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ScienceDirect European Journal of Protistology 50 (2014) 248–257
Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes Silke Ammermann, Helmut Hillebrand, Erhard Rhiel∗ Planktologie, ICBM, Carl-von-Ossietzky-Universität Oldenburg, P.O.B. 2503, D-26129 Oldenburg, Germany Received 6 January 2014; received in revised form 25 February 2014; accepted 27 February 2014 Available online 18 March 2014
Abstract Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6 M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6 M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19 kDa and two additional polypeptides of 23 and 26 kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Ejectisome; IgG fractions; Prasinophyte; Pyramimonas grossii; Reconstitution experiments; Transmission electron microscopy
Introduction Prasinophytes comprise a heterogeneous group of primitive, unicellular green algae. Beside motile, flagellated, scale-bearing genera, non-motile coccoid genera without body scales have been described. Phylogenetic analyses of SSU rRNA sequences of 13 prasinophyte species representing eight genera revealed four independent clades with the phylogenetic position of Pycnococcus provasoli being unresolved (Nakayama et al. 1998).
∗ Corresponding
author. Tel.: +49 441 798 3389; fax: +49 441 798 3151. E-mail address: [email protected] (E. Rhiel).
http://dx.doi.org/10.1016/j.ejop.2014.02.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
Based on ultrastructural data, six subgenera are actually believed to constitute the genus Pyramimonas, i.e. Pyramimonas McFadden, Vestigifera McFadden, Trichocystis McFadden, Punctatae McFadden, Hexactis Hori, Moestrup et Hoffman, and Macrura Hori et Moestrup (Hori et al. 1995; McFadden et al. 1986, 1987; Pienaar and Sym 2002). A common feature of species of the subgenera Trichocystis and Hexactis is that they harbor projectile organelles, i.e. ejectisomes which closely resemble R-bodies of several bacteria as both are built up by single coiled ribbons composed of protein. More complex ejectisomes have been described for cryptophytes and kathablepharids (Clay and Kugrens 1999a,b; Kugrens et al. 1994; Okamoto and Inouye 2006; Okamoto et al. 2009; Santore 1985; Vørs 1992; Wehrmeyer 1970; and references cited therein).
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The ejectisomes of the prasinophyte Pyramimonas grossii have been first described by Manton (1969). Non-discharged ejectisomes were located at the cells anterior ends and consisted of coiled ribbons which measured 5 nm in thickness and 500 nm in width. After discharge, the ejectisomes formed left-handed, single-coiled hollow tubes which measured up to 35 m in length and 100 nm in width. Morrall and Greenwood (1980) investigated the ejectisomes of Pyramimonas parkeae and found that non-discharged coils exhibited ribbon-like layers with thicknesses of 4.9–5.5 nm. Each ribbon consisted of highly ordered subunits which were obvious as light and dark substructures. The authors measured subunit intervals of 2.8–3.3 nm for both, non-discharged and discharged ejectisomes. These data have been confirmed in a recent study (Rhiel et al. 2013). In a previous study, the ejectisomes of three cryptophyte species have been isolated and the first data on the constituting polypeptides were presented. Furthermore, the chemical stability of cryptophycean ejectisome fragments was investigated, and they were successfully isolated by Percoll gradient centrifugation. Afterwards they were disassembled and reconstituted by treatment with guanidine hydrochloride followed by dialysis. Finally, an antiserum against the polypeptides was raised which immuno-labelled discharged ejectisomes of the cryptophytes (Ammermann et al. 2013). Investigations on the stability of the ejectisomes of Pyramimonas grossii have not been performed, and Percoll gradient centrifugation was not applied in our previous study (Rhiel et al. 2013). Additionally, no antiserum had been raised against the constituting polypeptides, and no dissociation/reconstitution experiments had been performed. Therefore, the current work focuses on the following three topics. First, the protocol for the isolation of discharged ejectisomes of Pyramimonas grossii was modified, and Percoll density gradient centrifugation and pH treatments were applied. Second, experiments with guanidine hydrochloride were performed to see if ejectisomes of Pyramimonas grossii could be successfully disintegrated and afterwards reassembled. Third, an antiserum was raised against a fraction enriched in ejectisome fragments of Pyramimonas grossii and used in immunogold-labelling electron microscopy experiments.
Material and Methods Cultures and growth conditions The prasinophyte Pyramimonas grossii was obtained from the Scandinavian Culture Collection of Algae and Protozoa (SCCAP, University of Copenhagen, Denmark, strain-no. K0253) and was cultured as described by Rhiel et al. (2013).
Isolation of ejectisome fragments and sodium dodecyl sulfate (SDS)-solubilisation The isolation and SDS-solubilisation of ejectisome fragments followed the protocols outlined by Rhiel et al. (2013)
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with some modifications. The cells (200–250 ml culture volume, 7–10 d culture age) were harvested by centrifugation at 3220 g for 15 min in an Eppendorf 5810R refrigerating centrifuge equipped with an A-4-62 swinging bucket rotor (Eppendorf, Hamburg, Germany). The cells were resuspended in 9 ml buffer (5 mM Tris/HCl, pH 7.5) and lysed by adding 1 ml of 10% (v/v) Triton X-100 (1% final concentration). The insoluble material consisting of contaminating bacteria, starch grains, body scales, and ejectisome fragments was pelleted from the supernatant by centrifugation at 3220 g for 15 min, resuspended in 4 ml buffer and again centrifuged. After incubation in 2–4 ml of 5% (w/v) EDTA, pH 7.1 for 4 h at room temperature, the body scales were dissolved and removed by two subsequent washing/centrifugation steps (washing in distilled water). The resulting ejectisome fragment-enriched fractions were kept either frozen or immediately used for experiments. For SDS solubilisation, ejectisome fragment-enriched fractions were pelleted by centrifugation and resuspended in 200–400 l distilled water. Fifty to one hundred (50–100) l aliquots of 10% (w/v) SDS were added (2% final concentration). Then, the mixtures were centrifuged (Heraeus Biofuge pico table top centrifuge for 10 min at 16000 g, Kendro Laboratory Products GmbH, Langenselbold, Germany). The SDS-insoluble pellets were subjected to two additional washing/centrifugation steps with distilled water before they were used for negative staining transmission electron microscopy and SDS-PAGE, whereas the supernatants were subjected to SDS-PAGE.
Percoll density gradient centrifugation Percoll density gradient centrifugation followed the protocol outlined by Ammermann et al. (2013). An ejectisome fragment-enriched fraction was diluted with 6 ml buffer consisting of 5 mM Tris/HCl pH 7.5 and 150 mM NaCl. Then, a mixture of 4.5 ml Percoll (Sigma, Munich, Germany) with 0.5 ml of 1.5 M NaCl was added. The solution was filled into a re-sealable polyallomer ultracentrifugation tube (Science Services, Munich, Germany), and the tube was sealed and centrifugated in a Beckman L8-55M ultracentrifuge (Beckman Coulter, Munich, Germany) equipped with a NVT65 rotor for 1 h at 27400 g and 20 ◦ C. The ejectisome fragmentcontaining bands were collected using needle tipped syringes, diluted with distilled water, and subjected to two washing/centrifugation steps (Heraeus Biofuge pico). Then, the fractions were used for SDS-PAGE.
pH treatment Five hundred (500) l of an ejectisome fragment-enriched fraction was pelleted by centrifugation (Heraeus Biofuge pico, 10 min, 16000 g) and incubated either in 500 l of 100 mM Tris/HCl pH 2.5 or in 500 l of 100 mM Tris/HCl pH 10.8. Then, the mixture was centrifuged, and the pellet was
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resuspended in 200 l distilled water and used for negative staining transmission electron microscopy and SDS-PAGE; whereas, the supernatant was used for SDS-PAGE.
