Protoplasma (2015) 252:271–281 DOI 10.1007/s00709-014-0675-3

ORIGINAL ARTICLE

Isolation and characterisation of the trichocysts of the dinophyte Prorocentrum micans Martin Westermann & Frank Steiniger & Nils Gülzow & Helmut Hillebrand & Erhard Rhiel

Received: 28 April 2014 / Accepted: 27 June 2014 / Published online: 17 July 2014 # Springer-Verlag Wien 2014

Abstract Trichocyst-enriched fractions were isolated from the marine dinophyte Prorocentrum micans. Transmission electron microscopy revealed that most of the trichocysts were discharged and had elongated to long filaments. Some trichocysts were still condensed. Fragments of discharged trichocysts measured up to 20 μm in length and 260 nm in width, those still condensed measured up to 1 μm in width and 16 μm in length. A distinct banding pattern with a transversal periodicity of approximately 16–18 nm and a periodic longitudinal striation of 3–4 nm could be measured along the trichocyst filaments. At higher magnifications, a fragile, alveolated, net-like organisation became obvious which resembled the one shown for the trichocysts of ciliates. When trichocyst-enriched fractions were treated with sodium dodecyl sulfate and centrifuged subsequently, no trichocysts were registered any longer in the sodium dodecyl sulfate-insoluble fraction by electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of trichocyst-enriched fractions and of the SDS-soluble fractions revealed a protein banding pattern which was dominated by polypeptides of 50–30, 12.5, and approximately 8.5 kDa. The polypeptide banding pattern Handling Editor: Reimer Stick This paper is dedicated to Renate Kort on the occasion of her 65th birthday. Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0675-3) contains supplementary material, which is available to authorized users. N. Gülzow : H. Hillebrand : E. Rhiel (*) Planktologie, ICBM, Carl-von-Ossietzky-Universität Oldenburg, P.O.B. 2503, 26129 Oldenburg, Germany e-mail: [email protected] M. Westermann : F. Steiniger Elektronenmikroskopisches Zentrum am Klinikum der Friedrich-Schiller-Universität Jena, Ziegelmühlenweg 1, 07743 Jena, Germany

deviated significantly from those registered for ejectisomes of cryptophytes and of the prasinophyte Pyramimonas grossii, for the Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis, and also from the banding pattern of trichocysts of Paramecium. An antiserum directed against trichocysts of Paramecium did not cross-react with the polypeptides present in the trichocyst-enriched fraction of Prorocentrum micans. Keywords Trichocyst . Dinophyte . Dinoflagellate . Prorocentrum micans . Transmission electron microscopy

Introduction Dinophytes comprise a heterogeneous group of photosynthetic, mixotrophic, or heterotrophic protists. Most species belong to the marine plankton, less occur in freshwater, and some are known to live benthic, parasitic, or as endosymbionts, i.e. as zooxanthellae (Dodge 1971, 1973; Taylor 1990). Dinophytes have several unique features in common. Their cell covering consists of a theca (amphiesma) which is composed of thecal (amphiesmal) plates and intercalary bands. Two flagella, a transverse and a longitudinal flagellum, are found if species are motile. They are either located in surface grooves (dinokont flagellation) or not (desmokont flagellation). The chromosomes within the unique nucleus, the dinokaryon, stay condensed throughout interphase and mitosis. Among the vacuome, two highly specialized vacuoles, the pusules, exist. They arise from ducts which open at the flagellar bases and probably function in osmoregulation. Finally, dinophytes harbour extrusomes, i.e. nematocysts, mucocysts, and trichocysts (Dodge 1973; Hoppenrath and Saldarriaga 2012). Nematocysts, mucocysts, and trichocysts occur simultaneously in species belonging to the pseudocolonial dinoflagellate genus Polykrikos (Hoppenrath and Leander 2007; Hoppenrath

