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Cell Motility and the Cytoskeleton 43:72–81 (1999)
Centrin-Like Filaments in the Cytopharyngeal
Apparatus of the Ciliates Nassula
and Furgasonia: Evidence for a Relationship
With Microtubular Structures
Bernard Vigues,* Marie-Pierre Blanchard, and Philippe Bouchard
UPRES A CNRS 6023, Laboratoire de Biologie Comparée des Protistes,
Université Blaise Pascal Clermont-Ferrand II, Aubière Cedex, France
The cytopharyngeal apparatus in the Nassulinid ciliates Nassula and Furgasonia is
a highly specialized microtubular/filamentous organelle designed for ingestion of
organisms such as filamentous bacteria. From studies on living cells, it was
previously shown that this organelle, also called ‘‘feeding basket,’’ guides the
filamentous bacteria and manipulates them to some extent during the early steps of
ingestion. This results in a complex sequence of movements where the basket is
successively dilated and constricted in its upper part. Whereas some of these
movements (dilation) seem to be intrinsic to the microtubular components of the
basket, others (constriction) are believed to be mediated by contractile filamentous
structures [Tucker, 1968: J. Cell Sci. 3:493–514]. In this study, we have used
antibodies raised against ciliate centrins to demonstrate these proteins by Western
blot and immunocytochemical methods in Nassula and Furgasonia. In both
ciliates, a 20-kDa centrin immunoanalog was localized in the upper (contractile)
part of the cytopharyngeal apparatus. Immunoelectron microscopy revealed that
cytopharyngeal centrin is engaged in filamentous material, forming a sphincterlike structure possibly involved in the movements of contraction. Interestingly,
physical links were noted between filaments labeled for centrin and cytopharyngeal microtubules. The mechanistic implications of these findings are discussed.
Cell Motil. Cytoskeleton 43:72–81, 1999. r 1999 Wiley-Liss, Inc.
Key words: centrin; contractility; microtubules; cell motility; endocytosis; ciliates; Protozoa
INTRODUCTION
Centrin is a 20-kDa Ca⫹⫹-binding protein that
belongs to the EF-hand protein superfamily. Despite its
structural similarities to other EF-hand proteins, centrin is
functionally unique in that it is believed to be directly
responsible for contraction of calcium-sensitive filamentous systems mediating various cell motility processes in
unicellular eukaryotes. Centrin-based filamentous systems were initially discovered in the flagellar apparatus of
unicellular green algae where they connect basal bodies
to one another and to the nuclear envelope [for a review
see, e.g., Melkonian et al., 1992]. Since this pionnering
work, molecular cloning and/or immunolocalization experiments have indicated that centrin is a ubiquitous
protein in eukaryotic cells. A common property of arrays
r 1999 Wiley-Liss, Inc.
of centrin in a number of cell types is their association
with microtubule organizing centers (MTOCs). Immunolocalization experiments indicate centrin in the spindle
pole body of yeast [Spang et al., 1993], mammalian
centrosome [see, e.g., Baron et al., 1992; Lee and Huang,
1993; Errabolu et al., 1994; Middendorp et al., 1997], and
MTOCs associated with basal bodies of cilia and eukary-
Contract grant sponsor: Centre National de la Recherche Scientifique.
*Correspondence to: Bernard Vigues, UPRES A CNRS 6023, Laboratoire de Biologie Comparée des Protistes, Université Blaise Pascal
Clermont-Ferrand II, 63177 Aubière Cedex, France.
E-mail: vigues@cicsun.univ-bpclermont.fr
Received 23 November 1998; accepted 14 January 1999
Centrin in the Feeding Basket of Nassulinid Ciliates
otic flagella [Salisbury, 1989; Levy et al., 1996]. In
mammalian centrosomes, centrin has been localized in
the core of centrioles and in the pericentriolar matrix
which organizes the network of cytoplasmic microtubules
and mitotic spindle during cell division. Attempts to find
some contribution, even indirect, of centrin to the process
of centrosome-dependent assembly of microtubules were,
however, unsuccessful [Moudjou et al., 1991]. From
more recent studies, further colocalizations with microtubular structures were noted, notably with microtubules of
the mid-body, a transient structure assembled in the
intercellular bridge of dividing cells [Paoletti et al.,
1996]. In no case, however, has the functional significance of centrin-microtubule colocalizations been elucidated.
