Europ. J. Protistol. 39, 416–422 (2003)
© Urban & Fischer Verlag
http://www.urbanfischer.de/journals/ejp
Dynamin involvement in Paramecium phagocytosis
Jolanta Wiejak, Liliana Surmacz and Elzbieta Wyroba*
Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093 Warsaw, Poland; E-mail:e.wyroba@nencki.gov.pl
Received: 1 September 2003; 17 September 2003.
Accepted: 20 September 2003
The role of dynamin in Paramecium phagocytosis w as examined by quantitative evaluation of immunoblots and ultrastructural detection by immunogold labelling w ith specific anti-dynamin antibodies.
Western blot analysis w as performed on the subcellular fractions isolated from cells internalising latex
beads or exposed to pharmacological compounds that inhibit phagocytic activity. Upon onset of phagocytosis the dynamin level w as significantly increased by 30% after 5 min of bead uptake but it did not
differ from the control value follow ing 15 min exposure to latex beads. Inhibition of phagocytosis w ith lpropranolol (150 µM ) evoked a ~ 2-fold decrease in dynamin expression, w hereas the effect of trifluoroperazine (20 µM ) w as less pronounced (14% decrease). In electron microscopic studies dynamin w as localized at the phagosomal and cytopharyngeal membranes and in discoidal vesicles w hich are indispensable for phagosome formation. After cessation of phagocytic activity no immunogold labelling of dynamin w as detected in the majority of discoidal vesicles. These observations may indicate that dynamin
is involved in Paramecium phagocytosis at its initial stage.
Key w ords: Dynamin; Paramecium ; Phagocytosis; Digestive vacuoles; Propranolol; Trifluoroperazine.
Introduction
The dynamin family of large GTP-ases is involved in the fission process, at various membranes
during endocytosis and in the formation of transport vesicles (McNiven 1998; Hinshaw 2000).
There is a growing body of evidence that dynamin
also participates in phagocytosis. These data come
from studies on mammalian macrophages (Gold
et al. 1999; Di et al. 2003) – professional phagocytes possessing the unique ability to internalise
and eliminate pathogenic microorganisms (Gold et
al. 1999; Desjardins and Griffiths 2003). The first
report on the role of dynamin in phagocytosis concerned murine resident peritoneal macrophages in
which dynamin 2 was shown to be enriched on
early phagosomes (Gold et al. 1999). In cells expressing a dominant-negative mutant of dynamin 2
phagocytosis of particles was inhibited at the stage
* corresponding author
0932-4739/03/39/04-416 $ 15.00/0
of extension of membranes around them. O n the
other hand, Tse et al. (2003) observed that in a dynamin 1 dominant-negative mutant there was no
inhibitory effect on phagocytosis. They suggested
that the observation of Gold et al. (1999) was a result of a block in the delivery of endomembranes
to the cell surface. This effect is not observed in
cells expressing mutant dynamin 1 because its localisation is restricted only to the surface membrane, whereas dynamin 2 is present on both surface and intracellular membranes (Cao et al. 1998;
Tse et al. 2003). Recent studies confirm the suggestions that dynamin 2 – a ubiquitously expressed
dynamin isoform – (but not dynamin 1) is involved
in formation and/or movement of the vesicles
from intracellular organelles to the cell surface to
deliver membranes needed for the formation of the
phagocytic cup (Di et al. 2003). In RAW 264.7 cells
transiently transfected with mutant dynamin
Paramecium dynamin in phagocytosis
(K44A), phagocytosis of IgG-coated red blood
cells was inhibited by 85% (Tse et al. 2003). In
nonphagocytic cells, invasion by Trypanosoma
cruzi was completely abolished by overexpression
of a dominant negative mutant of dynamin
(Wilkowsky et al. 2002).
In unicellular organisms phagocytosis is a
source of nutrients, but there are no previous reports in the literature on dynamin function in
phagocytosis by ciliates. We have recently cloned
and sequenced the N-terminal GTP-ase domain of
Paramecium dynamin (Surmacz et al. 2001; Wiejak
and Wyroba 2002) sharing a higher homology to
mammalian dynamin 2 than to dynamin 1: the
identity of the deduced amino acid sequence
reached 61% and 57–58% , respectively (our unpublished results). We further demonstrated, using
antibodies against the C-terminal region of human
dynamin 2, that Paramecium dynamin was localised to the endosomes in which transferrin, a
marker of receptor-mediated endocytosis, had
been internalised (Surmacz et al. 2003). It was of
interest therefore to examine whether dynamin is
involved in phagocytic activity of ciliates.