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et al. 2010; Westfall et al. 1983). Mucocysts and trichocysts are found, for example, in several Prorocentrum species, in Ostreopsis cf. ovata Fukuyo, and in Bispinodinium angelaceum (Honsell et al. 2013; Hoppenrath et al. 2013; Yamada et al. 2013). Many dinoflagellate species harbour exclusively either mucocysts or trichocysts (e.g. Hansen and Moestrup 1998; Hansen 2001; Hoppenrath et al. 2013; Yoon et al. 2012). Nematocysts resemble cnidocysts of cnidarians with respect to ultrastructure, mode of release, and function in prey capture (Hoppenrath and Leander 2007; Hoppenrath et al. 2010; Hwang et al. 2008; Westfall et al. 1983). Mucocysts are vesicles with granular or fibrous contents which represent most likely polysaccharides. Several planktonic and benthic dinophyte species are known to produce large amounts of mucilage which is composed of acidic polysaccharides and most probably functions in defense against grazing, regulation of buoyancy, or attachment to surfaces (Honsell et al. 2013, and references cited therein). Trichocysts are the most common type of extrusome in dinophytes. They are membraneenclosed organelles, synthesized in the Golgi, and finally positioned in the amphiesma perpendicular to the cell surface. Generally, they resemble the spindle trichocysts of ciliates. In the resting stage, they consist of a dense, crystalline core, i.e. proteinaceous rod, and tubular elements and fibres at the anterior end. Fibrous material may also be found between the core and the enclosing membrane. Cross-sectioned cores resemble squares or rhombes, whereas in longitudinal sections they show a regular, periodic striation and taper towards the ends. In Oxyrrhis marina, non-discharged trichocysts measure 1.5–2 μm in length and 300–400 nm in width. For Prorocentrum, length values of up to 4 μm have been registered (Bouck and Sweeney 1966; Hausmann 1973; Livolant 1982). As a peculiarity, Peridiniella catenata synthesizes small trichocysts, 1 μm in length and 180–230 nm in diameter, and large ones of 3–5 μm length and 650–900 nm in diameter (Hansen and Moestrup 1998). Ejected trichocysts elongate and measure up to 200 μm in length (Bouck and Sweeney 1966). They show, as a general characteristic, a regular crossstriation of the extruded shafts in form of alternating electrondense and electron-translucent bands. For O. marina, a periodicity of 68 nm was measured which was further subdivided into 16–17 nm subperiods (Hausmann 1973, 1978). For Prorocentrum micans, Livolant (1982) measured a periodicity of 67 nm which dropped to a mean value of 50 nm in sectioned material. Similar periodicities were measured by other authors and for other dinophyte species (see Table I in Livolant 1982). Studies on the isolation of trichocysts of dinophytes are rare and mainly mentioned in the context of descriptions of their ultrastructure. Drastic changes of the salinity of the growth media induced trichocyst discharge, and successive low speed and high speed centrifugation steps led to removal

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of the cells and to trichocyst-enriched fractions (Hausmann 1973; Messer and Ben-Shaul 1971; Ukeles and Sweeney 1969). Livolant (1982) induced trichocyst discharge in Prorocentrum micans by squashing the cells and collecting the extruded shafts by centrifugation. Data on the constituting polypeptides are almost completely missing and restricted to a single analysis of Messer and Ben-Shaul (1971) who registered that the amino acid profile of isolated trichocysts of Peridinium westii was not similar to that of collagen. More recently, Hwang et al. (2008) suggested that part of the nematocysts of Polykrikos might be assembled with a polypeptide similar to minicollagen of Hydra. Taken together, the ultrastructure of trichocysts of dinophytes has been described in detail and protocols for the isolation of these organelles exist. Data on the constituting polypeptides and information on the genes encoding them are still missing. The current work represents a first step in broadening our knowledge about these organelles and focuses on the following topics. (1) We elaborated a working protocol for the isolation of large amounts of discharged trichocysts. (2) We compared the data obtained from these isolated discharged trichocysts by electron microscopy with those of previous studies. (3) We present the first protein banding patterns of trichocyst-enriched fractions of a dinophyte and compare them with the banding pattern of ejectisomes of cryptophytes and of the prasinophyte Pyramimonas grossii, of the Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis, and of isolated trichocysts of Paramecium. (4) We checked if trichocyst-enriched fractions of Prorocentrum micans cross-react with an antiserum against trichocysts of Paramecium, and (5) we discuss these data in the context of those obtained for ejectisomes of cryptophytes and prasinophytes, for the Reb polypeptides, and for trichocysts of ciliates, i.e. of Paramecium.

Material and methods Cultures and growth conditions The dinophyte Prorocentrum micans was isolated by one of us (N. G.) from a North Sea water sample (Wilhelmshaven, Nassauer Harbour, Germany; 53° 30.82′ N, 008° 09.07′ E) by using an aspirator tube and micropipets (Drummond Scientific). Afterwards, several dilution series were conducted in six-well culture plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) to remove any other microalgae contamination. Once samples were unialgal, the alga was grown in f/2 medium (Guillard and Ryther 1962) at 17 °C in Erlenmeyer flasks of 250– 1,000 ml culture volume without aeration. The photon flux density was measured with an Almemo 2290-2 measuring instrument equipped with a FLA613-PSM sensor (Ahlborn Mess- und Regelungstechnik GmbH, Holzkirchen, Germany) and adjusted to 11 μmol photons m−2 s−1. The light/dark