GuHCl solubilisation/reconstitution experiments Ejectisome fragment-enriched fractions were pelleted by centrifugation (Heraeus Biofuge pico, 10 min, 16000 g) and incubated in 500–1000 l of 6 M GuHCl for 10–15 min at room temperature. After centrifugation of the clarified solutions for 20 min (Heraeus Biofuge pico), the GuHCl-insoluble material was pelleted. The clear supernatants were then dialysed against distilled water for approximately 2–4 h at room temperature without stirring, using Membra-Cel dialysis membranes (Serva, Heidelberg, Germany, MWCO 3500). Re-aggregation, visible as opaquewhite flocs, occurred during that time. The reconstituted ejectisome-like filaments were pelleted by centrifugation and used for negative staining transmission electron microscopy and SDS-PAGE; whereas, the GuHCl-insoluble pellets were subjected to two additional washing/centrifugation steps with distilled water before they were used for negative staining transmission electron microscopy and SDS-PAGE.
SDS-PAGE, Western-immunoblotting, and documentation SDS-PAGE was performed as described by Bathke et al. (1999). Generally, 50–100 l aliquots of the various fractions mentioned above were mixed with loading buffer, heat-denatured, and loaded onto 17.5% polyacrylamide gels using the buffer system of Laemmli (1970). Electrophoresis and Coomassie-staining of gels was performed as described by Ammermann et al. (2013). Ejectisome fragment-enriched fractions of Pyramimonas grossii were tested for cross-reactivity with the antiserum raised against them. They were subjected to SDS-PAGE followed by Western-immunoblotting according to Towbin et al. (1979). The antiserum was used in dilutions of up to 1:2000. Coomassie-stained gels, Western-immunoblots, and Percoll density gradient tubes were documented with an Olympus C3030 Zoom digital still CCD camera.
Antiserum production and selective isolation of IgG fractions The rabbit derived antiserum directed against an ejectisome fragment-enriched fraction was ordered commercially (BioScience, Göttingen, Germany). Preimmunserum was taken before the first immunisation. Four weeks after the final immunisation, the antiserum was obtained. For the isolation of distinct IgG fractions, ejectisome fragment-enriched fractions were subjected to SDS-PAGE followed by Western blotting. Then, the blot was stained with Ponceau S to visualise the polypeptides. Distinct stripes of the blot, comprising
either the three polypeptides with relative molecular weights of 12.5, 16, and 19 kDa (fraction termed #A) or those with relative molecular weights of 23 and 26 kDa (fraction termed #B) were cut out, blocked with 1% (w/v) BSA in TBS, and incubated with either preimmunserum (PS) or antiserum (AS) for 1 h. Then, the nitrocellulose strips were washed in TBS, and the IgG fractions absorbed to these polypeptides were desorbed by addition of 200–400 l of 100 mM Tris/HCl, pH 2.5 to the nitrocellulose stripes. Finally, 20–40 l 1 M Tris/HCl pH 7.5 was added to these IgG fractions, resulting in the fractions IgG-#A absorbed to preimmunserum (further named IgG-#A-PS), IgG-#A absorbed to antiserum (further named IgG-#A-AS), IgG-#B absorbed to preimmunserum (further named IgG-#B-PS), and IgG-#B absorbed to antiserum (further named IgG-#B-AS).
Negative staining transmission electron microscopy For negative staining transmission electron microscopy, 50 l droplets of the ejectisome fragment-enriched fractions, of the SDS-insoluble pellets, of the pH 2.5 treated ejectisome fragments, of reconstituted ejectisome-like filament fractions, and of the GuHCl-insoluble fractions were used. In some cases, prior to negative staining, the fractions were diluted in distilled water. The particles were allowed to adsorb for 5 min onto Formvar-coated grids. Then, the grids were stained for 1 min with 1% (w/v) uranyl acetate and washed in two drops of distilled water. Samples were examined with a Zeiss EM 902A electron microscope (Zeiss, Oberkochen, Germany) operated at 80 kV. Digitized images were taken with a 1k Proscan High Speed SSCCD camera (Proscan elektronische Systeme GmbH, Lagerlechfeld, Germany) operated by the iTEM Five software (Olympus Soft Imaging System GmbH, Münster, Germany).
Immuno-electron microscopy For immuno-electron microscopy, ejectisome fragmentenriched fractions were allowed to adsorb for 5 to 10 min onto Formvar-coated grids. Then, the grids were floated for 1 h onto 50 l drops of 1% of BSA dissolved in TBS, incubated for 1 h on 50 l drops of either preimmunserum, or antiserum, or IgG fractions. The preimmunserum and the antiserum were diluted 1:200 in TBS containing 0.1% BSA; whereas, the IgG fractions were used non-diluted. Then, the grids were washed in 10 drops of TBS and incubated for 1 h in gold-labelled goat-anti rabbit IgG conjugate (British Biocell International, Cardiff, UK, diluted 1:100 in TBS containing 1% BSA, 15 nm gold particles). After washing with 10 drops of TBS, the grids were stained with uranyl acetate, washed with two drops of distilled water, and used for transmission electron microscopy.
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Quantification of the immuno-label and statistics
Percoll density gradient centrifugation
The amounts of gold particles positioned on or tightly to ejectisome fragments were enumerated on digitized images. Then, the lengths of the fragments were measured, and the amounts of gold particles per 1 m fragment length were quantified. We performed a univariate Analysis of Variance (ANOVA) testing for treatment effects on the amount of gold particles which had to be log-transformed to achieve homogeneity of variances. Significant differences between treatment levels were detected using Tukey’s HSD posthoc test. All statistics were done using the R software package (R Development Core Team 2011).
Percoll density gradient centrifugation resulted in either one area or two areas of white, flocculent material (Fig. 1C). When subjected to SDS-PAGE, both showed the same banding pattern as the fraction prior to the density gradient centrifugation (Fig. 2, lanes 8 and 9 vs. lane 7).
Results Isolation of ejectisome fragments and SDS-solubilisation Fractions highly enriched in ejectisome fragments were achieved by following the protocol outlined by Rhiel et al. (2013) with some modifications. Negative staining transmission electron microscopy of those fractions mainly showed ejectisome fragments of various lengths (Figs 3A, 4A), and Coomassie-stained gels revealed three dominating polypeptides of 12.5, 16, and 19 kDa (marked with arrows in Fig. 1A, and see also Fig. 2, lanes 1, 4, 7, and 10). Two additional polypeptides of 23 and 26 kDa stained somewhat weaker (marked with arrowheads in Fig. 1A). Furthermore, a faint smear was registered along the entire lane. SDSsolubilisation of those fractions gave rise to an almost identical polypeptide pattern of the SDS-soluble fraction (Fig. 2, lane 2); whereas, the SDS-insoluble material showed a molecular polypeptide of approximately 80 kDa (marked with an asterisks in Figs 1A, 2A, lane 3). Transmission electron microscopy revealed that the SDS-insoluble material mainly consisted of bacterial shells, other cell debris, and starch grains (not shown). The ejectisome fragment-enriched fractions were thus judged to be appropriate starting material for further experiments.
pH treatment Incubation of ejectisome fragment-enriched fractions in 100 mM Tris/HCl pH 2.5 did not result in solubilisation or detachment of loosely bound polypeptides. The ejectisome fragments could be pelleted by centrifugation, and their structure resembled the one registered for those prior to pH 2.5 treatment (Fig. 3A vs. B). Supporting this observation, SDS-PAGE demonstrated that all protein was pelleted by centrifugation (Fig. 2, lane 6) and that no protein could be detected in the resulting supernatant (Fig. 2, lane 5). Similar results were obtained for the pH 10.8 treatment (not shown).
