED GmbH, Niederelbert,
Germany) (for TAP/2), or 60 mM or 120 mM sucrose was added for TAP-S and
TAP-SS, respectively. The osmolarity of all media was determined using a
freezing point depression osmometer (Osmomat 010, Gonotec, Berlin, Germany).
All transformants were always kept under selection pressure by addition of
antibiotics to the medium and transferred into new media at least every 6 weeks.
2894
Journal of Cell Science 125 (12)
Cells were cultured at 21 ˚C with a photon flux of 70 mmol/m2 s and a 14 hour:10
hour light:dark cycle. In all experiments 5-day-old cultures (6 1 day) were used
with a cell density of 106–107cells/ml.
Transformation of Chlamydomonas cells
All transformations were performed using Kindle’s glass bead method (Kindle,
1990). For the insertional mutagenesis Chlamydomonas CC3395 cells were
transformed with the HindIII cassette of pHyg3 (Berthold et al., 2002). Cells were
allowed to recover for 2 hours in TAP followed by 16 hours in TAP-S in the dark
and plated on TAP-S plates containing 10 mg/ml hygromycin B (Roth, Karlsruhe,
Germany). After transformation of UVM4 and Osmo75 with the CreMIP1-GFP
fusion construct cells were recovered in the appropriate media and plated onto
plates containing paromomycin (Sigma, St. Louis, MO; 10 g/ml). Transformed
cells were screened for GFP fluorescence using a fluorescence microscope (see
below).
Screening for osmoregulatory mutants
Individual clones were picked and transferred into 96-well plates containing, in
each well, 200 ml TAP-S. After 2–3 weeks aliquots were transferred into new
microtiter plates containing either TAP-S or TAP medium. Cell lines that showed a
different growth in TAP compared with TAP-S were selected and the screening
process was performed in triplicate.
Determination of the insertion site
Journal of Cell Science
The insertion flanking regions were determined using the RESDA-PCR protocol of
Gonzáles-Ballester et al. and specific primers for the HindIII fragment of pHyg3
developed by Matsuo et al. (Gonzáles-Ballester et al., 2005; Matsuo et al., 2008).
Nikon Eclipse 800 (Nikon GmbH, Düsseldorf, Germany) microscope equipped
with a mercury short lamp (Osram, Düsseldorf, Germany), Uniblitz shutter control
(Vincent Associates, Rochester, NY), a GFP filter set (480/40; 505; 535/50) and a
Spot RT CCD digital camera (Diagnostic Instruments, Sterling Heights, MI). The
images and videos were analyzed with Metamorph imaging software, version 6.3r4
(Universal Imaging, Corp., Bedford Hills, NY).
Electron microscopy
Cells were concentrated by centrifuging at 500 g at 20 ˚C for 15 minutes and
resuspended in high salt medium (HSM) with an appropriate amount of sucrose
added to reach the respective osmotic strength, and additional HEPES (3 mM final
concentration) before fixation simultaneously with glutaraldehyde and aqueous
osmium tetroxide (final concentration 1.25% and 1%). The first minute of fixation
was at room temperature and the additional 30 minutes on ice. After fixation the
cells were washed once with fresh medium. To allow easier handling during the
dehydration procedure, the cell pellets obtained by centrifugation were cross linked
with BSA as follows. Cells were resuspended in BSA solution (30% in medium)
and transferred into BEEM capsules (Plano, Marburg, Germany), pelleted (500 g,
at room temperature for 15 minutes) and overlaid with glutaraldehyde solution
(2.5% in medium). Samples were incubated for 30 minutes on ice before removal
of the pellets. The cell pellets were incubated overnight in a 1% aqueous uranyl
acetate solution at 4 ˚C. Samples were washed and dehydrated in an ethanol series
and embedded in Epon 812. Ultrathin sections (60 nm) were cut with a Leika
microtome EM UC7 and a diamante knife (diatome, 45 ˚ angle). Sections were
stained with 2% aqueous uranyl acetate and lead citrate (Reynolds, 1963).
Micrographs were taken with a transmission electron microscope (CM 10, Phillips,
Eindhoven, The Netherlands) and a digital camera (Orius SC200W 1; Gatan,
Pleasanton, CA). Images were analyzed with Digital Micrograph and Adobe
Photoshop CS4.