In ciliates, centrin or closely related proteins have
been reported in basal bodies [Klotz et al., 1997] and in
cortical filamentous networks displaying calcium-dependent contractile properties [Garreau de Loubresse et al.,
1991; David and Vigues, 1994; Levy et al., 1996]. We
previously showed that some of these networks may be
part of cytoskeletal complexes that include microtubular
structures [Vigues and Grolière, 1985; Vigues and David,
1994]. It seemed, therefore, that more detailed studies of
such complexes might be pertinent for our understanding
of centrin-microtubule relationships in general.
In the present study, we have investigated the
problem of centrin-microtubule relationships in the cytopharyngeal apparatus of Nassula aurea and Furgasonia
blochmanni, two ciliates that feed on filamentous bacteria. The fine structure of the cytopharyngeal apparatus,
also called the ‘‘feeding basket,’’ has been described in
detail by others [Tucker, 1968; Eisler, 1988]. In both
ciliates, the feeding baskets are similar in organization,
consisting mostly of microtubular structures. These include (1) the nemadesmata, which are arranged in a tube
through which the filamentous bacteria enter the organism and (2) crest-shaped structures attached to the
nemadesmata in the upper part of the basket and that
spiral around the outside of the tube in the lower part.
During ingestion, the basket guides the filamentous
bacteria and manipulates it to some extent. This results in
a complex sequence of movements, the so-called ‘‘feeding movements,’’ where the basket is successively dilated
and constricted in its upper part (see insets in Fig. 3).
Whereas some of these movements (dilation) seem to be
intrinsic to the microtubular components of the basket,
others (constriction) are believed to be mediated by
contractile filamentous structures [Tucker, 1968, 1978].
We report evidence suggesting that a 20-kDa centrin
immunoanalog may be functionally important for constriction of the upper part of the basket. An immunoelectron
microscopic study of this region is presented, showing the
73
20-kDa protein is a component of a sphincter-like filamentous structure that interconnects adjacent nemadesmata.
Accordingly, we suggest that colocalization of centrinbased filaments and microtubules, as shown in a number
of cells, may be more than circumstantial and might
reflect physical and possibly functional interactions, as
explored in the Discussion.
MATERIALS AND METHODS
Cells and Cell Fractions
The ciliates Nassula aurea and Furgasonia blochmanni were used as cell models for this study. The
method used for culturing both ciliates and their food, the
cyanobacteria Phormidium inundatum, have been described by others [Tucker, 1968; Eisler, 1988]. For cell
ghost preparation, cells were starved for 2 days in sterile
Volvic water and then collected by three successive
low-speed centrifugations in a GGT oil centrifuge (500g
for Nassula; 1,500g for Furgasonia; 5-min runs). Cell
pellets were suspended and gently shaken for 10 min in 5
volumes of ice cold SEMT solution (1M sucrose, 1 mM
EDTA, 0.1% 2-mercaptoethanol in 10 mM Tris-HCl
buffer, pH 8.6). The non-ionic detergent Triton X-100
(0.25 vol of a 10% [v/v] stock solution) and protease
inhibitors (1 mM PMSF, 10 mM TAME, 1 mM leupeptin,
0.1 mM pepstatin-A) were added in sequence with
continuous agitation. Cilia generally were disrupted at
this stage. Removal of endoplasmic material was monitored by phase contrast light microscopy. The released
cell ghosts were washed using two successive centrifugations (3,500g, 15 min) in PE buffer (10 mM sodium
phosphate, pH 6.9, 1 mM EDTA) containing 1 mM
PMSF.