GuHCl-treatment and reconstitution Incubation of pelleted ejectisome fragment-enriched fractions in 6 M GuHCl resulted in clarifying. SDS-PAGE of the GuHCl-insoluble material obtained after centrifugation did show minor amounts of polypeptides (Fig. 2, lane 12), and negative staining transmission electron microscopy revealed minor amounts of ejectisome fragments which had not been solubilised (Fig. 4B). Dialysis of the GuHCl solubilised, clarified supernatants resulted in the appearance of fine white flocs in the dialysis tubes which could be concentrated by centrifugation. The polypeptide pattern of these flocs matched the one registered for ejectisome fragments when subjected to SDS-PAGE (Fig. 2, lane 11). Negative staining electron microscopy of these flocs showed larger and smaller filaments (Fig. 4C). Widths of 133 ± 22 nm (n = 26) and 27.1 ± 7.6 nm (n = 36) were measured for the larger and smaller filaments, respectively. The larger filaments seemed to be composed of the smaller ones, as their ends often split up into several smaller filaments indicating a non-proper reassembly (arrows in Fig. 4C, and Fig. 4D). The smaller filaments were often spirally twisted (Fig. 4E).
Antiserum, IgG fractions, and immuno-electron microscopy Western-immunoblotting revealed that the antiserum against an ejectisome fragment-enriched fraction weakly cross-reacted with the 12.5–19 kDa, the 23 kDa, and the 26 kDa polypeptides of Pyramimonas grossii (Fig. 1B, lane marked “AS”). Rather, the antiserum labelled polypeptides with relative molecular weights of approximately 80, 66, 50, 36, and 8 kDa. No immune-reaction was observed using the preimmunserum (Fig. 1B, lane marked “PS”). Immunoelectron microscopy using either the preimmunserum or the antiserum also did not result in significant differences of gold particles per m filament length (Table 1). By using IgG fractions of the antiserum and preimmunserum however, significant differences in the amounts of gold particles per m filament length were registered (Fig. 5). Thus, a 3-fold higher label was registered for IgG-#A-AS in comparison to IgG#A-PS; whereas, IgG-#B-AS gave rise to a 6-fold higher label than IgG-#B-PS (Table 1). Treatment effects on the amount of gold particles per m filament length were highly significant (ANOVA, F(5;251) = 87.67, p < 0.0001). All pairwise comparisons were significant (Tukey’s HSD, p < 0.05), with the exception of IgG-PS-3B versus IgG-# PS-3B, and preimmunserum versus antiserum.
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Fig. 1. (A) SDS-PAGE of an ejectisome fragment-enriched fraction. The polypeptides marked with asterisk, arrows, and arrowheads are described in the Result section. The relative molecular masses of the marker proteins (kDa) are indicated on the left. (B) shows lanes of a gel after Western blotting and immuno-decoration with either the preimmunserum (PS) or the antiserum (AS) The brackets marked “#A” and “#B” indicate the blotted polypeptides with relative molecular weights of 12.5, 16, and 19 kDa (fraction #A) and those with relative molecular weights of 23 and 26 kDa (fraction #B), used for “IgG fishing”. For better display of the bands, the digitized pictures were adjusted for brightness and contrast. (C) Ultracentrifugation tubes after Percoll density gradient centrifugation showing either one or two bands enriched in ejectisome fragments.
Table 1. Compilation of the results obtained by immuno-electron microscopy, showing the amounts of gold particles per 1 m fragment length (means ± SD) being attached to ejectisome fragments of Pyramimonas grossii. Treatment
Gold particles per 1 m fragment length
Preimmunserum Antiserum IgG-#A-PS IgG-#A-AS IgG-#B-PS IgG-#B-AS
2.98 2.78 1.32 4.78 1.79 11.87
± ± ± ± ± ±
1.70 1.23 0.92 2.42 0.94 5.92
Discussion
SDS polyacrylamide gels resulted in Coomassie-stained gels, which showed the polypeptides of SDS-solubilised ejectisome fragments more clearly. The deviations measured for the relative molecular weights of the three dominating polypeptides (Rhiel et al. 2013) most probably result from the higher polyacrylamide concentration used. The observed polypeptide pattern deviated from those registered for the ejectisomes of limnic and marine cryptophytes (Ammermann et al. 2013; Yamagishi et al. 2012) and for R-bodies of the endosymbiotic bacterium Caedibacter taeniospiralis (Heruth et al. 1994; Kanabrocki et al. 1986), respectively. Actually, no further data are available for polypeptides constituting the ejectisomes of prasinophytes, such as Pyramimonas parkeae, P. pseudoparkeae, P. cirolanae, or P. virginica which are known to harbor ejectisomes (Hori et al. 1995; McFadden et al. 1986).
Isolation of ejectisome fragments and SDS-solubilisation Significant improvements were achieved by minor modifications of the previous isolation protocol (Rhiel et al. 2013). Thus, less culture volumes, younger cultures, shorter periods of EDTA-treatment, and the use of 17.5%
pH treatment and Percoll density gradient centrifugation Incubation of ejectisome fragments in either 100 mM Tris/HCl pH 2.5 or 100 mM Tris/HCl pH 10.8 did not
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Fig. 2. SDS-PAGE of fractions obtained during SDS-solubilisation (lanes 1–3), during pH 2.5 treatment (lanes 4–6), during Percoll gradient centrifugation (lanes 7–9), and during GuHCl dissociation/reconstitution experiments (lanes 10–12). Lanes 1, 4, 7, and 10 show ejectisome fragment-enriched fractions prior to the different treatments. Lane 2: supernatant after SDS-solubilisation; lane 3: SDS-insoluble pellet. The supernatant after pH 2.5 treatment and the resulting pellet are shown in lanes 5 and 6. The upper band and the lower band after Percoll gradient centrifugation are shown in lanes 8 and 9. Lane 11 shows the flocs obtained from the GuHCl-soluble fraction after dialysis against distilled water. The GuHCl-insoluble fraction is shown in lane 12.The relative molecular masses of the marker proteins (kDa) are indicated on the left and in the middle. For better display of the bands, the digitized pictures were adjusted for brightness and contrast.
Fig. 3. Micrographs of ejectisome fragment-enriched fractions prior to pH 2.5 treatment (A) and after pH 2.5 treatment (B) after negative staining. The structure of ejectisome fragments after pH 2.5 resembled the one registered for those prior to pH 2.5 treatment. Scale bars are 2 m.
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Fig. 4. Micrographs obtained by negative staining electron microscopy. In (A), an ejectisome fragment-enriched fraction is shown. A micrograph of the GuHCl-insoluble fraction obtained after centrifugation and washing is shown in (B). An overview and detailed views of reconstituted ejectisome-like filaments after GuHCl solubilisation and dialysis are given in (C), (D), and (E). Note that the larger filaments seemed to be composed of smaller ones as their ends often split up into several smaller filaments indicating a non-proper reassembly (arrows in C, and D showing the end of a filament). The smaller filaments were often spirally twisted (arrows in E). Scale bars are 2 m in A, B, and C, 500 nm in D, and 200 nm in E.
result in the dissociation of ejectisome fragments or loss of probably loosely attached polypeptides. Thus, the fragments obtained after centrifugation remained intact as visualized by negative staining electron microscopy, and no polypeptides were detected when the supernatants after pH 2.5 or pH 10.8 treatment were subjected to SDS-PAGE. No improvement but rather the same banding pattern was observed after Percoll density gradient centrifugation. Percoll density gradient centrifugation improved the purity of ejectisome fractions of cryptophytes significantly. A major disadvantage with respect to negative staining electron microscopy, however, was the finding that Percoll particles were still present, even after additional washing steps (Ammermann et al. 2013).