GFP fusion constructs
Total RNA was isolated using TRI REAGENT (MRC, Cincinnati, OH) following
the manufacturer’s instructions. cDNA was synthesized with the Revert Aid First
Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). In all PCR reactions
DreamTaq (Fermentas) was used in combination with an enhancer for GC-rich
templates (Ralser et al., 2006). The complete cDNA of CreSEC6 (Cre20.g759900)
and CreMIP1 (Cre12.g549300) was amplified and cloned into pGEM-T-easy
(Promega, Madison, WI). Subsequently, NdeI restriction sites were added to the
coding sequences by PCR, and ligated into the NdeI restriction site of pJR38
(Neupert et al., 2009) resulting in CreSEC6-GFP and CreMIP1-GFP fusion
constructs (primer, sequences and vector maps are presented) (supplementary
material Table S1; Figs S1, S2, S3).
Acknowledgements
The authors thank R. Bock (Golm, Germany) for providing plasmid
pJR38 and the UVM4 strain, and W. Mages (Regensburg, Germany)
for the plasmid pHyg3 and M. Schroda (Golm, Germany), K.-F.
Lechtreck (Athens, GA, USA) and J. Brown (Worcester, MA, USA)
for helpful discussions. In addition, we thank the following students:
D. Langenbach, R. M. Benstein, A.-K. Alteköster and K. Kehl, who
helped to characterize the Osmo75 mutant.
Funding
Mutant rescue
For rescue of the mutant phenotype, Osmo75 cells were transformed with
CreSEC6-GFP. After transformation cells were allowed to recover in TAP
medium before plating the cells on solid TAP medium without additional sucrose.
RT-PCR
Total RNA was isolated using the peqGOLD Plant RNA kit (Peqlab, Erlangen,
Germany). cDNA synthesis was performed with the Revert Aid First Strand cDNA
Synthesis kit (Fermentas) using 1 mg total RNA. In the PCR reactions DreamTaq
(Fermentas) was used in combination with an enhancer for GC-rich templates
(Ralser et al., 2006). The primers were designed using quantprime (http://www.
biomedcentral.com/1471-2105/9/465) and were specific for cDNA (primers are
listed in supplementary material Table S1).
Growth in different media
Cells for the determination of growth curves where cultured under reduced light
(20–30 mMol/m2/second). Two biological replicates were counted four times each
using a Neubauer hematocytometer.
Growth on media of different osmotic strengths was also analyzed using a plate
assay. The number of cells in a culture was counted using a Neubauer
hematocytometer. Cells were then diluted with TAP medium to a concentration
of 0.376106 cells/ml. The diluted cell suspension (3 ml) was dropped onto agar
plates with different osmotic strength (without antibiotics), in triplicate. Cells were
grown for 21 days.
Light microscopy
Light microscopy was performed as described by Buchmann and Becker, except
that we used 7 ml cell suspension on each slide (Buchmann and Becker, 2009). For
video microscopy images were taken every 0.5 seconds. At least three cycles were
analyzed per CV. The surface and volume of the CVs and cells were calculated as
a prolate spheroid. The efflux rate per cell was calculated using the size and period
of the cell investigated. To measure the flagella length, cells were fixed with 5%
Lugol’s iodine. Linear regression analysis and significance tests (Student’s t-test
with Welch correction) were done using the GraphPad Prism 5 software (GraphPad
Software Inc., La Jolla, CA). Fluorescence microscopy was performed using a
This work was supported by the Deutsche Forschungsgemeinschaft
[grant number Be1779/12-1 to B.B.].
Note added in proof
After acceptance of this paper a new release of the Chlamydomonas
genome became available (v5.3, 8 June 2012). In the new release the
former scaffold 20 has been mapped to chromosome 17 and therefore
all gene IDs have been changed as follow: Cre20.g759750.t1 Phytoene
dehydrogenase; new gene ID: g18016.t1; Cre20.g759800.t1 SpoU
rRNA methylase family, new gene ID: g1801t.t1; Cre20.g759850.t1
CGI-12 protein related, new gene ID: g18014.t1; Cre20.g759900.t1
Exocyst complex component Sec6, new gene ID: g18013.t1;
Cre20.g759950.t1 Protein of unknown function (DUF789), new
gene ID: g18012.t1, and Cre20.g760000.t1 No annotation, new gene
ID: g18011.t1.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.099184/-/DC1
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