1 University of Konstanz, Department of Biology, PO Box 5560, 78457 Konstanz,
Germany
2 Centre de Génétique Moléculaire, CNRS, Avenue de la
Terrasse, 91198 Gif-sur-Yvette Cedex, France
Author for correspondence (e-mail:
roland.kissmehl{at}uni-konstanz.de)
Accepted 1 August 2002
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Summary |
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Key words: Ciliates, Paramecium, Secretion, Phagocytosis, Golgi, Endoplasmic reticulum
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Introduction |
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A NSF protomer contains three distinct domains
(May et al., 2001;
Whiteheart et al., 2001
): an
N-terminal domain (N) responsible for interaction with the
-SNAP-SNARE
complex and two homologous ATP-binding domains (D1 and D2). The ability of the
D1 domain to hydrolyze ATP is required for NSF activity, while the D2 domain
is required for hexamerization. The sequences of NSF-D1 and NSF-D2 place NSF
in the AAA (ATPases associated with various cellular
activities) superfamily
(Neuwald et al., 1999
). Such
proteins contain at least one copy of a conserved,
230 amino acid
cassette with a characteristic phosphate-binding P-loop (Walker A) and a metal
ion-binding DEXX box (Walker B) nucleotide-binding sequence.
In a Paramecium cell, several clearly defined vesicle transport
routes exist (Allen, 1988;
Plattner, 1993
). The import
routes include coated vesicle-mediated endocytosis and non-coated phagocytosis
(Allen and Fok, 2000
). Several
export routes exist, including constitutive and stimulated exocytosis of dense
core-vesicles (trichocysts) (Plattner et
al., 1991
; Plattner,
1993
; Vayssié et al.,
2000
), defecation of spent phagosomes at the cytoproct
(Allen and Fok, 2000
), and
fluid release by the osmoregulatory contractile vacuole
(Allen, 2000
). Internal fusion
processes involve the route endoplasmic reticulum (ER)
Golgi apparatus
[e.g. for the biogenesis of constitutive secretory vesicles
(Flötenmeyer et al.,
1999
) and the biogenesis of trichocysts
(Momayezi et al., 1993
;
Gautier et al., 1994
)],
delivery from endocytotic vesicles via early endosomes to the compartments of
the digestive cycle (Allen et al.,
1992
; Flötenmeyer et al.,
1999
), membrane recycling from the cytoproct to the nascent food
vacuole (Allen et al., 1995
),
fusion and retrieval of different vesicle types along the digestive cycle, as
well as any membrane recycling along the precisely coordinated intracellular
digestive cycle (Allen and Fok,
2000
).
In the present study, starting from the recent identification of a NSF
partial sequence in a pilot genome project
(Dessen et al., 2001;
Sperling et al., 2002
), we
cloned two PtNSF genes from Paramecium tetraurelia. Gene
silencing, light and electron microscope (EM) and immunolocalization analyses
reveal a ubiquitous involvement of PtNSF in vesicle traffic occurring in
Paramecium, which thus shows its advantages for studies of NSF
functions.
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Materials and Methods |
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Cell fractionation
Cell surface complexes (isolated `cortices') were prepared as described
(Vilmart-Seuwen et al., 1986).
Other cell fractions were prepared from sterile cell cultures as previously
described (Kissmehl et al.,
1998
). Protein concentrations were determined with BSA as a
standard (Bradford, 1976
).
PCR of genomic DNA
Total wild-type DNA for PCR was prepared from log-phase cultures as
described (Duharcourt et al.,
1995). The short probe consisted of a 336 bp PCR amplification
product with PtNSF1 specific primers:
Each PCR reaction (50 µl) contained 150 ng of DNA, 50 pmol of each primer, 0.2 mM of each dNTP and 2.5 U of Taq DNA polymerase (Roche Diagnostics). Reactions were carried out for one cycle of denaturation (1 minute, 92°C), and 30 cycles of denaturation (30 seconds, 92°C), annealing (45 seconds, 54°C) and extension (90 seconds, 72°C), with a final extension step (10 minutes, 72°C).