When larger amounts of basket material were
required, cells from large-scale cultures were concentrated by centrifugation and Triton X-100 was 2% (v/v) in
SEMT buffer instead of 0.6% in the usual conditions,
resulting in noticeable enrichment in the feeding basket
due to disintegration of pellicular structures. Following
two successive centrifugations (4,000g; 15 min) in PE
buffer, pellets were dissolved in 6M urea, 0.2% Nonidet
P-40, 10 mM Tris-HCl, pH 8.6. Extractable proteins were
concentrated in a microprodicon vacuum concentrator
(Bioblock Scientific, Illkirch, France) filled with 6M
urea. The final concentrate was diluted in an equivalent
volume of 8M urea, 0.4% Nonidet P-40, and ampholines
at the concentration required for isoelectric focalization
experiments (see electrophoresis).
Antibodies
Anti-EEB antiserum was raised against 22/23 kDaCa⫹⫹-binding proteins of the ectoendoplasmic boundary,
74
Vigues et al.
a major centrin-like filamentous system in the ciliate
Isotricha prostoma. Specificity of this antiserum for
ciliate centrins was reported elsewhere [Vigues et al.,
1984; Garreau de Loubresse et al., 1991, see also
Madeddu et al., 1996 for molecular characterization of
the antigens as centrins]. A69 monoclonal antibody (mAb
A69) was raised against calmyonemin, a 23-kDa centrin
homolog involved in various cell motility processes in
entodiniomorphid ciliates [David and Vigues, 1994;
Vigues and David, 1994]. Anti-tubulin antibody, raised
against ␤-tubulin from Tetrahymena pyriformis, has been
described elsewhere [Bouchard et al., 1998]. In all
labeling experiments, i.e., immunocytochemistry and
immunoblottings (see below), anti-EEB antiserum was
diluted 1/250 and monoclonal antibodies were used as
crude hybridomas culture supernatants.
Secondary antibodies used in this study were as
follows: fluorescein-conjugated goat anti-mouse or rabbit
antibodies were from Sigma (Sigma Chemical Co., St.
Louis, MO) and used at a 1/200 dilution. Horseradish
peroxidase goat anti-mouse or rabbit antibodies were
from Promega (Promega Corporation, Madison, WI) and
used at a 1/5,000 dilution. Goat anti-mouse or rabbit
antibodies conjugated with 15-nm colloidal particles
were purchased from Sigma and used at 1/200.
Electrophoresis and Immunoblotting Techniques
SDS-PAGE was performed using 15% acrylamide
gels under standard conditions (Laemmli, 1970). Onedimensional gels were routinely stained using Coomassie
Blue. For 2-D gels, isoelectric focusing was performed by
the method of O’Farell [1975] on 4% acrylamide gels
containing 0.8% of 3–10 ampholines and 1.2% of 4–6
ampholines (Pharmacia, Uppsala, Sweden). Seconddimension gels were silver stained according to Merril et
al. [1981].
Western transfers of material from gels to PVDF
membranes (Millipore, Bedford, MA) followed the protocol of Towbin et al. [1979]. Before immunolabeling,
membranes were shaken for 30 min in PBS (10 mM
sodium phosphate, pH 7.2, 0.17 M NaCl) containing 5%
w/v dry milk, then briefly washed in PBS plus 0.5% dry
milk before incubation for 1 h with primary antibodies.
Excess of primary antibody was removed by washes in
PBS-0.5% nonfat dry milk before incubation for 1 h with
horseradish peroxidase-conjugated second antibody. After removal of excess secondary antibody by copious
washes, the reaction was either developed using 0.001%
hydrogen peroxide and 0.5 mg/ml diamino benzidine as
substrates or processed for chemoluminescence labeling
using an ECL kit according to the instructions of the
manufacturer (Amersham International, Amersham, England).
Immunofluorescence
For immunofluorescence microscopy, starved cells
were deciliated by successive transfers in 2% (w/v)
manganese chloride and permeabilized for 2 min in
PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM
EGTA, 2 mM MgCl2) containing 1% Triton X-100. Cells
were fixed for 30 min with 2% (w/v) paraformaldehyde in
PHEM buffer, copiously washed in PHEM and then
incubated in TBS-T (10 mM Tris-HCl, pH 7.4, 0.15 M
NaCl, 0.1% [v/v] Tween 20) containing 3% (w/v) bovine
serum albumin (BSA). TBS-T buffer was used in all
steps: incubation with primary antibody, washes, incubation with secondary antibody, and final washes. Cells
were finally mounted in 50% (v/v) glycerol and observed
under an Olympus BH2 epifluorescence microscope.