On the trichocysts of Prorocentrum micans

regime was 14 h:10 h. The cryptophytes Chroomonas LT, Cryptomonas S2, and Cryptomonas LT, and the prasinophyte Pyramimonas grossii were cultivated as described earlier (Rhiel and Westermann 2012; Rhiel et al. 2013). Preparation of trichocyst-enriched fractions The cells (culture volume, 500–1,000 ml; culture age, 14–21 days) were harvested by centrifugation at 200 × g for 6 min in an Eppendorf 5810R refrigerating centrifuge equipped with an A-4-62 swinging bucket rotor (Eppendorf, Hamburg, Germany). The resulting cell pellet was resuspended in 10 ml f/2 medium. Trichocyst discharge was induced by the addition of 2 ml of ice-cold ethanol. After an incubation period of 10 min, 24 ml distilled water and 4 ml of 10 % (v/v) Triton X-100 (1 % final concentration) were added. Then, the cells were pelleted from the supernatant (S1) by low-speed centrifugation (7 min, 50×g, Eppendorf 5810R refrigerating centrifuge equipped with a F34-6-38 fixed angle rotor), resuspended in 40 ml distilled water, and again centrifuged. The resulting supernatants (S1, S2) mainly consisted of discharged trichocysts and contained minor amounts of intact and lysed cells, cell debris, and contaminating bacteria. They were centrifuged for 20 min at 12,850×g (Eppendorf 5810R refrigerating centrifuge, F34-6-38 fixed angle rotor). The pelleted material was pooled and resuspended in 4 ml distilled water. Intact and lysed cells were removed by an additional low-speed centrifugation step for 3 min at 30×g, using conical 5 ml Eppendorf PCR tubes (Eppendorf 5810R refrigerating centrifuge, A-4-62 swinging bucket rotor). Then, the trichocysts were pelleted from the supernatant by centrifugation for 15 min at 3,220×g, washed twice (washing in distilled water, centrifugation for 15 min at 3220×g, Eppendorf 5810R refrigerating centrifuge, A-4-62 swinging bucket rotor), and finally dissolved in 200–400 μl distilled water. Aliquots of those trichocyst-enriched fractions were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), for transmission electron microscopy, and for sodium dodecyl sulfate (SDS) solubilisation experiments. Preparation of ejectisome-enriched fractions and sources of Rbodies, trichocysts of Paramecium, and of an antiserum against trichocyst matrix polypeptides of Paramecium Ejectisomeenriched fractions of the three cryptophytes Chroomonas LT, Cryptomonas S2, and Cryptomonas LT, and of the prasinophyte Pyramimonas grossii were obtained as described earlier (Ammermann et al. 2013; Rhiel et al. 2013). A mixture of recombinant R-body polypeptides was kindly provided by Dr. Martina Schrallhammer (University of Freiburg, Germany). Isolated trichocysts of Paramecium and an antiserum directed against them (originally produced by Prof. Dr. Helmut Plattner, formerly University of Konstanz, Konstanz, Germany) were kindly

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provided by Dr. Martin Simon (Saarland University, Saarbrücken, Germany). SDS solubilisation experiments For SDS-solubilisation, trichocyst-enriched fractions of Prorocentrum micans were pelleted by centrifugation (Heraeus Biofuge pico, 10 min, 16,000×g) and resuspended in 100–400 μl distilled water. Twenty-five to 100 μl aliquots of 10 % (w/v) SDS were added (2 % final concentration). Then, the mixtures were centrifuged. The SDS-insoluble pellets were subjected to two additional washing/centrifugation steps before they were used for transmission electron microscopy and SDS-PAGE, whereas the supernatants were either kept frozen or subjected immediately to SDS-PAGE. SDS-PAGE and Western immunoblotting SDS-PAGE was performed as described (Bathke et al. 1999). Aliquots of the trichocyst-enriched fractions and of the SDS-soluble and SDS-insoluble fractions, of the ejectisome-enriched fractions, of the recombinant R-body polypeptides, and of the isolated trichocysts of Paramecium were mixed with loading buffer, heat-denatured, and loaded onto 15 % polyacrylamide gels using the buffer system of Laemmli (1970). Electrophoresis and Coomassie-staining of gels were performed as described by Ammermann et al. (2013). Trichocyst-enriched fractions of Prorocentrum micans were tested for cross-reactivity with an antisera directed against trichocysts of Paramecium. They were subjected to SDS-PAGE together with isolated trichocysts of Paramecium. Western immunoblotting was performed according to Towbin et al. (1979). The antiserum directed against trichocysts of Paramecium was used in dilutions of 1:2,000. The Coomassie-stained gels and Western immunoblots were documented with an Olympus C3030 Zoom digital still CCD camera. Negative staining transmission electron microscopy The trichocyst-enriched fractions and the SDS-insoluble pellets were used for negative staining transmission electron microscopy. Fifty microlitre droplets of the fractions mentioned above were used. If appropriate, 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. Most samples were examined with a Zeiss EM 902A electron microscope (Zeiss, Oberkochen, Germany) operated at 80 kV. Digitized images were taken with a 1 k Proscan High Speed SSCCD camera (Proscan elektronische Systeme GmbH, Lagerlechfeld, Germany) operated by the iTEM Five software (Olympus Soft Imaging System GmbH, Münster, Germany). Some samples were examined in a Jeol JEM2100F electron microscope (Jeol Ltd., Tokyo, Japan) operated at 120 kV. The microscope was equipped with Orius SC200D and Orius SC600 CCD