GuHCl-treatment and reconstitution Similar to the results obtained for the ejectisomes of cryptophytes (Ammermann et al. 2013; Rhiel and Westermann 2012), those of the prasinophyte Pyramimonas grossii did not dissolve in the presence of Triton X-100 and resist 8 M urea treatment (not shown). The ejectisomes of Pyramimonas grossii differ from those of cryptophytes in that they were easily dissolved in the presence of SDS (Rhiel et al. 2013; current data). Dissociation of ejectisome fragments was achieved solely by 6 M GuHCl in both cryptophytes and Pyramimonas grossii. As observed for cryptophytes (Ammermann et al. 2013), the rather slow removal of GuHCl
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Fig. 5. Micrographs of isolated, negative-stained ejectisome fragments of Pyramimonas grossii immunogold-labelled with either the IgG fractions of the preimmunserum (A, C) or antiserum (B, D) absorbed to either the three polypeptides with relative molecular weights of 23 and 26 kDa (fraction #B) or to those with relative molecular weights of 12.5, 16, and 19 kDa (fraction #A). Significant differences in labelling were registered for the IgG fractions gained from the antiserum IgG-#A-AS (D) and IgG-#B-AS (B) in comparison to the IgG fractions gained from the preimmunserum IgG-#A-PS (C) and IgG-#B-PS (A). For further details see Materials and Methods, and Table 1. Scale bars are 500 nm.
by dialysis against distilled water without stirring caused reaggregation into ejectisome-like filaments in Pyramimonas grossii. The fact that the polypeptide patterns of ejectisome fraction-enriched fractions, of the supernatant obtained after SDS-solubilization, of the bands obtained by Percoll density gradient centrifugation, and of reconstituted, ejectisome-like filaments did not deviate significantly from one another strengthen the assumption that the polypeptides with relative molecular weights of 12.5–19, 23, and 26 kDa are major components of the ejectisomes of Pyramimonas grossii.
In this context, it is worth to be mentioned that reconstituted ejectisome-like filaments of cryptophytes form tube-like filaments which closely resemble native, discharged ejectisome fragments; whereas, reconstituted ejectisome-like filaments of Pyramimonas grossii do not. In cryptophytes, discharged ejectisomes form long tubes with bended tips. The tubes themselves result from bending of the margins of the ribbons. Similarly, reconstituted ejectisome-like filaments are more or less transversally curved or rolled-up until a tubular structure is reached (Ammermann et al. 2013).
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Discharged, native ejectisomes of prasinophytes, on the contrary, form spirally twisted tubes. The spiral twist is reflected by the overlaps visible at the surfaces of the tubes (Manton 1969). Ejectisome widths of up to 200 nm were measured when subjected to negative staining electron microscopy compared to smaller diameters of approximately 150 nm for those subjected to freeze-fracture or Cryo-transmission electron microscopy (Rhiel et al. 2013). Reconstituted ejectisome-like filaments of Pyramimonas grossii showed larger filaments with a mean width of 133 nm and smaller filaments of approximately 27 nm. The larger filaments did not show the spiral twist of native, unfurled ejectisomes. They rather seemed to be composed of several smaller filaments giving rise to frayed endings. Spiral twists were observed for the small-sized reconstituted filaments. Thus, the reassembly of polypeptides which constitute prasinophyte ejectisomes seems to be more complicated than the one registered for cryptophytes.
Antiserum, IgG fractions, and immuno-electron microscopy The antiserum raised against an ejectisome fragmentenriched fraction did not satisfy the expectations. In Western-immunoblots, it labeled high molecular weight polypeptides and weakly cross-reacted with the dominating polypeptides, and almost no differences were observed between preimmunserum and antiserum by immuno-electron microscopy. Obviously, contaminants which were still present in the fraction caused higher immune responses in rabbit than the ejectisome fragments. The approach of “IgG fishing” with stripes of nitrocellulose with either the 12.5–19 kDa or the 23 and 26 kDa polypeptides resulted in IgG fractions which significantly immuno-labelled ejectisome fragments. These stripes were cut from Western-blotted ejectisome fragment-enriched fractions after SDS-PAGE. A quite common procedure for the enrichment of immunoglobulins specific to defined antigenic polypeptides is affinity chromatography. In this case, the antigen is coupled to a column matrix, and the antiserum is allowed to pass the column. Following several washing steps, the immunoglobulins bound to the polypeptides are eluted from the column, concentrated, and used in further experiments. The enrichment of IgGs described in the current study is a somewhat modified version of this approach and was successfully applied in earlier studies on the photosynthetic apparatus of the marine cryptophyte Cryptomonas maculata (Rhiel et al. 1989).
Perspective In the past, research on extrusomes of non-ciliate protists mainly focused on their morphology whereas studies on trichocysts of ciliates such as Paramecium included analyses of their ultrastructure, the characterization of the constituting
polypeptides, and the isolation of the corresponding genes (Gautier et al. 1996; Hausmann 1978; Kugrens et al. 1994; Rosati and Modeo 2003; and references cited therein). Quite recently, the ejectisomes of cryptophytes have been investigated in detail, giving first data on their polypeptide composition and the genes encoding them (Ammermann et al. 2013; Yamagishi et al. 2012). To our knowledge, for prasinophytes no other literature is known that could be cited in this context except our previous publication (Rhiel et al. 2013). Further experiments such as N-terminal amino acid sequencing, MALDI-TOF analyses of trypsin- or chymotrypsin-digested polypeptides, and gene cloning are needed to gain further information about these fascinating projectile organelles.
Acknowledgements The authors express their gratitude to Dr. Paul Frost and Clay Prater (Trent University, Peterborough, Ontario, Canada) for critical reading of the manuscript.
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Kugrens, P., Lee, R.E., Corliss, J.O., 1994. Ultrastructure, biogenesis, and functions of extrusive organelles in selected non-ciliate protists. Protoplasma 181, 164–190. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of bacteriophage T7. Nature 227, 680–685. Manton, I., 1969. Tubular trichocysts in a species of Pyramimonas (P. grossii PARKE). Österreichische Botanische Zeitschrift 116, 378–392. McFadden, G.I., Hill, D.R.A., Wetherbee, R., 1986. A study of the genus Pyramimonas (Prasinophyceae) from south-eastern Australia. Nordic J. Bot. 6, 209–234. McFadden, G.I., Hill, D.R.A., Wetherbee, R., 1987. Electron microscopic observations on Pyramimonas olivacea N. Carter (Prasinophyceae, Chlorophyta). Phycologia 26, 322–327. Morrall, S., Greenwood, A.D., 1980. A comparison of the periodic substructure of the trichocysts of the Cryptophyceae and Prasinophyceae. Biosystems 12, 71–83. Nakayama, T., Marin, B., Kranz, H.D., Surek, B., Huss, V.A., Inouye, I., Melkonian, M., 1998. The basal position of scaly green flagellates among the green algae (Chlorophyta) is revealed by analyses of nuclear-encoded SSU rRNA sequences. Protist 149, 367–380. Okamoto, N., Inouye, I., 2006. Hatena arenicola gen. et sp. nov., a katablepharid undergoing probable plastid acquisition. Protist 157, 401–419. Okamoto, N., Chantangsi, C., Horák, A., Leander, B.S., Keeling, P.J., 2009. Molecular phylogeny and description of the novel katablepharid Roombia truncata gen. et sp. nov., and establishment of the Hacrobia taxon nov. PLoS One 4 (9), e7080. Pienaar, R.N., Sym, S.D., 2002. The genus Pyramimonas (Prasinophyceae) from southern African inshore waters. S. Afric. J. Bot. 68, 283–298. R Development Core Team, 2011. R: A Language and Environment for Statistical Computing. R Foundation for
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Statistical Computing, Vienna, Austria, ISBN 3-900051-07-0 http://www.R-project.org/ Rhiel, E., Westermann, M., 2012. Isolation, purification and some ultrastructural details of discharged ejectisomes of cryptophytes. Protoplasma 249, 107–115. Rhiel, E., Kunz, J., Wehrmeyer, W., 1989. Immunocytochemical localization of phycoerythrin-545 and of a chlorophyll a/c light harvesting complex in Cryptomonas maculata (Cryptophyceae). Bot. Acta 102, 46–53. Rhiel, E., Westermann, M., Steiniger, F., Kirchhoff, C., 2013. Isolation and characterization of the ejectisomes of the prasinophyte Pyramimonas grossii. Protoplasma 250, 1351–1361. Rosati, G., Modeo, L., 2003. Extrusomes in ciliates: diversification, distribution, and phylogenetic implications. J. Eukaryot. Microbiol. 50, 383–402. Santore, U.J., 1985. A cytological survey of the genus Cryptomonas (Cryptophyceae) with comments on its taxonomy. Arch. Protistenkd. 130, 1–52. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354. Vørs, N., 1992. Ultrastructure and autecology of the marine, heterotrophic flagellate Leucocryptos marina (Braarud) Butcher 1967 (Katablepharidaceae/Kathablepharidaceae), with a discussion of the genera Leucocryptos and Katablepharis/Kathablepharis. Eur. J. Protistol. 28, 369–389. Wehrmeyer, W., 1970. Struktur, Entwicklung und Abbau von Trichocysten in Cryptomonas und Hemiselmis (Cryptophyceae). Protoplasma 70, 295–315. Yamagishi, T., Kai, A., Kawai, H., 2012. Trichocyst ribbons of a cryptomonads are constituted of homologs of R-body proteins produced by the intracellular parasitic bacterium of Paramecium. J. Mol. Evol. 74, 147–157.