The long probe, consists of a 2310 bp amplification product with PtNSF1 specific primers:
The product was obtained with the kit Expand Long Template PCR System (Roche Diagnostics). Each reaction (50 µl), adjusted to a concentration of nucleotides corresponding to the Paramecium A+T rich genome composition (740 nM of dATP and dTTP; 260 nM of dCTP and dGTP), contained 150 ng of DNA, 50 pmol of each primer and 3 U of polymerase mix. Amplification was performed with one cycle of denaturation (92°C, 2 minutes), 10 cycles of denaturation (92°C, 10 seconds), annealing (55°C, 30 seconds), extension (68°C, 210 seconds), then 20 cycles of denaturation (92°C, 10 seconds), annealing (55°C, 30 seconds), extension (68°C, 210 seconds plus 15 seconds/cycle) with a final extension step (68°C, 7 minutes).
PCR of cDNAs
The open reading frames (ORFs) of PtNSF1 and PtNSF2 were
amplified from a P. tetraurelia 51S cDNA library
(Klumpp et al., 1994) in
ZAP Express (Stratagene GmbH, Heidelberg, Germany) using polymerase
chain reaction (PCR) performed with the Advantage 2 PCR Kit (Clontech,
Heidelberg, Germany). The reaction (50 µl) contained 200 µM each of the
four nucleotides (dATP, dTTP, dCTP, dGTP), 3 µl of the cDNA library
(3x105 plaque-forming units), 20 pmol of each primer and 1
µl of Advantage 2 polymerase mix. In the case of PtNSF1 the
primers used were:
both containing artificial restriction sites added at their 5'-ends (SpeI and BamHI, respectively). The same is true for oligonucleotides 7 and 8, which are specific for PtNSF2:
Amplification of PtNSF1 was performed with one cycle of denaturation (95°C, 1 minute), 35 cycles of denaturation (95°C, 30 seconds), annealing (54°C, 45 seconds) and extension (68°C, 3 minutes), followed by a final extension step at 68°C for 3 minutes. In the case of PtNSF2, the annealing temperature was 62°C and the number of cycles was increased to 55.
PtNSF1-specific products were purified using the QIAquick PCR
Purification Kit (Qiagen) and digested by SpeI and BamHI (10
units each, 2 hours, 37°C). Double-digested cDNA was then extracted from
low-melt TAE agarose gels using the QIAquick gel extraction kit (Qiagen) and
ligated into the plasmid pBluescript II SK- (Stratagene GmbH),
digested with the same enzymes. PtNSF2-specific PCR products were
cloned into the plasmid pTAdv by using the AdvanTAgeTM Cloning Kit
(Clontech) according to the manufacturer's instructions. After transformation
into E. coli (DH5 cells or TOP10F' cells), positive
clones were sequenced as described below.
Homology-dependent gene silencing
For silencing experiments, PCR products amplified in the same conditions as
for the long probe were purified by QIAquick PCR Purification Kit (Qiagen,
Hilden, Germany), filtered on Millex-GV (0.22 µm) (Millipore, Bedford, MA),
precipitated and resuspended in water at a final concentration between 10 and
30 µg/µl. Before microinjection, wild-type cells were treated with a
solution of aminoethyldextran (Plattner et
al., 1984) at 0.01% to stimulate trichocyst exocytosis before
microinjection, thus avoiding any further discharge that could disturb
microinjection. This does not interfere with the analysis of exocytosis since
a full complement of trichocysts is resynthesized within less than 7 hours
(Plattner et al., 1993
),
whereas the first signs of silencing appear after at least 16 hours at
27°C. DNA microinjections were made under an inverted Nikon phase-contrast
microscope, using a Narishige micromanipulation device, and an Eppendorf
air-pressure microinjector as described
(Ruiz et al., 1998
;
Galvani and Sperling,
2001
).
The PtNSF silencing effect on cells clonally derived from
microinjected cells can be recognised after 24 hours and 48 hours of growth at
27°C by reduced phagocytic activity before cell lethality occurs (see
Results). Therefore, the efficiency of silencing was tested by adding India
ink to the medium to follow food vacuole formation over a 10 minute period.
NSF-silenced cells do not form any food vacuoles, whereas 10 are formed
in normal cells over a 10 minute period.