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cameras, both operated by Digital Micrograph software (Gatan, Pleasanton, CA, USA). Tilt series were taken by using the TEMography software packages Recorder, Composer and Visualizer kai (System in Frontier Inc., Tokyo, Japan). Cryo transmission electron microscopy A trichocyst-enriched fraction was used for Cryo transmission electron microscopy (Cryo-TEM). One small droplet (5 μl) was placed on a copper grid covered by a holey carbon film (Quantifoil R 1.2/1.3, pore size 1.2 μm, 400 mesh, Quantifoil Micro Tools, Jena, Germany). Excess liquid was blotted for 3 s between two strips of filter paper. Subsequently, the sample was rapidly plunged into liquid ethane, cooled to about −180 °C with liquid nitrogen in a Cryobox (Zeiss, Oberkochen, Germany). The frozen specimen was transferred with a cryo-holder Gatan 626-DH (Gatan Inc., Pleasanton, CA, USA) into a pre-cooled Philips CM 120 cryo transmission electron microscope (Philips, Eindhoven, Netherlands) operated at 120 kV and viewed under low dose conditions. Cryo-TEM images were recorded with a 1 k CCD Camera (FastScan F114, Software EMMENU V3.0, TVIPS, Munich, Germany). Freeze-fracture transmission electron microscopy For freezefracture electron microscopy, glutardialdehyde-fixed cells (2.5 % (v/v) glutardialdehyde in growth medium, 1 h) and aliquots of a trichocyst-enriched fraction were concentrated by centrifugation in a Heraeus Biofuge stratos centrifuge (Kendro Laboratory Products GmbH, Langenselbold, Germany, 10 min, 5,000×g, room temperature). Small aliquots of the pelleted material (1–2 μl) were enclosed between two 0.1mm-thick copper profiles as used for the sandwich doublereplica technique. Rapid plunge freezing and freeze-fracturing followed the protocol outlined by Ammermann et al. (2013). Freeze-etching was achieved at −110 °C for 30 s. The obtained replicas were transferred to a Proteinase K “cleaning” solution (400 μg/ml proteinase K, 0.5 % (w/v) SDS, 0.5 % (w/v) CaCl2, Tris/HCl 50 mM, pH 7.5) at 60 °C for 1 h, followed by a treatment in 65 % (v/v) nitric acid for 15 min at 60 °C. Then, the replicas were washed three times in distilled water and transferred onto uncoated EM-grids for examination in a Zeiss EM 902A electron microscope (Zeiss, Oberkochen, Germany) using a 1 k FastScan-CCDcamera (TVIPS camera and software, Munich, Germany). Fast Fourier transformation (FFT) was performed as already described (Rhiel and Westermann 2012). Scanning electron microscopy For scanning electron microscopy, cells were harvested by centrifugation, fixed in 4 % (v/v) glutaraldehyde in 100 mM Na/K-phosphate buffer (pH 7), washed in buffer, and dehydrated in a graded ethanol series. After critical point drying in a Balzers CPD 030, samples were coated with 2–5 nm gold in a Balzers SCD 005 device (BALTEC GmbH, Schalksmühle, Germany). Specimens were

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examined with a Hitachi S-3200 N scanning electron microscope operated at 20 kV (Hitachi High Technologies Europe GmbH, Krefeld, Germany). Digitized pictures were taken with the DISS and DIPS software packages (Point Electronic GmbH, Halle, Germany).