ScienceDirect European Journal of Protistology 50 (2014) 248–257
Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes Silke Ammermann, Helmut Hillebrand, Erhard Rhiel∗ Planktologie, ICBM, Carl-von-Ossietzky-Universität Oldenburg, P.O.B. 2503, D-26129 Oldenburg, Germany Received 6 January 2014; received in revised form 25 February 2014; accepted 27 February 2014 Available online 18 March 2014
Abstract Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6 M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6 M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19 kDa and two additional polypeptides of 23 and 26 kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum. © 2014 Elsevier GmbH. All rights reserved.
Keywords: Ejectisome; IgG fractions; Prasinophyte; Pyramimonas grossii; Reconstitution experiments; Transmission electron microscopy
Introduction Prasinophytes comprise a heterogeneous group of primitive, unicellular green algae. Beside motile, flagellated, scale-bearing genera, non-motile coccoid genera without body scales have been described. Phylogenetic analyses of SSU rRNA sequences of 13 prasinophyte species representing eight genera revealed four independent clades with the phylogenetic position of Pycnococcus provasoli being unresolved (Nakayama et al. 1998).
∗ Corresponding
author. Tel.: +49 441 798 3389; fax: +49 441 798 3151. E-mail address: [email protected] (E. Rhiel).
http://dx.doi.org/10.1016/j.ejop.2014.02.001 0932-4739/© 2014 Elsevier GmbH. All rights reserved.
Based on ultrastructural data, six subgenera are actually believed to constitute the genus Pyramimonas, i.e. Pyramimonas McFadden, Vestigifera McFadden, Trichocystis McFadden, Punctatae McFadden, Hexactis Hori, Moestrup et Hoffman, and Macrura Hori et Moestrup (Hori et al. 1995; McFadden et al. 1986, 1987; Pienaar and Sym 2002). A common feature of species of the subgenera Trichocystis and Hexactis is that they harbor projectile organelles, i.e. ejectisomes which closely resemble R-bodies of several bacteria as both are built up by single coiled ribbons composed of protein. More complex ejectisomes have been described for cryptophytes and kathablepharids (Clay and Kugrens 1999a,b; Kugrens et al. 1994; Okamoto and Inouye 2006; Okamoto et al. 2009; Santore 1985; Vørs 1992; Wehrmeyer 1970; and references cited therein).
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The ejectisomes of the prasinophyte Pyramimonas grossii have been first described by Manton (1969). Non-discharged ejectisomes were located at the cells anterior ends and consisted of coiled ribbons which measured 5 nm in thickness and 500 nm in width. After discharge, the ejectisomes formed left-handed, single-coiled hollow tubes which measured up to 35 m in length and 100 nm in width. Morrall and Greenwood (1980) investigated the ejectisomes of Pyramimonas parkeae and found that non-discharged coils exhibited ribbon-like layers with thicknesses of 4.9–5.5 nm. Each ribbon consisted of highly ordered subunits which were obvious as light and dark substructures. The authors measured subunit intervals of 2.8–3.3 nm for both, non-discharged and discharged ejectisomes. These data have been confirmed in a recent study (Rhiel et al. 2013). In a previous study, the ejectisomes of three cryptophyte species have been isolated and the first data on the constituting polypeptides were presented. Furthermore, the chemical stability of cryptophycean ejectisome fragments was investigated, and they were successfully isolated by Percoll gradient centrifugation. Afterwards they were disassembled and reconstituted by treatment with guanidine hydrochloride followed by dialysis. Finally, an antiserum against the polypeptides was raised which immuno-labelled discharged ejectisomes of the cryptophytes (Ammermann et al. 2013). Investigations on the stability of the ejectisomes of Pyramimonas grossii have not been performed, and Percoll gradient centrifugation was not applied in our previous study (Rhiel et al. 2013). Additionally, no antiserum had been raised against the constituting polypeptides, and no dissociation/reconstitution experiments had been performed. Therefore, the current work focuses on the following three topics. First, the protocol for the isolation of discharged ejectisomes of Pyramimonas grossii was modified, and Percoll density gradient centrifugation and pH treatments were applied. Second, experiments with guanidine hydrochloride were performed to see if ejectisomes of Pyramimonas grossii could be successfully disintegrated and afterwards reassembled. Third, an antiserum was raised against a fraction enriched in ejectisome fragments of Pyramimonas grossii and used in immunogold-labelling electron microscopy experiments.
Material and Methods Cultures and growth conditions The prasinophyte Pyramimonas grossii was obtained from the Scandinavian Culture Collection of Algae and Protozoa (SCCAP, University of Copenhagen, Denmark, strain-no. K0253) and was cultured as described by Rhiel et al. (2013).
Isolation of ejectisome fragments and sodium dodecyl sulfate (SDS)-solubilisation The isolation and SDS-solubilisation of ejectisome fragments followed the protocols outlined by Rhiel et al. (2013)
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with some modifications. The cells (200–250 ml culture volume, 7–10 d culture age) were harvested by centrifugation at 3220 g for 15 min in an Eppendorf 5810R refrigerating centrifuge equipped with an A-4-62 swinging bucket rotor (Eppendorf, Hamburg, Germany). The cells were resuspended in 9 ml buffer (5 mM Tris/HCl, pH 7.5) and lysed by adding 1 ml of 10% (v/v) Triton X-100 (1% final concentration). The insoluble material consisting of contaminating bacteria, starch grains, body scales, and ejectisome fragments was pelleted from the supernatant by centrifugation at 3220 g for 15 min, resuspended in 4 ml buffer and again centrifuged. After incubation in 2–4 ml of 5% (w/v) EDTA, pH 7.1 for 4 h at room temperature, the body scales were dissolved and removed by two subsequent washing/centrifugation steps (washing in distilled water). The resulting ejectisome fragment-enriched fractions were kept either frozen or immediately used for experiments. For SDS solubilisation, ejectisome fragment-enriched fractions were pelleted by centrifugation and resuspended in 200–400 l distilled water. Fifty to one hundred (50–100) l aliquots of 10% (w/v) SDS were added (2% final concentration). Then, the mixtures were centrifuged (Heraeus Biofuge pico table top centrifuge for 10 min at 16000 g, Kendro Laboratory Products GmbH, Langenselbold, Germany). The SDS-insoluble pellets were subjected to two additional washing/centrifugation steps with distilled water before they were used for negative staining transmission electron microscopy and SDS-PAGE, whereas the supernatants were subjected to SDS-PAGE.