Electron Microscopy
Immunolocalizations at the ultrastructural level
were performed using the colloidal gold method. Cells
were aldehyde fixed for 1 h in 3.6% glutaraldehyde plus
0.4% paraformaldehyde in 0.1M sodium phosphate buffer
pH 7.2, at 4°C in the presence (Nassula) or absence
(Furgasonia) of 0.2% tannic acid [Begg et al., 1978].
Cells were then dehydrated and infiltrated with the polar
embedding medium Lowicryl K4M at ⫺35°C. The resin
was then polymerized overnight by U.V. irradiation at
⫺35°C. Thin sections were collected on nickel grids,
saturated with 3% (w/v) BSA in PBS and processed for
immunogold labeling as described elsewhere [Vigues and
Grolière, 1985].
For conventional electron microscopy, cells were
fixed for 30 min in 2% glutaraldehyde and post-fixed in
1% OsO4 both at 4°C in phosphate buffer. Samples were
embedded in Epon 812. Sections were examined under a
Jeol JEM-1200X transmission electron microscope.
RESULTS
Immunofluorescence Localization
of a Cytopharyngeal Centrin-Immunoanalog
in the Ciliates Nassula and Furgasonia
Indirect immunofluorescence labeling was performed on aldehyde-fixed cells previously deciliated and
permeabilized in the presence of the non-ionic detergent
Triton-X100. Figure 1A shows the typical fluorescence
pattern given by anti-tubulin antibody in these conditions.
Cilia have disintegrated but basal bodies and the feeding
basket persist following this treatment. The staining
pattern of Nassula cells by A69 monoclonal antibody is
shown in Figure 1B. Immunoreactive material is restricted to the anterior part of the basket. This material
forms adjacent polygonal meshes that seem precisely
fitted to the transverse section of nemadesmata. Identical
Centrin in the Feeding Basket of Nassulinid Ciliates
75
Fig. 1. Immunofluorescence microscopy of Triton-X100 permeabilized, deciliated cells of Nassula (A, B, and C) and Furgasonia (D and
E) stained with anti-tubulin (A), mAb A69 (B and D), and anti-EEB
antiserum (C and E). Tubulin labeling mainly decorates basal bodies
and microtubular components of the basket (*). mAb A69 and
anti-EEB antiserum provide similar staining patterns, resulting in a
collar of adjacent polygonal meshes near the top of the basket. Scale
bars: A ⫽ 40 µm; B ⫽ 15 µm; C ⫽ 7 µm; D and E ⫽ 10 µm.
results were obtained when cells were treated by antiEEB antiserum (Fig. 1C). From a series of micrographs of
N. aurea cells processed for immunofluorescence microscopy, we calculated a number of adjacent polygonal
meshes in agreement with the number (variable; from 25
to 34) of nemadesmata in this species.
The basket structure, which reacted in Nassula, was
also successfully identified in cells of Furgasonia using
the two antibodies (Fig. 1D, E). The size of this structure
is different due to the lower size of Furgasonia cells.
Fifteen to eighteen polygonal adjacent meshes could be
counted on the more characteristic micrographs, which,
here again, corresponds to the number of nemadesmata in
F. blochmanni. For each ciliate, control experiments were
performed in which the primary antibody was omitted
(mAb A69) or replaced by the corresponding preimmune
serum (anti-EEB antiserum). Under these conditions the
secondary antibodies failed to detect any cell structure
(data not shown).