Results Prorocentrum micans cells discharge trichocysts often in bundles Scanning electron microscopy revealed that cells of Prorocentrum micans measured approximately 38 μm in length and 26 μm in width. The main characteristics, i.e. the thecal plates, the intercalary band, the thecal pores, and the spine were identified (Fig. 1a, c, Online Resource 1). Discharged trichocysts were mainly registered in bundles outside the cells (Fig. 1b, d), and those which had been arrested in the process of becoming discharged were recognised within the pores (Fig. 1c, Online Resource 1). Similar to this finding, trichocysts which obviously withstood the replica cleaning with proteinase K and hot nitric acid were registered for freeze-fractured cells. Thus, residues of elongated trichocysts with four-sided pyramidal tips were still attached to the platinum-carbon cast (Fig. 2a, b), and at their basis, the fractures of the crystalline cores were visible (Fig. 2b). Trichocysts are easy to isolate Generally, the cells of Prorocentrum micans could be easily pelleted by low-speed centrifugation steps while the discharged trichocysts mainly remained in the resulting supernatants. The treatment with 16– 17 % cold ethanol caused a massive discharge of trichocysts, and the addition of distilled water and 1 % Triton X-100 afterwards caused further release of trichocysts without major effects on the overall integrity and morphology of the cells. As some amount of the trichocysts was pelleted with the cells during the subsequent low-speed centrifugation, a second washing/low-speed centrifugation step of the cells with distilled water was conducted. This treatment increased the yield of trichocysts. Electron microscopy revealed that subsequent low-speed and high-speed centrifugation and washing steps of the two supernatants (S1, S2) finally led to fractions which mainly consisted of trichocysts and fragments thereof, and to less extent of bacteria, starch grains, and other cell debris (Fig. 3a). Discharged trichocysts resemble tapering square section steel More likely, fragments of discharged trichocysts were isolated. They measured up to 20 μm in length and up to 260 nm in width in negative contrast, and generally resembled straight, rigid filaments which tapered towards one end (Figs. 3a, d, and 4d). Smoothly bended trichocysts and, less

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Fig. 1 Scanning electron microscopy micrographs of a cell of Prorocentrum micans (a), of pores showing trichocysts in the process of being protruded (c), and of discharged, aggregated trichocysts positioned in between the cells (b, d). Arrows in b and d indicate the square-shaped profiles of trichocyst filaments. Scale bars are indicated

often, even kinked ones were also registered. As the trichocysts tend to aggregate into bundles, longer and wider filaments were seen, and it was difficult to follow a single trichocyst from its beginning to its end. Tilt series revealed that they had collapsed somewhat due to drying (see movie of Online Resources 3). However, their square-shaped profiles

Fig. 2 Trichocysts in freezefractured cells of Prorocentrum micans (a, b) and isolated trichocysts imaged by freezeetching (c) and Cryo-TEM (d). After replica cleaning with proteinase K and hot nitric acid, some residues of unfolded trichocysts keep attached to the platinum-carbon cast (a). The residues of partially unfolded trichocysts are visible as black needle-like structures (a). At their basis, the fracture of the crystalline core is visible (b). After freeze-etching, the surface of isolated trichocysts shows a striated pattern with a frequency of 16–18 nm (c). In Cryo-TEM view (d), the striation is also visible with a frequency of 16 nm. Scale bars are indicated

were still visible in 3-D reconstructions (see arrows in Online Resource 4B and D) and in micrographs obtained by scanning electron microscopy (marked with arrowheads in Fig. 1b, d). Maximum diameter values of 260 nm were measured for freeze-fractured trichocysts and those subjected to CryoTEM (Fig. 2c, d). The regular cross-striation in form of

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Fig. 3 Micrographs of negativestained, discharged trichocysts of Prorocentrum micans. a Overview of a trichocyst-enriched fraction showing unfolded trichocysts (marked with arrowheads) and those which are still folded (marked with stars). Some cell debris is also visible. b Detail view of unfolded, aggregated trichocysts. c Folded trichocyst. d, e Overview and detail of a trichocyst which was arrested in the process of unfolding. Scale bars are indicated

electron-dense and electron-transparent areas along the trichocyst filaments was registered by all three electron microscopy techniques. Thus, frequencies of the cross-striation of approximately 16, 16–18, and 16 nm were deduced by negative

staining, freeze fracture, and Cryo-TEM, respectively (Figs. 2 and 4, and Online Resource 2). High-resolution micrographs obtained by negative staining and Cryo-TEM revealed an additional longitudinal striation with a frequency

Fig. 4 a High-resolution Cryo-TEM micrograph of an isolated discharged trichocyst of Prorocentrum micans. A faint longitudinal striation of 3.7 nm frequency and a prominent transversal striation pattern of bright stripes of 16.0 nm periodicity were detected and analyzed by FFT (see FFT of A in Online Resource 2B). The longitudinal and transversal pattern are visualized in b by an overlay of a over the inverse FFT of the filtered 3.7 and 16.0 nm frequencies found in a. In some micrographs of

negative-stained isolated discharged trichocysts, a similar type of a bright transversal (c) and longitudinal (d) striation are visible with frequencies of about 3.4 nm for the longitudinal and 13.8 nm for the transversal pattern. The arrows in a and c indicate dark electron dense bands that are separated by three less dark appearing double-lined interbands. The distance of the dark bands is in Cryo-TEM view 68 nm and measures 54 nm in negative-stained preparations. The scale bars are indicated