Percoll density gradient centrifugation Percoll density gradient centrifugation followed the protocol outlined by Ammermann et al. (2013). An ejectisome fragment-enriched fraction was diluted with 6 ml buffer consisting of 5 mM Tris/HCl pH 7.5 and 150 mM NaCl. Then, a mixture of 4.5 ml Percoll (Sigma, Munich, Germany) with 0.5 ml of 1.5 M NaCl was added. The solution was filled into a re-sealable polyallomer ultracentrifugation tube (Science Services, Munich, Germany), and the tube was sealed and centrifugated in a Beckman L8-55M ultracentrifuge (Beckman Coulter, Munich, Germany) equipped with a NVT65 rotor for 1 h at 27400 g and 20 ◦ C. The ejectisome fragmentcontaining bands were collected using needle tipped syringes, diluted with distilled water, and subjected to two washing/centrifugation steps (Heraeus Biofuge pico). Then, the fractions were used for SDS-PAGE.
pH treatment Five hundred (500) l of an ejectisome fragment-enriched fraction was pelleted by centrifugation (Heraeus Biofuge pico, 10 min, 16000 g) and incubated either in 500 l of 100 mM Tris/HCl pH 2.5 or in 500 l of 100 mM Tris/HCl pH 10.8. Then, the mixture was centrifuged, and the pellet was
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resuspended in 200 l distilled water and used for negative staining transmission electron microscopy and SDS-PAGE; whereas, the supernatant was used for SDS-PAGE.
GuHCl solubilisation/reconstitution experiments Ejectisome fragment-enriched fractions were pelleted by centrifugation (Heraeus Biofuge pico, 10 min, 16000 g) and incubated in 500–1000 l of 6 M GuHCl for 10–15 min at room temperature. After centrifugation of the clarified solutions for 20 min (Heraeus Biofuge pico), the GuHCl-insoluble material was pelleted. The clear supernatants were then dialysed against distilled water for approximately 2–4 h at room temperature without stirring, using Membra-Cel dialysis membranes (Serva, Heidelberg, Germany, MWCO 3500). Re-aggregation, visible as opaquewhite flocs, occurred during that time. The reconstituted ejectisome-like filaments were pelleted by centrifugation and used for negative staining transmission electron microscopy and SDS-PAGE; whereas, the GuHCl-insoluble pellets were subjected to two additional washing/centrifugation steps with distilled water before they were used for negative staining transmission electron microscopy and SDS-PAGE.
SDS-PAGE, Western-immunoblotting, and documentation SDS-PAGE was performed as described by Bathke et al. (1999). Generally, 50–100 l aliquots of the various fractions mentioned above were mixed with loading buffer, heat-denatured, and loaded onto 17.5% polyacrylamide gels using the buffer system of Laemmli (1970). Electrophoresis and Coomassie-staining of gels was performed as described by Ammermann et al. (2013). Ejectisome fragment-enriched fractions of Pyramimonas grossii were tested for cross-reactivity with the antiserum raised against them. They were subjected to SDS-PAGE followed by Western-immunoblotting according to Towbin et al. (1979). The antiserum was used in dilutions of up to 1:2000. Coomassie-stained gels, Western-immunoblots, and Percoll density gradient tubes were documented with an Olympus C3030 Zoom digital still CCD camera.
Antiserum production and selective isolation of IgG fractions The rabbit derived antiserum directed against an ejectisome fragment-enriched fraction was ordered commercially (BioScience, Göttingen, Germany). Preimmunserum was taken before the first immunisation. Four weeks after the final immunisation, the antiserum was obtained. For the isolation of distinct IgG fractions, ejectisome fragment-enriched fractions were subjected to SDS-PAGE followed by Western blotting. Then, the blot was stained with Ponceau S to visualise the polypeptides. Distinct stripes of the blot, comprising
either the three polypeptides with relative molecular weights of 12.5, 16, and 19 kDa (fraction termed #A) or those with relative molecular weights of 23 and 26 kDa (fraction termed #B) were cut out, blocked with 1% (w/v) BSA in TBS, and incubated with either preimmunserum (PS) or antiserum (AS) for 1 h. Then, the nitrocellulose strips were washed in TBS, and the IgG fractions absorbed to these polypeptides were desorbed by addition of 200–400 l of 100 mM Tris/HCl, pH 2.5 to the nitrocellulose stripes. Finally, 20–40 l 1 M Tris/HCl pH 7.5 was added to these IgG fractions, resulting in the fractions IgG-#A absorbed to preimmunserum (further named IgG-#A-PS), IgG-#A absorbed to antiserum (further named IgG-#A-AS), IgG-#B absorbed to preimmunserum (further named IgG-#B-PS), and IgG-#B absorbed to antiserum (further named IgG-#B-AS).
Negative staining transmission electron microscopy For negative staining transmission electron microscopy, 50 l droplets of the ejectisome fragment-enriched fractions, of the SDS-insoluble pellets, of the pH 2.5 treated ejectisome fragments, of reconstituted ejectisome-like filament fractions, and of the GuHCl-insoluble fractions were used. In some cases, prior to negative staining, the fractions were diluted in distilled water. The particles were allowed to adsorb for 5 min onto Formvar-coated grids. Then, the grids were stained for 1 min with 1% (w/v) uranyl acetate and washed in two drops of distilled water. Samples were examined with a Zeiss EM 902A electron microscope (Zeiss, Oberkochen, Germany) operated at 80 kV. Digitized images were taken with a 1k Proscan High Speed SSCCD camera (Proscan elektronische Systeme GmbH, Lagerlechfeld, Germany) operated by the iTEM Five software (Olympus Soft Imaging System GmbH, Münster, Germany).
Immuno-electron microscopy For immuno-electron microscopy, ejectisome fragmentenriched fractions were allowed to adsorb for 5 to 10 min onto Formvar-coated grids. Then, the grids were floated for 1 h onto 50 l drops of 1% of BSA dissolved in TBS, incubated for 1 h on 50 l drops of either preimmunserum, or antiserum, or IgG fractions. The preimmunserum and the antiserum were diluted 1:200 in TBS containing 0.1% BSA; whereas, the IgG fractions were used non-diluted. Then, the grids were washed in 10 drops of TBS and incubated for 1 h in gold-labelled goat-anti rabbit IgG conjugate (British Biocell International, Cardiff, UK, diluted 1:100 in TBS containing 1% BSA, 15 nm gold particles). After washing with 10 drops of TBS, the grids were stained with uranyl acetate, washed with two drops of distilled water, and used for transmission electron microscopy.
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Quantification of the immuno-label and statistics
Percoll density gradient centrifugation
The amounts of gold particles positioned on or tightly to ejectisome fragments were enumerated on digitized images. Then, the lengths of the fragments were measured, and the amounts of gold particles per 1 m fragment length were quantified. We performed a univariate Analysis of Variance (ANOVA) testing for treatment effects on the amount of gold particles which had to be log-transformed to achieve homogeneity of variances. Significant differences between treatment levels were detected using Tukey’s HSD posthoc test. All statistics were done using the R software package (R Development Core Team 2011).