oped. In the procedure used, cells of Nassula were
aldehyde-fixed in the presence of tannic acid, a method
that allows a better visualization of filamentous structures
[Begg et al., 1978]. For quantitative evaluation of the
antigen(s), a positive control was run using an antitubulin antibody previously found to afford strong labeling of microtubular structures in ciliates. When thin
sections of the anterior part of the basket were treated
with this antibody, gold particle deposits were found on
microtubules of nemadesmata (Fig. 2). Crest microtubules, basal bodies and cilia were also stained by the
antibody (not shown). Figure 3A and B shows sections of
the same region of the basket stained by anti-EEB
antiserum and mAb A69, respectively. With both antibodies, the label was confined to the area occupied by
filamentous material of the apical contractile annulus, a
filamentous structure initially described by Tucker [1968],
and thought to mediate constriction of the basket during
feeding movements. It is noteworthy that labeling of the
filamentous annulus was comparable in intensity to that
of nemadesmata stained with anti-tubulin antibody. We
also note that many filaments abut against the wall of
nemadesmata. No significant staining was observed in
control experiments in which the first antibody was left
out or replaced by preimmune serum (not shown).
Similar ultrastructural labelings were obtained in
Furgasonia cells (Fig. 4). In this case, tannic acid was
absent from the fixative solution, resulting in noticeable
loss of definition with respect to apical annulus substructure. The discrepancy is particularly evident when comparing, for instance, ultrastructure of the apical annulus in
either of the two ciliates (compare, e.g., Fig. 3B with Fig.
Immunoelectron Localization of Anti-EEB
Antiserum and mAb A69 Immunoreactivity
A variety of methods were experimented with to
localize anti-EEB/mAb A69 antibody immunoreactivity
at the ultrastructural level. All attempts to use preembedding labeling procedures such as those described in
previous studies [see, e.g., Vigues et al., 1984; Garreau de
Loubresse et al., 1988] were unsatisfactory because gold
particles, even of very small diameter, were unable to
penetrate into the bulk of cytoskeletal structures found in
the anterior part of the basket. For this reason, a thin
section post-embedding labeling procedure was devel-
76
Vigues et al.
Fig. 2. Immunocytochemical labeling of the anterior part of Nassula
feeding basket using anti-tubulin antibody. The section cuts through the
top of nemadesmata (nd) and shows the filamentous system termed
the apical filamentous annulus (fa). Nemadesmata are heavily stained;
the latter pass through the annulus and are connected to each other by
the filamentous material. Note that most of the filaments are attached
by end-on associations to the sides of nemadesmata (arrows). Inset:
Schematic representation of a resting basket showing the position of
the annulus (arrow) with regard to the palisade of nemadesmata. Scale
bar ⫽ 0.5 µm.
4B). On the other hand, electron micrographs obtained in
these conditions revealed an interesting aspect of microtubule-filament structural interactions. As shown in Figure
4B, plates of electron dense material are interposed
between nemadesmata and filaments of the annulus.
These plates were consistently found in the zones of
contact and absent lower down in the basket, suggesting a
role of the dense material in the attachment of filaments to
the wall of nemadesmal microtubules.
All the other bands were faintly stained by conventional
procedures. Small-sized polypeptides were notably present in fairly low proportions, in a narrow MW range
between 16 and 20 kDa.
When Western blotting was carried out on Nassula
cell ghost proteins, anti-EEB antiserum and mAb A69
both reacted with the same polypeptide at 20 kDa (Fig.
5C). The signal given by chemoluminescence was slightly
less intense in the case of the monoclonal antibody. In
both cases, an additional band was also evident, though
weakly stained, near the bottom of the gel.
For further biochemical characterization of the
20-kDa antigen, 2% Triton X-100 was used in the
extraction buffer, a method that generally disintegrates
the pellicle leading to an enrichment of oral structures
[Wolfe, 1970]. Feeding basket–enriched preparations
obtained in such conditions were solubilized in 6 M
urea-2% Nonidet P-40. Soluble proteins were then concentrated by vacuum dialysis, resuspended in IEF sample
buffer and processed for 2-D gel electrophoresis as
described in Materials and Methods. In these conditions,
the 20-kDa antigen was split into two spots easily
distinguishable by their isoelectric point (Fig. 5D,1).