On the trichocysts of Prorocentrum micans

of approximately 3.4–3.7 nm. Both the transverse and the longitudinal striation patterns could be easily visualized by overlay of an original micrograph over the inverse FFT of the filtered frequencies which were found in the original micrograph (see Fig. 4 and Online Resource 2). Occasionally, a repetitive cross-striation of thicker and thinner electron-dense areas was registered by all three techniques. The darker electron dense bands were separated by three less dark appearing double-lined interbands. The distance of the dark bands was approximately 68 nm in Cryo-TEM and 54 nm in negativestained preparations (marked with arrowheads in Figs. 2c, d and 4a, c). In some instances, micrographs of negative-stained trichocysts which were taken at higher magnifications revealed a fragile, alveolated, net-like organisation (Fig. 4c, d). Non-discharged trichocysts look furred Beside discharged trichocysts, occasionally non-discharged ones were registered in negative-stained samples. They measured up to 16 μm in length and up to 1 μm in width, and left the impression of being furred on their outsides (Fig. 3a, c). The trichocyst shown in Fig. 3d obviously became arrested in the process of discharge as the cross-striated filament protruded at its one end (shown in detail in Fig. 3e). Trichocysts show a complex polypeptide banding pattern and become solubilised by SDS A Coomassie-stained gel onto which fractions obtained during the isolation of trichocysts of Prorocentrum micans had been loaded is shown in Fig. 5. The banding pattern of cells (lane 1) showed an intense smear along the entire lane and major protein bands with relative molecular weights of approximately 67, 32, 23, and 21 kDa. An almost similar banding pattern was registered for the pellet obtained by the additional low-speed centrifugation step for 3 min at 30×g (lane 2, and described in “Preparation of trichocyst-enriched fractions”). The major protein bands of trichocyst-enriched fractions of Prorocentrum micans exhibited relative molecular weights of approximately 47, 41, 34, 30, 12.5, and 8.5 kDa (marked with stars in Fig. 5). Sometimes, additional bands, i.e. those larger than 70 kDa, were observed (see Fig. 5, lanes 3, 4, and 11). Furthermore, a faint smear was registered along the entire lanes. Trichocystenriched fractions which had been treated with SDS gave rise to an identical polypeptide pattern of the SDS-soluble fraction (lane 4) in comparison to the fraction prior to SDS treatment (lane 3). No polypeptides were registered for the SDSinsoluble material (lane 5) although transmission electron microscopy revealed that the SDS-insoluble material consisted of cell debris and bacteria (not shown). The polypeptide banding pattern of trichocysts deviated from those of ejectisomes, R-bodies, and trichocysts of Paramecium The polypeptide banding pattern obtained for trichocyst-enriched fractions of Prorocentrum micans

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(Fig. 5, lane 11) deviated significantly from those registered for ejectisome-enriched fractions of cryptophytes and of Pyramimonas grossii, and for recombinant R-bodies of C. taeniospiralis (Fig. 5, lanes 6–10 and 11). The ejectisomes of cryptophytes were dominated by polypeptides with relative molecular weights of approximately 6–7 kDa (Fig. 5, lanes 7– 9). An ejectisome-enriched fraction of Pyramimonas grossii showed mainly polypeptides of 16–20 kDa (Fig. 5, lane 10), and the recombinant R-bodies gave rise to a ladder-like banding pattern, starting with polypeptides of approximately 17–18 kDa up to those which even remained in the stacking gel (Fig. 5, lane 6). The trichocyst samples of Paramecium were rather old but still showed major protein bands with relative molecular weights of approximately 37.5, 30, 22, 18, and 14.5 kDa. Two larger polypeptides of approximately 84 and 65 kDa were also registered (Fig. 5, lane 12). The polypeptides of trichocysts of Prorocentrum micans are immunologically not related to the trichocyst polypeptides of Paramecium When a trichocyst-enriched fraction of Prorocentrum micans and trichocysts of Paramecium as positive control were subjected to SDS-PAGE followed by Western immunoblotting, the antiserum against the trichocysts of Paramecium did not cross-react with any of the polypeptides present in the trichocystenriched fractions of the dinophyte (Fig. 5, lanes 13), whereas immunoreactions were exclusively observed in the lane containing the Paramecium trichocyst fraction (Fig. 5, lane 14). For the latter, the 37–30-kDa polypeptides were labelled. Weak labelling was also observed for the 22-, 18-, and 14.5-kDa polypeptides.