Preparation of radioactive probes
Probes were synthesised by [-32P]dATP incorporation using
a Random Primers Labeling System (Gibco-BRL, Cergy-Pontoise, France),
according to the supplier's protocol.
Southern blots
Paramecium DNA was digested by restriction enzymes according to
the manufacturer's instructions (New England Biolabs, Beverly, MA), then
fractionated by electrophoresis on 1% agarose gels and transferred to
Hybond-N+ filters. Hybridizations were carried out as described
(Church and Gilbert, 1984), at
60°C. The membranes were then washed at the same temperature with
decreasing concentrations of SSC, in the presence of 0.1% SDS as follows:
2x SSC for 30 minutes and 0.2x SSC for 30-45 minutes
(Sambrook et al., 1989
).
Images were obtained by using a Phosphorimager (Molecular Dynamics, Sunnyvale,
CA). Hybridizations were quantified by ImageQuant Software (Molecular
Dynamics).
RNA extractions and northern blots
Total RNA was prepared essentially as described
(Chomczynski and Sacchi, 1987)
by using the Trizol reagent (Gibco BRL), except that the cells were lysed by
vortexing in the presence of glass beads. Total RNA was fractionated on
formaldehyde/1.25% agarose gels, and transferred to positively charged nylon
membranes (Ambion, Austin, TX) by capillarity. Hybridization was carried out
at 50°C in 6x SSC, 2x Denhardt's solution and 0.1% SDS
(Sambrook et al., 1989
); the
filters were then washed and imaged as described for a Southern blot.
Sequencing
The sequencing was either made on an AbI 310 sequencer using the BigDye
Primer Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA) or
made by MWG Biotech custom sequencing service. Overview sequencing of
different library clones hybridizing with a PtNSF1 probe was made
with a single primer: oligonucleotide 9:
5'-GAAGGAGCCATAATCAATC-3'. DNA sequences were aligned either by
the CLUSTAL W or by the JOTUN HEIN method, both integrated in the DNASTAR
Lasergene software package (Madison, WI).
Peptide synthesis and antibody preparation
Synthesis and immunisation of a peptide corresponding to the amino acid
sequence of Paramecium PtNSF1
(NH2-CF464QKKLNKQDELKVKRSDF481-CONH2)
was carried out by Pineda (Antikörper-Service, Berlin, Germany). The
peptide also contains an additional cysteine at the N-terminus. For
immunisation the peptide was coupled to keyhole-limpet haemocyanin and
injected into rabbits and guinea pigs as previously described
(Hauser et al., 1998).
Antibodies (Abs) were affinity-purified using the same peptide immobilised on
CNBr-activated Sepharose 4B (
3 ml, at about 3 mg/ml).
SDS-PAGE and immunoblotting
Protein samples were denatured by boiling for 3 minutes in SDS sample
buffer, subjected to electrophoresis either on 10% SDS polyacrylamide gels
using a discontinuous buffer system described previously
(Laemmli, 1970). The replicas
were electroblotted to nitrocellulose membranes and immuno reactions were
carried out as described (Kissmehl et al.,
1997
) by using affinity-purified Abs against yeast NSF, Sec18p
(Mayer et al., 1996
), or
against a peptide of Paramecium PtNSF1 (see above). Bound Abs were
detected with the corresponding peroxidase-conjugated secondary Ab
(anti-rabbit IgG or anti-guinea pig IgG) using the Amersham enhanced
chemiluminescence (ECL) detection system.
Immunofluorescence localisation
Living cells suspended in Pipes/HCl buffer (5 mM, pH 7.2) with KCl and
CaCl2 added (1 mM each) were exposed for up to 30 minutes to 0.01%
saponin in the presence of MgCl2, ATP--S and NEM (1 mM
each), in the same buffer solution. Then, when the cells were still viable,
they were fixed at 22°C for 30 minutes in 8% formaldehyde (in
phosphate-buffered saline, PBS) plus 0.1% Triton X-100, washed in PBS and then
twice in PBS supplemented with 50 mM glycine, and finally in PBS plus bovine
serum albumin (1%). Then they were exposed to anti-NSF Abs, followed by
secondary Ab-FITC. In controls, the additives mentioned or primary Abs were
omitted.