Biochemical Characterization of Filamentous
Annulus Antigen
As shown in Figure 5A, cell ghosts obtained in the
presence of 1% Triton X-100 are devoid of cilia but retain
the feeding basket. The electrophoretic pattern of Nassula
cell ghosts is shown in Figure 5B. Even though cilia have
disintegrated, tubulins occur in fairly high proportion,
probably owing to the persistence of basal bodies,
associated fibers, and microtubules of the basket. The
band of highest intensity migrates with an apparent MW
of 33 kDa and probably corresponds to a major protein
component of the pellicle. Five bands of medium intensity are evident with MW of 60, 50, 45, 43, and 35 kDa.
Fig. 3. Immunogold labeling of the anterior part of the Nassula feeding
basket using anti-EEB antiserum (A) and A69 monoclonal antibody
(B). Both antibodies produce intense decoration of the apical filamentous annulus (fa and arrows). Nemadesmata are not stained, so that the
pattern provided could be ‘‘negatively’’ imaged in electromicrographs
obtained with anti-tubulin antibody (compare with Fig. 2). Scale
bars ⫽ 0.3 µm. Inset: Schematic representations of the feeding process
steps in Nassula [Tucker, 1968]. The top of the basket dilates (1) as it is
closely applied to the filamentous bacteria (fb). Subsequent feeding
movements include bending of the filamentous bacteria into a hair-pin
shape (2), and then constriction of the top of the basket, mediated by
the apical annulus (3).
78
Vigues et al.
Fig. 4. Apical filamentous annulus in Furgasonia as viewed by
conventional (A) and immuno-electron microscopy (B,C) following
staining with anti-EEB (B) and mAb A69 (C). As shown in Nassula,
gold particles are found in regions occupied by filaments of the annulus
(fa). Labeling of the annulus is slightly less intense with mAb A69.
Note that microtubules of nemadesmata (nd) are embedded in electron
dense material (arrowheads) as they pass through the filament annulus.
Accumulation of such dense material is evident in the zones of contact
with filaments of the apical annulus. Scale bars: A ⫽ 0.5 µm; B and C ⫽
1 µm.
Centrin in the Feeding Basket of Nassulinid Ciliates
79
Fig. 5. Anti-EEB antiserum and mAb A69 are both reactive with the
same 20-kDa protein. A: Light micrograph of a cell ghost obtained
from Nassula. Cilia have disintegrated. The feeding basket is conserved (arrow) and retains most if not all the nemadesmata (arrowheads). Scale bar ⫽ 35 µm. B: SDS-PAGE, gels were stained with
Coomassie Brilliant Blue. Lane 1: Standard proteins from Bio-Rad.
Lane 2: Nassula cell ghost protein components. The main bands are
represented by tubulins (tu) and a 33-kDa polypeptide (*). C: Western
blot analysis. Cell ghost proteins were transferred to filter membranes
then reacted with anti-EEB antiserum (lane 1) or mAb A69 (lane 2). In
the two cases, a 20-kDa band was antibody-stained. D: Portions of 2-D
Western blots of the 20-kDa antigen. 1: Silver staining. Note that the
antigen is revealed as two spots with acidic pI between 4.8 and 4.9. 2:
Immunodetection with anti-EEB antiserum. The two spots are reactive
to the antiserum. 3: Immunodetection with mAb A69. Only spot a,
which is the more acidic one, is reactive to the monoclonal antibody.
Spot b is not stained.
Immunoblots performed on replicates of 2-D electrophoregrams gave different results depending on the antibody
tested. Using anti-EEB antiserum, isoelectric variants A
and B were both immunoreactive as shown in Figure
5D,2. Using mAb A69 (Fig. 5D,3), immunoreactivity was
restricted to variant A, i.e., the more acidic isoelectric
variant of the 20-kDa protein antigen, suggesting that for
mAb A69 immunoreactivity, variants A and B are antigenically distinct polypeptides.
mAb A69 revealed two main arrays of centrin in Entodinium cytoalimentary system: a contractile lip-like structure and a filamentous sheath extending between and
parallel to the nemadesmata [Vigues and David, 1994].