Percoll density gradient centrifugation resulted in either one area or two areas of white, flocculent material (Fig. 1C). When subjected to SDS-PAGE, both showed the same banding pattern as the fraction prior to the density gradient centrifugation (Fig. 2, lanes 8 and 9 vs. lane 7).
Results Isolation of ejectisome fragments and SDS-solubilisation Fractions highly enriched in ejectisome fragments were achieved by following the protocol outlined by Rhiel et al. (2013) with some modifications. Negative staining transmission electron microscopy of those fractions mainly showed ejectisome fragments of various lengths (Figs 3A, 4A), and Coomassie-stained gels revealed three dominating polypeptides of 12.5, 16, and 19 kDa (marked with arrows in Fig. 1A, and see also Fig. 2, lanes 1, 4, 7, and 10). Two additional polypeptides of 23 and 26 kDa stained somewhat weaker (marked with arrowheads in Fig. 1A). Furthermore, a faint smear was registered along the entire lane. SDSsolubilisation of those fractions gave rise to an almost identical polypeptide pattern of the SDS-soluble fraction (Fig. 2, lane 2); whereas, the SDS-insoluble material showed a molecular polypeptide of approximately 80 kDa (marked with an asterisks in Figs 1A, 2A, lane 3). Transmission electron microscopy revealed that the SDS-insoluble material mainly consisted of bacterial shells, other cell debris, and starch grains (not shown). The ejectisome fragment-enriched fractions were thus judged to be appropriate starting material for further experiments.
pH treatment Incubation of ejectisome fragment-enriched fractions in 100 mM Tris/HCl pH 2.5 did not result in solubilisation or detachment of loosely bound polypeptides. The ejectisome fragments could be pelleted by centrifugation, and their structure resembled the one registered for those prior to pH 2.5 treatment (Fig. 3A vs. B). Supporting this observation, SDS-PAGE demonstrated that all protein was pelleted by centrifugation (Fig. 2, lane 6) and that no protein could be detected in the resulting supernatant (Fig. 2, lane 5). Similar results were obtained for the pH 10.8 treatment (not shown).
GuHCl-treatment and reconstitution Incubation of pelleted ejectisome fragment-enriched fractions in 6 M GuHCl resulted in clarifying. SDS-PAGE of the GuHCl-insoluble material obtained after centrifugation did show minor amounts of polypeptides (Fig. 2, lane 12), and negative staining transmission electron microscopy revealed minor amounts of ejectisome fragments which had not been solubilised (Fig. 4B). Dialysis of the GuHCl solubilised, clarified supernatants resulted in the appearance of fine white flocs in the dialysis tubes which could be concentrated by centrifugation. The polypeptide pattern of these flocs matched the one registered for ejectisome fragments when subjected to SDS-PAGE (Fig. 2, lane 11). Negative staining electron microscopy of these flocs showed larger and smaller filaments (Fig. 4C). Widths of 133 ± 22 nm (n = 26) and 27.1 ± 7.6 nm (n = 36) were measured for the larger and smaller filaments, respectively. The larger filaments seemed to be composed of the smaller ones, as their ends often split up into several smaller filaments indicating a non-proper reassembly (arrows in Fig. 4C, and Fig. 4D). The smaller filaments were often spirally twisted (Fig. 4E).
Antiserum, IgG fractions, and immuno-electron microscopy Western-immunoblotting revealed that the antiserum against an ejectisome fragment-enriched fraction weakly cross-reacted with the 12.5–19 kDa, the 23 kDa, and the 26 kDa polypeptides of Pyramimonas grossii (Fig. 1B, lane marked “AS”). Rather, the antiserum labelled polypeptides with relative molecular weights of approximately 80, 66, 50, 36, and 8 kDa. No immune-reaction was observed using the preimmunserum (Fig. 1B, lane marked “PS”). Immunoelectron microscopy using either the preimmunserum or the antiserum also did not result in significant differences of gold particles per m filament length (Table 1). By using IgG fractions of the antiserum and preimmunserum however, significant differences in the amounts of gold particles per m filament length were registered (Fig. 5). Thus, a 3-fold higher label was registered for IgG-#A-AS in comparison to IgG#A-PS; whereas, IgG-#B-AS gave rise to a 6-fold higher label than IgG-#B-PS (Table 1). Treatment effects on the amount of gold particles per m filament length were highly significant (ANOVA, F(5;251) = 87.67, p < 0.0001). All pairwise comparisons were significant (Tukey’s HSD, p < 0.05), with the exception of IgG-PS-3B versus IgG-# PS-3B, and preimmunserum versus antiserum.
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Fig. 1. (A) SDS-PAGE of an ejectisome fragment-enriched fraction. The polypeptides marked with asterisk, arrows, and arrowheads are described in the Result section. The relative molecular masses of the marker proteins (kDa) are indicated on the left. (B) shows lanes of a gel after Western blotting and immuno-decoration with either the preimmunserum (PS) or the antiserum (AS) The brackets marked “#A” and “#B” indicate the blotted polypeptides with relative molecular weights of 12.5, 16, and 19 kDa (fraction #A) and those with relative molecular weights of 23 and 26 kDa (fraction #B), used for “IgG fishing”. For better display of the bands, the digitized pictures were adjusted for brightness and contrast. (C) Ultracentrifugation tubes after Percoll density gradient centrifugation showing either one or two bands enriched in ejectisome fragments.
Table 1. Compilation of the results obtained by immuno-electron microscopy, showing the amounts of gold particles per 1 m fragment length (means ± SD) being attached to ejectisome fragments of Pyramimonas grossii. Treatment
Gold particles per 1 m fragment length
Preimmunserum Antiserum IgG-#A-PS IgG-#A-AS IgG-#B-PS IgG-#B-AS
2.98 2.78 1.32 4.78 1.79 11.87
± ± ± ± ± ±
1.70 1.23 0.92 2.42 0.94 5.92
Discussion
SDS polyacrylamide gels resulted in Coomassie-stained gels, which showed the polypeptides of SDS-solubilised ejectisome fragments more clearly. The deviations measured for the relative molecular weights of the three dominating polypeptides (Rhiel et al. 2013) most probably result from the higher polyacrylamide concentration used. The observed polypeptide pattern deviated from those registered for the ejectisomes of limnic and marine cryptophytes (Ammermann et al. 2013; Yamagishi et al. 2012) and for R-bodies of the endosymbiotic bacterium Caedibacter taeniospiralis (Heruth et al. 1994; Kanabrocki et al. 1986), respectively. Actually, no further data are available for polypeptides constituting the ejectisomes of prasinophytes, such as Pyramimonas parkeae, P. pseudoparkeae, P. cirolanae, or P. virginica which are known to harbor ejectisomes (Hori et al. 1995; McFadden et al. 1986).
Isolation of ejectisome fragments and SDS-solubilisation Significant improvements were achieved by minor modifications of the previous isolation protocol (Rhiel et al. 2013). Thus, less culture volumes, younger cultures, shorter periods of EDTA-treatment, and the use of 17.5%
pH treatment and Percoll density gradient centrifugation Incubation of ejectisome fragments in either 100 mM Tris/HCl pH 2.5 or 100 mM Tris/HCl pH 10.8 did not
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Fig. 2. SDS-PAGE of fractions obtained during SDS-solubilisation (lanes 1–3), during pH 2.5 treatment (lanes 4–6), during Percoll gradient centrifugation (lanes 7–9), and during GuHCl dissociation/reconstitution experiments (lanes 10–12). Lanes 1, 4, 7, and 10 show ejectisome fragment-enriched fractions prior to the different treatments. Lane 2: supernatant after SDS-solubilisation; lane 3: SDS-insoluble pellet. The supernatant after pH 2.5 treatment and the resulting pellet are shown in lanes 5 and 6. The upper band and the lower band after Percoll gradient centrifugation are shown in lanes 8 and 9. Lane 11 shows the flocs obtained from the GuHCl-soluble fraction after dialysis against distilled water. The GuHCl-insoluble fraction is shown in lane 12.The relative molecular masses of the marker proteins (kDa) are indicated on the left and in the middle. For better display of the bands, the digitized pictures were adjusted for brightness and contrast.