Discussion Trichocyst isolation An intense study of the older literature revealed that several approaches have been described for the enrichment of trichocysts of dinoflagellates, mainly for electron microscopical studies for which less amounts of cells are necessary. Thus, Ukeles and Sweeney (1969) induced trichocyst discharge in Prorocentrum micans by addition of NaCl to finally double the salinity of the medium. Afterwards, the cells were removed by low-speed centrifugation and the trichocysts were pelleted from the supernatant by a high-speed centrifugation step. Messer and Ben-Shaul (1971) achieved trichocyst discharge in Peridinium westii by washing the cells in distilled water, followed by centrifugation steps at 1,000, 3,000, 10,000, and 25,000×g. The 10,000–25,000×g fraction contained the discharged trichocysts. A similar approach was used by Hausmann (1973) for the isolation of intact and extruded trichocysts of O. marina. Livolant (1982) induced trichocyst

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Fig. 5 Left, SDS-PAGE of fractions obtained during the isolation of trichocysts of Prorocentrum micans. Lane 1, polypeptide pattern of total cell protein; lane 2, polypeptide pattern of the residual cells which were present within the trichocyst-enriched fraction and pelleted by a 30×g centrifugation step; lane 3, trichocyst-enriched fraction; lane 4, supernatant obtained after SDS treatment of the trichocyst-enriched fraction; lane 5, washed pellet which was not solubilized by the treatment with SDS. The major polypeptide bands of the trichocyst-enriched fraction are marked with asterisks between lanes 3 and 4. The relative molecular masses of the marker proteins (kDa) are indicated on the left. For better display of the bands, the digitized pictures were adjusted for brightness and contrast. Middle, SDS-PAGE of recombinant R-bodies of C. taeniospiralis (lane 6), of ejectisome-enriched fractions of the three cryptophytes Chroomonas LT (lane 7), Cryptomonas LT (lane 8), and Cryptomonas S2 (lane 9), of the prasinophyte Pyramimonas grossii (lane 10), and of trichocyst-enriched fractions of Prorocentrum micans (lane 11) and Paramecium (lane 12).

The major polypeptides of the trichocyst-enriched fraction of Prorocentrum micans are marked with asterisks between lanes 10 and 11. The relative molecular masses of the marker proteins (kDa) are indicated on the left. For better display of the bands, the digitized pictures were adjusted for brightness and contrast. Right, Western blotting and immunodecoration with the antiserum directed against trichocyst polypeptides of Paramecium. Aliquots of a trichocyst-enriched fraction of Prorocentrum micans and of trichocysts of Paramecium, equivalent to those of lanes 11 and 12, were loaded on lanes 13 and 14. The antiserum mainly reacted with the trichocyst polypeptides of the ciliate (lane 14, marked with asterisks on the right) and labelled the 84-, 65-, and 37–30-kDa polypeptides. The label of the 22-, 18-, and 14.5-kDa polypeptides was less pronounced. No crossreactivity was registered for polypeptides of the trichocyst-enriched fraction of Prorocentrum micans (lane 13). The relative molecular masses of the marker proteins (kDa) are indicated on the left. For better display of the bands, the digitized pictures were adjusted for brightness and contrast

discharge in Prorocentrum micans by squashing the cells and collecting the extruded shafts by centrifugation. Eperon and Peck (1988) isolated the extrusomes of the ciliate Pseudomicrothorax dubius. The authors induced trichocyst discharge mainly by adding ethanol to a final concentration of approximately 15 %. Taken together, squashing of the cells caused cell damage, release of soluble proteins and membrane fragments, and finally led to contaminations of the trichocyst fractions with other cellular compounds, whereas low and high salt treatment and the use of ethanol mainly left the cells intact. Our approach is finally a combination of ethanolinduced and distilled water-induced trichocyst discharge followed by repeated low-speed and high-speed centrifugation steps. In our hands, trichocyst discharge was most by combining ethanol and low salt treatment. The addition of Triton X-100 did not result in cell lysis but rather in solubilising bacterial cells and complete detachment of the trichocysts from the dinophyte cells.

micrographs have been published that show non-discharged/ resting/in toto trichocysts of dinoflagellates. Hausmann (1973) isolated resting stages of trichocysts of O. marina which measure 1.5–2 μm in length and 0.3–0.4 μm in width. Livolant (1982) showed micrographs of rotary platinum shadowed, in toto trichocyst preparations of Prorocentrum micans. The trichocyst which is shown in her Fig. 1d measures approximately 6–7 μm in length and 0.25–0.3 μm in width. The author states that portions of the shafts of those trichocysts are obviously in an amorphous state which either corresponds to an intermediate state between two crystalline phases or to an artificial disorganisation due to the preparation procedure. This type of non-discharged trichocysts was regularly registered in our preparations. As Livolant (1982) found these amorphous-looking trichocysts in preparations in which the cells were squashed, we assume that they arose in our experiments from cells which were in the process of lysis. Maximum lengths of 16 μm were measured for those nondischarged trichocysts, with most of them being in the range of 5–6 μm. The higher values probably reflect those which were in an advanced phase of discharge while the smaller values correspond to those which were not. Discharged trichocysts showed banding periodicities of 68 nm for the intense black transverse bands, of 16 nm for

Trichocyst ultrastructure Generally, the overall morphology and most of the values measured for non-discharged and discharged trichocysts of Prorocentrum micans are in line with those published earlier. To our knowledge, not many