Electron microscopy and immunolabelling of ultrathin sections
PtNSF-silenced cells and non-silenced controls were treated
identically as follows. For analysis of ultrastructural changes, cells were
processed routinely (i.e. fixed in 2.5% glutaraldehyde, followed by 1%
OsO4, ethanol dehydration and embedding in Spurr's resin) and
sections were sequentially stained with aqueous uranyl acetate followed by
lead citrate at pH 12.0.
For immunolocalization, PtNSF-gene-silenced cells were fixed in 8%
formaldehyde plus 0.1% glutaraldehyde (1 hour, 22°C) and then, for
transportation, in 8% formaldehyde only, essentially as published
(Flötenmeyer et al.,
1999; Hauser et al.,
2000
). This combined formaldehyde/glutaraldehyde fixation protocol
has been applied to allow for adequate fixation without too much loss of
antigenicity. For the latter reason, glutaraldehyde had to be deleted during
sample transport. We performed dehydration with ethanol by progressively
lowering the temperature, followed by LR Gold embedding and UV polymerization
at -35°C. After the usual washes, to avoid unspecific Ab binding,
ultrathin sections were exposed to Abs (against PtNSF1-derived peptide,
diluted 1:50) and then to protein A-Au6nm conjugate, followed by
section staining with aqueous uranyl acetate only. EM micrographs of defined
magnification collected from
10 cell sections of each sample type were
quantitatively evaluated by referring gold counts to area size analyzed, using
the hit-point method (Plattner and
Zingsheim, 1983
). Then labeling density was normalized to
cytoplasmic labeling.
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Results |
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After sequencing the complete PtNSF1 gene, a second round of hybridisation of the indexed library was made with a probe corresponding to the entire ORF. In addition to the 9 clones previously identified with the short probe, three new ones were identified (50e4, 55e3 and 56o4), thus revealing a second gene, designated PtNSF2. A restriction mapping analysis showed that the three clones correspond to the same sequence, with variable cloning position. Both extremities of the three inserts were sequenced. Clone 55e3 showed a sequence very similar to, but not identical to the 5' sequence of PtNSF1 close to the border of the insert. This plasmid was chosen for sequencing the entire PtNSF2 gene (accession number AJ347752).
To look for any other possible PtNSF genes, Southern blot analysis on total genomic DNA revealed bands compatible with the restriction map of the clones containing PtNSF1 when the short probe was used. Additional bands were observed with the long probe, in agreement with the restriction map obtained for the PtNSF2 library clones (Fig. 1B). This suggests that only two PtNSF genes are present in the Paramecium genome.
The two PtNSF genes were also cloned from a Paramecium
cDNA library, created from size-selected RNA (0.5-5 kb) of vegetative 51S
cells (Klumpp et al., 1994). A
comparison of the genomic sequences with their cDNA equivalents revealed that
both genes contain three short introns at the same positions that all display
the characteristics of Paramecium, bordered by 5'-GTA and
TAG-3' and of 25-28 nucleotides in size. The percentages of identity
between the corresponding introns are, respectively, 80, 64 and 83, suggesting
that duplication occurred recently.
Characteristics and expression of the Paramecium PtNSF
genes
The PtNSF1 gene consisting of 2234 bp encodes a protein of 751
amino acids, with a calculated molecular weight of 84,671. The overall
identities of its amino acid sequence with homologous genes from other species
range from between 38.5% for Drosophila melanogaster and 42.1% for
Nicotiana tabacum. In addition, the 2335 bp PtNSF2 gene
encodes a protein of 751 amino acids, with approximately the same calculated
molecular weight (84,661) as PtNSF1. In the case of PtNSF2
the overall identities at the amino acid level vary between 39.0% in D.
melanogaster and 42.5% in N. tabacum.