Paramecium phagocytosis was described in detail
by Allen and coworkers and particular stages of
phagosome (called digestive vacuoles – DV – in ciliates) formation were characterised. DVs differ in
their size, shape, membrane type and coat, pH and
enzymatic composition (Fok and Allen 1982; Allen
and Fok 1984; 2000). They are formed due to fusion
of discoidal vesicles with cytopharyngeal membrane, quickly followed by fusion of acidosomes to
the DV (Allen 1974; Allen and Fok 2000). We show
evidence that dynamin is localised to this compartment as based on immunogold ultrastructural studies with a specific antibody against the amino-terminal peptide of the cloned GTP-ase domain. By
using cell fractionation, SDS-PAGE and quantitative Western blot analysis we also demonstrate that
expression levels of dynamin may be correlated
with the phagocytic activity of Paramecium.
417
Cells were pretreated for 15 min with l-propranolol
(150 µM) or trifluoroperazine (20 µM) to inhibit phagocytic activity (Wyroba 1989a; 1989b; Surmacz et al.
2001), or phagocytosis was induced by feeding with
polystyrene monodispersed latex beads (0.95 µm in diameter) for 5 or 15 min. Efficiency of phagocytic activity and its inhibition by these pharmacological compounds was monitored prior to each experiment by DV
score (Wyroba 1991).
Equal aliquots of control and treated cells were transferred to 2 volumes of ice-cold homogenization solution (50 mM Tris-HCl, pH 7.4, 10 mM EDTA) containing protease inhibitors: pepstatin A, aprotinin, leupeptin (2 µg/ml each) and PMSF (4 µg/ml). Homogenization, fractionation, SDS-PAGE and Western blotting were performed as described previously (Surmacz
et al. 2001). Blots were stained in 0.5% Ponceau Red in
3% trichloroacetic acid before immunodetection.
Immunodetection and densitometric analysis
Immunodetection was performed using primary antibodies (Ab) specific for Paramecium dynamin (1:500,
overnight at 4 °C) followed by incubation with the
HRP-conjugated anti-rabbit IgG for 2 h (1:1000) and
processing for chemiluminescent detection with West
Pico (Pierce). Control experiments with pre-immuneserum or without the primary Ab produced no immunoreactive bands.
The specific rabbit primary antibodies (SigmaGenosys) used as antiserum were directed against the
synthetic peptide sequence spanning amino acids
residues 100–113 (DLPGITKNPVGDQ PC) of the
GTP-ase domain of Paramecium dynamin cloned by us
(Genbank Acc. # AF351193).
O ptical density readings for the dynamin immunoreactive bands (from the blots with an equal protein loading of 20 µg per lane) were determined using a computer-assisted Image Q uant program (Molecular Dynamics, Sunnyvale, USA). Protein determination was performed as described by Wiejak et al. (2001). Results are
the mean values from 3–10 experiments. Data were expressed as mean % of control ± SD (according to Student’s t-test).
Electron microscopy
M aterials and methods
Fractionation and Western blot analysis
Paramecium octaurelia cells of strain 299s were
grown in axenic medium (Soldo et al. 1966). 5-day-old
cultures were harvested and prepared as previously described (Wyroba 1991).
Immunolocalisation studies were carried out using
the post-embedding method described by Wiejak et al.
(2002). Detection was performed with the same antidynamin Ab (1: 250, overnight at RT) as in immunoblotting followed by incubation with the antirabbit IgG (1: 20) conjugated with colloidal gold (5 nm)
for 4 h at RT. In control experiments the primary Ab
was omitted. The sections were observed in JEM 1200
EX electron microscope.
418
J. Wiejak, L. Surmacz and E. Wyroba
Results
Dynamin function in Paramecium phagocytosis
was studied by analysis of the protein level and its
subcellular localisation during induction and inhibition of phagocytosis. Specific antibodies directed
against the synthetic peptide derived from the
cloned GTP-ase domain of Paramecium dynamin
were used.
Western blot analysis revealed a major immunoreactive band of ~116 kDa in a cytosolic (S2)
fraction (Fig. 1B, lane 1) which was obtained as a
result of cell fractionation at 100 000 g (Fig. 1A,
lane 1). We followed the expression levels of
Paramecium dynamin as analysed by immunoblotting of the subcellular fractions isolated
from the ciliates either underoging phagocytosis
(Fig. 1 A, B, lanes 2–3) or blocked in this process
(Fig.1 A, B, lanes 4–5).
