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Dynamin involvement in Paramecium phagocytosis

2003, European Journal of Protistology

The role of dynamin in Paramecium phagocytosis was examined by quantitative evaluation of immunoblots and ultrastructural detection by immunogold labelling with specific anti-dynamin antibodies. Western blot analysis was 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 was significantly increased by 30% after 5 min of bead uptake but it did not differ from the control value following 15 min exposure to latex beads. Inhibition of phagocytosis with lpropranolol (150 µM) evoked a ~2-fold decrease in dynamin expression, whereas the effect of trifluoroperazine (20 µM) was less pronounced (14% decrease). In electron microscopic studies dynamin was localized at the phagosomal and cytopharyngeal membranes and in discoidal vesicles which are indispensable for phagosome formation. After cessation of phagocytic activity no immunogold labelling of dynamin was detected in the majority of discoidal vesicles. These observations may indicate that dynamin is involved in Paramecium phagocytosis at its initial stage.

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. 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