PtNSF1 and PtNSF2 genes are very similar, with 87%
identity at the nucleotide sequence level and
93% at the protein sequence
level. Sequence alignments with NSF proteins from other species revealed an
average identity of
40% throughout all kingdoms. Clearly, highest
similarity occurs in functionally important domains
(Fig. 2). Respective
percentages of identity with PtNSF1 and PtNSF2 are:
Plasmodium falciparum (a member of the closely related phylum
Apicomplexa) (40.6/39.9); Dicytostelium discoideum (39.9/40.6);
Saccharomyces cerevisiae (40.5/40.2); D. melanogaster
(39.0/39.0 and 38.5/39.2 for DmNSF1 and DmNSF2,
respectively); Arabidopsis thaliana (40.7/41.3); and Homo
sapiens (38.9/39.2). The alignment shows a very strong conservation over
two-thirds of the molecule, particularly within the two blocks that interact
with ATP, D1 and D2. As members of a large family of AAA-ATPases, the two
PtNSF genes contain a 231 amino acid AAA domain centered in the
Walker A and B boxes (Fig. 2).
Similarities are particularly high for the functional domains, D1 and D2.
Beyond this, they also contain the so-called second homology domain, which
makes up part of the AAA cassette
(Latterich, 1998
). Owing to
these signatures, there is no doubt that these genes encode PtNSF
proteins.
|
The presence of the two genes in a cDNA library indicates that both
isoforms are expressed. The expression level of PtNSF1 and
PtNSF2 genes was also analyzed on northern blots by using the long
probe, revealing a 2.3 kb band (Fig.
1C), the size to be expected for both PtNSF
transcripts.
Identification of PtNSF proteins
To identify and localize PtNSF proteins, we used either a crossreactive
polyclonal Ab against NSF from yeast (affinity-purified IgGs against SEC18p)
or a peptide-specific Ab against a unique sequence of PtNSF1 located
at the end of D1 (see Materials and Methods). The peptide selected for Ab
production is remarkably different in NSF molecules from other species, when
compared, for example, to the corresponding site 497-513 in P.
falciparum or to 457-475 in H. sapiens. In western blots, the
peptide used for immunization was therefore easily recognized by these Abs
(data not shown), whereas no crossreactivity occurred when recombinant NSF
from yeast Sec18p was used (Fig.
3, lane 9). However, with both types of Abs, anti-Sec18 Abs and
anti-PtNSF1 Abs, we could detect PtNSF as a 84 kDa band not only in whole
cell homogenates but also in particulate fractions enriched in ER
(Fig. 3).
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Silencing of PtNSF genes
Homology-dependent gene silencing can be efficiently obtained in
Paramecium by microinjection into the macronucleus of large amounts
of DNA corresponding to the coding sequence, without flanking 5' and
3' sequences (Ruiz et al.,
1998; Bastin et al.,
2001
). We used the PtNSF1 sequence, which is supposed to
silence both PtNSF genes, since cosilencing is assumed for genes
sharing
85% identity in nucleotide sequence [extrapolated from RNAi
experiments in the nematode (Parrish et
al., 2000
) (see also Ruiz et
al., 1998
)].
After microinjection, cells underwent two fissions within 24 hours at
27°C, whereas controls made four fissions. Silenced cells displayed a
characteristic phenotype: small size, dark appearance, decreasing motion rate.
Whereas normal cells form approximately one digestive (food) vacuole per
minute, silenced cells decreased and finally stopped phagocytosis activity.
These criteria routinely served to identify silenced cells for subsequent
analysis. Cells eventually died within 48 hours at 27°C. The defects we
recognized after silencing were not simply general metabolic effects, since
control experiments (e.g. with -tubulin silenced cells) showed quite
different effects [i.e. they stopped duplicating their ciliary basal bodies
and decreased their cell size after division
(Ruiz et al., 1999
)], whereas
food vacuole formation was not affected
(Froissard et al., 2002
). All
this indicates that silencing yields specific responses depending on the type
of gene to be silenced.
We also found that the terminal step of stimulated trichocyst exocytosis is
not affected in silenced cells. However, an effect of PtNSF silencing
on the organization of microdomains in the exocytosis sites could be
documented using a conditional mutant, nd9-1, in which this event could be
decoupled from previous steps in trichocyst biogenesis
(Froissard et al., 2002).
Otherwise, PtNSF silencing has no effect on exocytosis sites, once
they are established. On the EM level, PtNSF-silenced cells display
massive changes, as documented in Fig.