Q uantitative evaluation of Western blots revealed that upon onset of latex phagocytosis there
was a significant (p < 0.05) increase in dynamin ex-
Fig. 1. Expression levels of dynamin homologue in Paramecium during modulation of phagocytosis as analysed by
Western blotting with dynamin-specific antibody. The dynamin level was quantified after induction of phagocytosis
by latex beads for 5 min (lane 2) and 15 min (lane 3) and inhibition by l-propranolol (lane 4) and TFP (lane 5) in comparison to the control (lane 1). A. SDS-PAGE of representative protein separations after staining with Ponceau Red.
B. Western blots of corresponding electrophoretic separations shown in A. Molecular mass markers (kDa) are shown
at the left. C. Q uantitative densitometric analysis of dynamin. Each lane contained the same amount of protein (20
µg). Data were expressed as mean % of control ± SD (p < 0.05). Lane 2 and 3: n = 6, lane 4: n = 4, lane 5: n = 10.
Paramecium dynamin in phagocytosis
pression reaching 30% above the control after
5 min of incubation (Fig. 1C, lane 2, cf. lane 1).
After longer incubation with beads (15 min) dynamin level did not differ from that observed in the
untreated cells (Fig. 1C, lane 3).
Phagocytosis inhibition – evoked by a 15 min
pretreatment of the cells with 150 µM of the betablocker l-propranolol – resulted in a 55% decrease
in dynamin level in comparison to untreated cells
(Fig. 1C, lane 4). In the cytosolic fractions isolated
from ciliates previously exposed to 25 µM TFP for
15 min to completely block phagocytic activity, a
14% decrease in dynamin expression was observed
(Fig. 1C, lane 5, p < 0.05).
Immunoelectron microscopic studies using immunogold detection (5 nm) revealed the presence
of dynamin in untreated cells at the cytopharyngeal membrane and discoidal vesicles (Fig. 2A).
During internalisation of latex beads Au label was
also associated with membranes surrounding the
beads (Fig. 2C–E, G) with which discoidal vesicles
containing dynamin were fusing (Fig. 2C). Labelling of these membranes was much more pronounced after 5 min of bead internalisation
(Fig. 2D) than after 15 min (Fig. 2E), whereas cytopharyngeal membranes seem not to differ in
gold label during the process of latex phagocytosis
(Fig. 2B and Fig. 2F). Discoidal vesicles labelled
with gold were observed also after 15 min of internalisation (Fig. 2G), though not fusing with the
phagosome membrane as observed at the beginning of uptake (Fig. 2C).
There was no dynamin in most of the discoidal
vesicles after pretreatment with l-propranolol
(Fig. 2I) and TFP (Fig. 2J), but gold label was observed along the cytopharyngeal membrane in both
cases (Fig. 2H and Fig. 2K, respectively). No gold
particles were observed in control samples from
which primary antibodies were omitted (Fig. 2L).
Discussion
In this study expression and localisation of
Paramecium dynamin was followed during phagocytosis utilising specific antibody against the
amino-terminal peptide of the cloned GTP-ase domain. We detected a dynamin immunoanalogue of
~116 kDa in Western blot analysis and performed
quantitative analysis of its expression during induction and inhibition of phagocytic activity of
Paramecium cells.
419
The increase in dynamin level after 5 min of incubation with latex beads may suggest that dynamin participates mainly at the first stage of
phagocytosis. These observations are in agreement
with results of Gold et al. (1999), who localised dynamin in forming phagocytic cups of rat
macrophages. Upon overexpression of GFP-dynamin 2, a motile dynamin pool was demonstrated
in RAW 264.7 macrophages by Di and coworkers
(2003). A fraction of this pool migrated toward the
sites of particle internalisation and transiently associated with the phagosomal cup (Di et al. 2003).
During the phagocytic cycle Paramecium food
vacuoles pass through at least four stages: up to
6 min DV of stage I are formed and their membranes are derived from discoidal vesicles (Fok
et al. 1982; Allen et al. 2000). Consistent with this
observation, we detected dynamin in the membrane surrounding internalised latex particles and
in discoidal vesicles fusing with it within the first
5 min of the phagocytic process in Paramecium.