The latter location was not found in this study. The
filamentous annulus might be the functional equivalent of
the contractile lip that contributes to ingestion of large
plant tissue fragments by entodiniomorphids. We also
note that centrin fibers may be associated with the
phagocytic process in some unicellular algae. However,
this cannot be directly correlated to the situation found in
Nassulinids since the role of these fibers seems limited to
food particle capture by flagella or more specialized
organelles as shown for instance in Ochromonas and
Pseudopedinella [Melkonian et al., 1992].
The biological function of the 20-kDa protein is not
known. It is tempting to speculate that it mediates
contraction of the filamentous annulus, possibly in response to an increase in local calcium concentration.
From observation of living feeding cells of Nassula and
ultrastructural studies of the basket, it has been proposed
that feeding movements result from antagonistic action of
crest microtubules and filaments of the apical annulus
[Tucker, 1968, 1978]. Whereas sliding of adjacent microtubules of the crests increases the distance between
adjacent nemadesmata and thereby dilates the top of the
basket, contraction of the fibrous annulus has the opposite
effect. If this interpretation is correct, our results indicate
the fibrous annulus is a specialization of centrin-mediated
cell motility designed to act antagonistically to a microtubule-sliding type of movement, a dual system that might
DISCUSSION
In this study we identified a 20-kDa antigen shared
by the two ciliates Nassula and Furgasonia and immunoreactive to antibodies raised against and specific for
non-centriolar forms of centrin in ciliated protozoans
[see, e.g., Garreau de Loubresse et al., 1988, 1991; David
and Vigues, 1994]. By indirect immunofluorescence
microscopy, it is shown that the 20-kDa protein is
restricted to the cytopharyngeal basket, a complex microtubular-filamentous organelle specialized for ingestion of
filamentous cyanobacteria. Immunoelectron microscopy
shows the 20-kDa protein in the apical filamentous
annulus that interconnects microtubular fibers (nemadesmata) in the upper part of the basket. A centrin immunoanalog in association with the cytoalimentary system of
ciliates has been found in previous studies on Entodinium
and its close relatives. In the so-called entodiniomorphids, a cytopharyngeal specialization occurs below the
cytostome [see, e.g., Furness and Butler, 1988], reminiscent of the feeding basket in Nassulinids. Labeling with
80
Vigues et al.
account for extreme changes in cell body length in the
large Heterotrich ciliates Stentor and Spirostomum [Huang
and Mazia, 1975]. In this respect, it is worth noting that
the contractile fibers (myonemes) that effect shortening of
Stentor and Spirostomum have been shown to be composed of spasmin [Ochiai et al., 1989], another calciumsensitive protein closely related to algal and mammalian
centrins [Melkonian et al., 1992; Levy et al., 1996].
Detectable amounts of the 20-kDa protein are not
present in regions occupied by basal bodies, which are
sites where one would also expect to find centrins in
ciliated Protozoa (see below). The simplest interpretation
that basal bodies of Nassula and Furgasonia lack centrin
is in obvious conflict with current concepts of evolutionary conservation of centriolar proteins. Since kinetosomal
centrin has been demonstrated in other ciliates, including
when cells were prepared for IF as in this study [see, e.g.,
Klotz et al., 1997], it is conceivable that the failure of our
antibodies to react with kinetosomes is due to their
specificity for non-kinetosomal forms of this protein.