4. The most salient changes are as follows. (1) Golgi fields
increasingly disappear and become fragmented and ER cisternae swell
(Fig. 4A,B). Owing to loss of
ribosomes from ER in some places, it is not always possible to clearly
identify the origin of each swollen cisterna, which may arise from ER, Golgi,
or endosomes, most of them localized in cortical regions
(Allen, 1988
). Frequently, many
small transport vesicles appear attached, as though `frozen', on such swollen
cisternae, particularly in the cortex (Fig.
4A); they often look as though they were trapped after rounding up
(Fig. 4B) but closure of such
cisternae has not been shown since this would require serial sectioning. (2)
Coated pits with clathrin [called `parasomal sacs' in Paramecium
(Allen, 1988
;
Allen et al., 1992
)], if still
present, may also appear `frozen', sometimes in an atypical oblique
attachment. Occasionally they are replaced by groups of smooth vesicles
(Fig. 4C). (3) Early endosomes
[`terminal cisternae' (Patterson,
1978
; Allen, 1988
)]
either appear swollen or are totally missing
(Fig. 4A). Since both these
structures may serve constitutive exo-/endocytosis
(Flötenmeyer et al.,
1999
), our observations imply disturbance of these processes. (4)
Furthermore, phagocytosis is increasingly depressed in the course of NSF
silencing (a criterion of successful silencing), as is (5) the formation
and/or transport of discoidal vesicles (not shown), which normally serve
membrane recycling, after defecation from spent vacuoles to the site of the
formation of new phagosomes (Allen et al.,
1995
; Allen and Fok,
2000
). (6) Aggregates of small vesicles sticking together around
fields of ER (with intense gold labeling, as shown in
Fig. 6D) occur at different
sites (Fig. 4D), whereas they
are absent in non-silenced cells. These regions contain ribosomes, thus
strongly suggesting their origin from ER
(Fig. 4D). Such forms of ER are
known to occur in Paramecium
(Allen, 1988
;
Fok and Allen, 1981
).
Identification of ER-related structures is supported by the higher
magnification of Fig. 6D in
Fig. 6E.
|
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|
Silencing experiments may also reveal biogenetic pathways, when transport
vesicles are `frozen' firmly attached at a target membrane
(Fig. 4C,
Fig. 5). This had to be
expected for the interaction of small vesicles with phagosomal membranes
(Allen and Fok, 1984) since
different types of vesicles bud and fuse during their life cycle. However, we
see for the first time occasional attachment of small vesicles,
30 nm in
diameter, at alveolar sacs, which may support their biogenesis via vesicle
flow, as discussed below.
Immunolabeling
Immuno-gold labeling data obtained by anti-PtNSF Abs after PtNSF
gene silencing are presented in Fig.
6A,B and Table 1.
It is not easy on such sections to discriminate between ER and Golgi areas
(owing to low contrast allowed by the fixation method) and different types of
small vesicles scattered in such ER-rich domains, which therefore are taken as
representative for general cytoplasmic labeling. (In non-silenced cells, these
domains are homogenously labeled; Fig.
6C.) The low values, with large s.e.m., found over trichocysts and
lipid droplets are not statistically significant. However, in silenced cells,
lysosomes (identified by their size of 1 µm, compact contents and
single membrane envelope) are slightly but significantly labeled
(Table 1). This indicates
ongoing degradation of PtNSF molecules, while formation of new ones is
inhibited, which results in the decrease in vesicle traffic described above.
Concomitantly, aggregates, several microns in size, of intimately clumped
cisternae occur (compare Fig.
6A,B with Fig. 4D).
Since they are 4.75-times more labeled than the average cytoplasm
(Table 1), this may indicate
inability to dissociate SNARE complexes and to drive vesicle transport and
fusion at this stage of silencing. Reduced vesicle traffic may, thus, also
explain decreasing cell size during NSF gene silencing.
|
To increase the chance of seeing PtNSF bound as a complex to target
membranes, we used NSF ATPase activity inhibitors such as ATP--S and
NEM in immunofluorescence experiments. Cells were carefully permeabilized and
association of PtNSF with fusogenic sites was maintained by adding the
inhibitors, followed by immuno-FITC labeling. This resulted in labeling of the
cytostome and cytoproct region (Fig.