Discoidal vesicles are largely formed from retrieved
DV-I and from the spent vacuole membrane at the
cytoproct, though the origin of them has not been
completely resolved (Allen 1974; 2000). During the
membrane retrieval from DV-I, a pool of membrane tubules is formed and it is later remodelled
into the discoidal vesicles that recycle back to the
cytopharynx (Allen and Fok 2000). In fact, in some
micrographs we have observed membrane tubules
with dynamin gold label (data not shown). O ur results suggest that dynamin is indispensable only for
engulfing the latex particle and it is next moved to a
tubular compartment presumably destined for
membrane retrieval, though the precise mechanism
of this process is not known. Therefore after 15 min
of bead uptake the density of dynamin labelling, as
revealed by immunogold detection, is less pronounced than at the onset of the phagocytosis.
During inhibition of phagocytic activity by
l-propranolol and TFP, the dynamin level was significantly decreased in comparison to the control
and the majority of discoidal vesicle profiles were
devoid of gold label. However, dynamin is still
found on the cytopharyngeal membrane suggesting that only the process of membrane retrieval
may be inhibited. It is tempting to speculate that,
under these conditions, the dynamin pool is not
retrieved via incorporation from the spent vacuoles into discoidal vesicles. This may resemble the
observations on macrophages discussed in the Introduction (Di et al. 2003; Tse et al. 2003) that in a
dynamin 2-negative mutant delivery of endomem-
420
J. Wiejak, L. Surmacz and E. Wyroba
Fig. 2. Electron microscopic immunolocalisation of dynamin in the Paramecium phagocytic compartment. Micrographs of untreated cells (A), during latex (marked with white L) internalisation for 5 (B–D) and 15 min (E–G) or after
blocking of phagocytic activity by l-propranolol (H–I) and TFP (J–K). No gold particles are seen where the primary
antibody was omitted (L). Arrows indicate the presence of dynamin in discoidal vesicles. Many latex beads are enclosed
in a single DV, which has a total diameter of about 6.7 µm. Bar represents 100 nm, except G, where it equals 200 nm.
branes to the cell surface was inhibited, thus blocking phagocytosis.
These two pharmacological compounds differ in
their action but the molecular mechanism of their
effect on phagocytosis remains obscure.
1. Trifluoperazine – a calmodulin antagonist –
inhibited the process of membrane retrieval (Fok
et al. 1985) and completely blocked digestive vacuole formation in P. caudatum (Fok et al. 1985)
and P. aurelia (Surmacz et al. 2001). It was also
Paramecium dynamin in phagocytosis
shown to evoke changes in swimming pattern and
inhibit secretion in Paramecium (Garofalo et al.
1983; O tter et al. 1984). The effect of TFP may be
due to calcium/calmodulin signal regulating formation of food vacuoles (Gonda et al. 2000). Both
calmodulin and calcium-dependent calmodulinbinding membrane proteins were found in Paramecium (Walter and Schultz 1981; Chan et al. 1999).
2. The effect of l-propranolol on phagocytosis
may be related to the presence of a Paramecium
homologue of the beta-adrenergic-receptor
evolved as a nutrient receptor in this cell (Wiejak
et al. 2002). Signalling through heterotrimeric
G proteins is also required for phagocytosis in
Dictyostelium discoideum, mainly for regulating
the actin cytoskeleton (Peracino et al. 1998). It is in
agreement with our previous results that actin may
be a primary target of pharmacological agents used
to inhibit Paramecium phagocytosis (Surmacz
et al. 2001). Interestingly, it was postulated that dynamin may also contribute to the assembly and remodelling of actin at the phagosomal cup (Tse et al.
2003). Recent data indicate that dynamin may
function at the interface between the membranes
and filamentous actin forming a polymeric contractile scaffold. Such a mode of its action is enabled by dynamin’s ability to form supramolecular
structures and interaction with actin-binding proteins (O rth and Mc Niven 2003). Up to now no
data are available on this problem in ciliates.
Ciliates have proved to be a useful model to follow different membrane events (Allen 1974;
Wyroba 1987; Chan et al. 1999), and continuing
study of dynamin in ciliates should prove valuable.
Acknowledgments: This work was supported by the
Committee for the Scientific Research (KBN) Grant
No. 3 PO 4C 095 22 and the statutory funds to the
Nencki Institute of Experimental Biology.
The technical assistance in electron microscopic
studies of Mr. H. Bilski, Mr. K. Krawczyk and Mr. R.
Strzalkowski is acknowledged.
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