Several reports describe heterogeneity in the current
centrin protein family; some based on the observation of
multiple bands or spots on one- or two-dimensional
protein gels [Vigues and Grolière, 1985; Garreau de
Loubresse et al., 1991; Levy et al., 1996; Paoletti et al.,
1996], others based on direct examination of centrin
genes themselves [Madeddu et al., 1996; Middendorp et
al., 1997]. On the basis of sequence comparison, two
divergent subfamilies of centrin proteins have been
proposed in mammalian cells possibly linked to their
implication in distinct centrosome-associated functions
[Middendorp et al., 1997]. The discriminative properties
of our antibodies could well reflect a similar dichotomy
between centrins involved in filamentous contractile
structures and kinetosomal forms of the protein. Consistent with this hypothesis, centrins previously characterized by mAb A69 and anti-EEB antibody have been
found in myonemes and/or related contractile fiber systems [Vigues et al., 1984; Vigues and Grolière, 1985;
Garreau de Loubresse et al., 1988; Hulays et al., 1991;
David and Vigues, 1994] and were absent from kinetosomes as shown in the present study. With the increasing
availability of centrin genes in ciliates, it would be
interesting to know where the epitopes reside on nonkinetosomal forms of centrin since this might provide
further insight into the molecular mechanisms that contribute to the functional diversity of these proteins in a
singled-cell organism.
Two isoeletric variants were disclosed in the case of
the 20-kDa protein, a situation that is more reminiscent of
that reported for centrin in algal flagellar apparatus. As far
as we know, algal centrin is a single gene product that
may exist as two isoforms that differ by the presence of a
phosphate group, Ca⫹⫹-binding being able to induce
dephosphorylation of centrin in vivo [Salisbury et al.,
1984]. Microheterogeneity of the 20-kDa protein could
well correspond to post-translational modification such as
phosphorylation, possibly associated with Ca⫹⫹-binding
activity. The suggestion of a unique type of posttranslationally regulated centrin in Nassulinid feeding
basket, however, conflicts with the following observations: first, Salisbury and coworkers [1984] showed that
the electrophoretic mobility of centrin is substantially
increased by the coupled effect of Ca⫹⫹-binding and
dephosphorylation, whereas in our experiments, variants
A and B were consistently found to be indistinguishable
otherwise than by their net charge as revealed by isofocalization. Second, for mAb A69 immunoreactivity, we
show here that variants A and B are antigenically distinct
polypeptides. Variant A stains with mAb A69 but variant
B does not. It cannot, however, be inferred that mAb A69
epitope is absent from variant B: the epitope could be
modified and/or masked in a special mode upon phosphorylation. On the other hand, assuming one of the two
variants is a phosphorylated form of the 20-kDa protein,
one would expect pretreatment of feeding baskets with
dephosphorylating agents to restore immunoreactivity of
variant B to mAb A69. We verified that pretreatment with
alkaline phosphatase, for instance, had no apparent effect
either on immunoreactivity or on the 2-D gel pattern of
the 20-kDa protein bands (data not shown). The relationship of the two proteins disclosed on 2-D gels is,
therefore, still unknown and further work is required to
determine whether the two proteins are derived one from
the other via post-translational modifications, or correspond to different gene products coexpressed in the same
cell.
Finally, the results presented in this study extend
our knowledge about the occurrence and functions of
centrins in different organisms. More interestingly, they
raise the question of whether physiological processes
relying on centrin-microtubule interactions may be a
more widespread phenomenon, particularly in mammalian centrosomes. This possibility is especially intriguing
since studies by different groups have identified centrin as
a permanent component of the pericentriolar matrix,
which is the main nucleating and anchoring site for
centrome-assembled microtubules. A good way for mammalian cells to control the number and orientation of
microtubules of the cytoplasmic microtubule complex
may be to influence and possibly modify the distribution
of their anchoring sites in the centrosome region. The
centrin array found in the pericentriolar matrix, whose
overall conformation can be altered by calcium in vitro
may have properties compatible with such a function
[Moudjou and Bornens, 1992; Paintrand et al., 1992;
Baron et al., 1994]. Whether physical links between
centrin containing filaments and microtubules, which are
Centrin in the Feeding Basket of Nassulinid Ciliates
particularly evident in the ciliate feeding basket, have
structural counterparts in mammalian centrosome remains to be determined. Biochemical and immunological
characterization of electron-dense cytopharyngeal material is in progress to explore this possibility.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. J. C. Roumagoux
for his kind help in the large-scale preparation of ciliate
cultures, Mrs M. Bérard and M. Dion for excellent
technical assitance, and Mr. J. L. Vincenot for the
photographic work.
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