7A,B), of the outlets of contractile vacuoles
(Fig. 7B), and of the onsets of
radial canals, as shown in Fig.
7B,C. Different labeling intensity of these sites with periodic
membrane (dis-)connection may indicate some asynchrony in their
membrane-to-membrane interaction. In control cells, subjected to the labeling
protocol without ATP-
-S and NEM (or without primary Ab), these
structures were not labeled (Fig.
7E,F). These data further support specificity of Ab binding, since
anti-PtNSF Abs bind to specific sites only when cautiously permeabilized cells
were exposed to conditions mediating irreversible NSF binding to potential
fusion sites. Beyond that, requirement of ATP for detachment of NSF from
membranes can also explain why a high percentage of PtNSF is membrane-bound in
western blots from cell fractions (Fig.
3).
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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In this work, we identified and studied two NSF genes in Paramecium. We combined cytological and ultrastructural studies with gene silencing experiments to gain insight into the function and site of action of NSF. To localize PtNSF in situ, we used anti-NSF Abs in conjunction with gold-labeling after silencing, and Ab-fluorescence labeling in normal cells. Thereby we took into account that PtNSF may be trapped at membrane-to-membrane interaction zones if its rapid dissociation after SNARE assembly is blocked by inhibiting its ATPase activity. Our approach was greatly facilitated by the occurrence of well established avenues of vesicle traffic in Paramecium. Together, we showed that PtNSF is involved in most, if not all, membrane traffic pathways recognizable in Paramecium. This is the first experimental evidence that, in these pathways, complex NSF/SNAP/SNARE machinery must operate, as is the case in other eukaryotes.
Two closely related NSF genes in Paramecium
The two PtNSF genes found have 87% nucleotide identity, each gene
encoding a protein of 84.7 kDa with 94% identity. The occurrence of at least
two expressed genes for the same kind of protein is frequent in P.
tetraurelia (Hauser et al.,
1997; Bernhard and Schlegel,
1998
; Kim et al.,
1998
; Chan et al.,
1999
).
The reason why Paramecium expresses two rather similar
NSF genes remains unclear. Searches in the Caenorhabditis
elegans and the human genome databases reveal only one NSF gene,
indicating that a single NSF gene can support all membrane traffic.
In rats, NSF transcripts undergo alternative splicing
(Puschel et al., 1994). In
D. melanogaster, which possesses two NSF genes
(Ordway et al., 1994
;
Pallanck et al., 1995
), the
two are expressed at different stages of development, despite similar
functional properties (Golby et al.,
2001
).
Effects of NSF gene silencing
The close resemblance of the two PtNSF genes in
Paramecium suggests that gene silencing experiments on one gene
affect both genes. However, it is not excluded that only PtNSF1 is
silenced and not PtNSF2. In this case, this would mean that
PtNSF1 by itself is (1) essential and (2) not replaceable by
PtNSF2.
Silencing of the PtNSF genes has a dramatic influence not only on
the growth and survival of clonal descendants, but also on their subcellular
organization (Table 2).
Different types of vesicle transport operate at different rates and,
therefore, may have a different sensitivity to PtNSF silencing.
According to our EM analysis, rough ER-to-Golgi delivery, early endosome
formation, and recycling of spent phagosomal membranes from the cytoproct to
the cytostome via discoidal vesicles, appear very sensitive to NSF
deprivation. Concomitantly, these structures either undergo deformation or
disappear. NSF gene knockout in yeast also disturbs ER to Golgi
vesicle delivery (Graham and Emr,
1991) and endocytotic vesicle trafficking
(Prescianotto-Baschong and Riezman,
1998
). In contrast, exocytosis of docked trichocysts can be
triggered in a normal manner in PtNSF-silenced cells. This may imply
that the sensitivity of an organelle to silencing may depend on the turnover
of intermembrane interactions. Once trichocysts are attached to the plasma
membrane, the situation is rather stable and requires no further rounds of
PtNSF interaction.