Fig. 3. Micrographs of ejectisome fragment-enriched fractions prior to pH 2.5 treatment (A) and after pH 2.5 treatment (B) after negative staining. The structure of ejectisome fragments after pH 2.5 resembled the one registered for those prior to pH 2.5 treatment. Scale bars are 2 m.
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Fig. 4. Micrographs obtained by negative staining electron microscopy. In (A), an ejectisome fragment-enriched fraction is shown. A micrograph of the GuHCl-insoluble fraction obtained after centrifugation and washing is shown in (B). An overview and detailed views of reconstituted ejectisome-like filaments after GuHCl solubilisation and dialysis are given in (C), (D), and (E). Note that the larger filaments seemed to be composed of smaller ones as their ends often split up into several smaller filaments indicating a non-proper reassembly (arrows in C, and D showing the end of a filament). The smaller filaments were often spirally twisted (arrows in E). Scale bars are 2 m in A, B, and C, 500 nm in D, and 200 nm in E.
result in the dissociation of ejectisome fragments or loss of probably loosely attached polypeptides. Thus, the fragments obtained after centrifugation remained intact as visualized by negative staining electron microscopy, and no polypeptides were detected when the supernatants after pH 2.5 or pH 10.8 treatment were subjected to SDS-PAGE. No improvement but rather the same banding pattern was observed after Percoll density gradient centrifugation. Percoll density gradient centrifugation improved the purity of ejectisome fractions of cryptophytes significantly. A major disadvantage with respect to negative staining electron microscopy, however, was the finding that Percoll particles were still present, even after additional washing steps (Ammermann et al. 2013).
GuHCl-treatment and reconstitution Similar to the results obtained for the ejectisomes of cryptophytes (Ammermann et al. 2013; Rhiel and Westermann 2012), those of the prasinophyte Pyramimonas grossii did not dissolve in the presence of Triton X-100 and resist 8 M urea treatment (not shown). The ejectisomes of Pyramimonas grossii differ from those of cryptophytes in that they were easily dissolved in the presence of SDS (Rhiel et al. 2013; current data). Dissociation of ejectisome fragments was achieved solely by 6 M GuHCl in both cryptophytes and Pyramimonas grossii. As observed for cryptophytes (Ammermann et al. 2013), the rather slow removal of GuHCl
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Fig. 5. Micrographs of isolated, negative-stained ejectisome fragments of Pyramimonas grossii immunogold-labelled with either the IgG fractions of the preimmunserum (A, C) or antiserum (B, D) absorbed to either the three polypeptides with relative molecular weights of 23 and 26 kDa (fraction #B) or to those with relative molecular weights of 12.5, 16, and 19 kDa (fraction #A). Significant differences in labelling were registered for the IgG fractions gained from the antiserum IgG-#A-AS (D) and IgG-#B-AS (B) in comparison to the IgG fractions gained from the preimmunserum IgG-#A-PS (C) and IgG-#B-PS (A). For further details see Materials and Methods, and Table 1. Scale bars are 500 nm.
by dialysis against distilled water without stirring caused reaggregation into ejectisome-like filaments in Pyramimonas grossii. The fact that the polypeptide patterns of ejectisome fraction-enriched fractions, of the supernatant obtained after SDS-solubilization, of the bands obtained by Percoll density gradient centrifugation, and of reconstituted, ejectisome-like filaments did not deviate significantly from one another strengthen the assumption that the polypeptides with relative molecular weights of 12.5–19, 23, and 26 kDa are major components of the ejectisomes of Pyramimonas grossii.
In this context, it is worth to be mentioned that reconstituted ejectisome-like filaments of cryptophytes form tube-like filaments which closely resemble native, discharged ejectisome fragments; whereas, reconstituted ejectisome-like filaments of Pyramimonas grossii do not. In cryptophytes, discharged ejectisomes form long tubes with bended tips. The tubes themselves result from bending of the margins of the ribbons. Similarly, reconstituted ejectisome-like filaments are more or less transversally curved or rolled-up until a tubular structure is reached (Ammermann et al. 2013).
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Discharged, native ejectisomes of prasinophytes, on the contrary, form spirally twisted tubes. The spiral twist is reflected by the overlaps visible at the surfaces of the tubes (Manton 1969). Ejectisome widths of up to 200 nm were measured when subjected to negative staining electron microscopy compared to smaller diameters of approximately 150 nm for those subjected to freeze-fracture or Cryo-transmission electron microscopy (Rhiel et al. 2013). Reconstituted ejectisome-like filaments of Pyramimonas grossii showed larger filaments with a mean width of 133 nm and smaller filaments of approximately 27 nm. The larger filaments did not show the spiral twist of native, unfurled ejectisomes. They rather seemed to be composed of several smaller filaments giving rise to frayed endings. Spiral twists were observed for the small-sized reconstituted filaments. Thus, the reassembly of polypeptides which constitute prasinophyte ejectisomes seems to be more complicated than the one registered for cryptophytes.
Antiserum, IgG fractions, and immuno-electron microscopy The antiserum raised against an ejectisome fragmentenriched fraction did not satisfy the expectations. In Western-immunoblots, it labeled high molecular weight polypeptides and weakly cross-reacted with the dominating polypeptides, and almost no differences were observed between preimmunserum and antiserum by immuno-electron microscopy. Obviously, contaminants which were still present in the fraction caused higher immune responses in rabbit than the ejectisome fragments. The approach of “IgG fishing” with stripes of nitrocellulose with either the 12.5–19 kDa or the 23 and 26 kDa polypeptides resulted in IgG fractions which significantly immuno-labelled ejectisome fragments. These stripes were cut from Western-blotted ejectisome fragment-enriched fractions after SDS-PAGE. A quite common procedure for the enrichment of immunoglobulins specific to defined antigenic polypeptides is affinity chromatography. In this case, the antigen is coupled to a column matrix, and the antiserum is allowed to pass the column. Following several washing steps, the immunoglobulins bound to the polypeptides are eluted from the column, concentrated, and used in further experiments. The enrichment of IgGs described in the current study is a somewhat modified version of this approach and was successfully applied in earlier studies on the photosynthetic apparatus of the marine cryptophyte Cryptomonas maculata (Rhiel et al. 1989).
Perspective In the past, research on extrusomes of non-ciliate protists mainly focused on their morphology whereas studies on trichocysts of ciliates such as Paramecium included analyses of their ultrastructure, the characterization of the constituting
polypeptides, and the isolation of the corresponding genes (Gautier et al. 1996; Hausmann 1978; Kugrens et al. 1994; Rosati and Modeo 2003; and references cited therein). Quite recently, the ejectisomes of cryptophytes have been investigated in detail, giving first data on their polypeptide composition and the genes encoding them (Ammermann et al. 2013; Yamagishi et al. 2012). To our knowledge, for prasinophytes no other literature is known that could be cited in this context except our previous publication (Rhiel et al. 2013). Further experiments such as N-terminal amino acid sequencing, MALDI-TOF analyses of trypsin- or chymotrypsin-digested polypeptides, and gene cloning are needed to gain further information about these fascinating projectile organelles.
Acknowledgements The authors express their gratitude to Dr. Paul Frost and Clay Prater (Trent University, Peterborough, Ontario, Canada) for critical reading of the manuscript.
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