|
After PtNSF silencing, many of the transport vesicles clump
together to form big aggregates, just as in yeast after NSF gene
knockout (Novick et al.,
1980). As we show, such vesicle aggregates are most heavily
labeled with anti-NSF Ab-gold conjugates. Some label clearly above general
cytoplasmic labeling, specifically of ER-rich zones, but well below that in
vesicle aggregates, also occurs in lysososmes, identified by their size, shape
and appearance of their contents. Later, lysosomal enzyme delivery and/or
retrieval from phagosomes (Allen and Fok,
2000
) may also be interrupted. Accordingly, some phagolysosomes
show unusual numbers of small vesicles attached after silencing. Both the
clustering of strongly labeled small vesicles in some areas of silenced cells
and the ongoing deformation of some of the organelles participating in
membrane traffic indicate increasing abolition of vesicle traffic. Clearly the
osmoregulatory system functions longer than the phagocytotic pathway, possibly
because it requires far fewer membrane fusion events
(Allen, 2000
), in contrast to
the multiple rounds of fusion events, each involving a multitude of vesicles
(acidosomal, discoidal, primary lysosomal, endocytotic), required during a
digestive cycle (Allen and Fok,
2000
).
Rare fusion events are trapped by inhibiting NSF dissociation
To visualize PtNSF at the whole cell level, we took advantage of the
general property of NSF to dissociate from the SNARE complex upon ATP
hydrolysis (Whiteheart et al.,
2001). We then developed a new protocol to visualize potential
membrane fusion zones by NSF Ab-fluorescence labeling. This includes careful
saponin permeabilization of viable cells in the presence of ATP-
-S and
NEM, to block PtNSF dissociation from membrane attachment/fusion sites. The
outcome was uncertain a priori for the following reason. On the one hand, in
addition to ATPase activity, an additional role had been assigned to NSF in
SNARE (dis-)assembly (Müller et al.,
1999
). On the other hand, a requirement of ATP in in vitro SNARE
disassembly had recently been shown (Wagner et al., 2001). With our approach,
we could clearly label low frequency docking zones under restrictive
conditions. Not only are the cytostome, the cytoproct and the outlet of the
contractile vacuole labeled, but also the attachments of radial canals to the
contractile vacuole. Their repeated disconnection and reconnection during
activity cycles has been a matter of debate
(Hausmann and Allen, 1977
).
Our data agree with the more recent indirect evidence from
electrophysiological recordings (Tominaga
et al., 1998a
; Tominaga et
al., 1998b
). Even occasional labeling of radial canals and of the
attached spongiome membranes would fit the eventual vesiculation and refusion
of these membranes, as reported previously
(Tominaga et al., 1998b
). Some
of the ubiquitous vacuoles and vesicles of different sizes, which are also
labeled, are more difficult to identify they are probably components
of different origin of the elaborate endo-/phago-/lysosomal system. This is
even more difficult for the widely branching bulk of ER components, with the
numerous Golgi fields in-between. In agreement with EM labeling analysis, the
firmly established docking sites of trichocysts are not labeled by this
procedure.
Unexpected aspects of organelle biogenesis
Membrane biogenesis frequently operates very inconspicuously and,
therefore, may not always be evident from EM micrographs. However, when halted
by PtNSF silencing, attachment of small vesicles can indicate such
biogenetic pathways. We show this first for digestive vacuoles for which such
a pathway is established (Allen and Fok,
2000), but then also for alveolar sacs, whose biogenesis was under
considerable debate until recently
(Capdeville et al., 1993
;
Flötenmeyer et al.,
1999
). After PtNSF gene silencing,
30 nm wide
vesicles are occasionally seen attached to alveolar sacs. Recently, both
expression of an ER-type Ca2+-ATPase (pump) as a fusion protein
with green fluorescent protein and staining by affinity probes in vivo have
suggested a biogenetic pathway by vesicle flow in Paramecium
(Hauser et al., 2000
).
However, it was previously difficult to imagine how even small vesicles could
reach alveolar sacs since their side facing the cell center is lined by an
electron dense epiplasmic layer.
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Conclusions |
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Acknowledgments |
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Footnotes |
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References |
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