On the trichocysts of Prorocentrum micans

the regular cross-striation, and of 3–4 nm for the additional longitudinal striation. These values are in line to those given by Hausmann (1973), Messer and Ben-Shaul (1971), and Livolant (1982). The length values indicate that fragments rather than intact trichocysts were isolated. Bouck and Sweeney (1966) stated that discharged trichocysts of Prorocentrum micans measured up to 200 μm in length. Comparable values were never observed in our experiments, not even by light microscopy of cells, immediately after inducing the discharge of trichocysts (not shown). Here, discharged trichocysts were approximately of cell length at most. Assuming that regular, non-discharged trichocysts measure approximately 5–6 μm in length, they would elongate about seven- to eightfold. We assume that rather bundles of trichocysts than single trichocyst filaments resulted in the observed length of 200 μm mentioned by Bouck and Sweeney (1966). Trichocyst polypeptide patterns It is a striking contrast that nothing is known about the polypeptides which constitute the trichocysts of dinophytes, while huge data sets exist for those of ciliates, i.e. of Paramecium. Tip and body of trichocysts of ciliates contain paracrystalline matrices which consist of trichocyst matrix proteins (TMPs). These TMPs are synthesized as 40–45-kDa precursors which are finally processed to two polypeptides of 15–20 kDa each by proteolytic maturation. Only the mature polypeptides build up the crystalline matrix (Gautier et al. 1996; Rosati and Modeo 2003; Sperling et al. 1987, and references cited therein). The three polypeptides of Paramecium with relative molecular weights of 22, 18, and 14.5 kDa (Fig. 5, lane 12) thus most likely represent TMPs, while those of 37.5–30 kDa most likely represent aggregates or precursors. Messer and Ben-Shaul (1971) investigated the amino acid profile of isolated trichocysts of Peridinium westii. The authors registered a low proline content and absence of hydroxyproline and state that the amino acid composition was not similar to that of collagen. Based on the amino acid composition given by Messer and Ben-Shaul (1971) and assuming that a single polypeptide would constitute the trichocysts of Peridinium westii, a theoretical pI value of 4.2 and a molecular weight of 10.2 kDa can be deduced. According to our findings, trichocysts of dinophytes are built up by several polypeptides of different molecular weights. Nematocysts of dinophytes obviously harbour polypeptides which are related to collagen. Thus, Hwang et al. (2008) showed in an immunofluorescence study that the anterior chamber of the nematocysts of Polykrikos most likely contained a polypeptide related to minicollagen. Minicollagen is located at the inner capsule wall of the nematocysts of Hydra. Taken together, the polypeptide pattern observed for trichocyst-enriched fractions of Prorocentrum micans deviates from those described for the trichocysts of ciliates, for the ejectisomes of cryptophytes and of the

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prasinophyte Pyramimonas grossii, and for the R-bodies of C. taeniospiralis (Ammermann et al. 2013; Rhiel et al. 2013; Yamagishi et al. 2012). Trichocysts of dinophytes, trichocysts of ciliates, ejectisomes, and R-bodies Some of the structural findings registered for the trichocysts of Prorocentrum micans pertain to spindle trichocysts of ciliates as well. First, undischarged trichocysts of Paramecium caudatum measure 4–6 μm in size and consist of several different components, i.e. envelope, various sheaths, microtubules, body, and tip (Bannister 1972; Sperling et al. 1987). They finally attach to distinct cortical docking sites. In the condensed state, the tip is 2 μm long and 0.3 μm in diameter. The cylindrical body measures 3–4 μm in length and 1 μm in diameter. Secondly, trichocysts of Paramecium elongate to about seven to eight times their initial length and resemble needle-shaped filaments when they come into contact with Ca2+ ions and water during exocytosis. Thirdly, the undischarged tips and bodies of trichocysts show regular transverse dense lines which are about 5 nm wide and spaced at intervals of approximately 16 nm. Fully extended trichocyst bodies show a characteristic, repetitive transversal banding pattern with 50–60 nm repeats. Finally, Bannister (1972) registered hexagonal units composed of fine filaments which branch and anastomose in a regular pattern. The author distinguished two sub-patterns: (1) filaments of approximately 3 nm either formed hexagonal arrays with each hexagon being connected in the longitudinal direction to slightly thicker (5 nm) filaments, and (2) instead of hexagons, the thin filaments were arranged in a transverse zig zag line with the angles of the zig zack line being connect via thick filaments. Similar highly ordered structures were observed for Paramecium tetraurelia (Peterson et al. 1987; Sperling et al. 1987) and for the compound trichocysts of the ciliate Drepanomonas dentata (Hausmann and Mignot 1975). Micrographs of negative-stained trichocysts of Prorocentrum micans taken at higher magnifications revealed a net-like organisation of filaments which slightly resembled the highly ordered patterns shown by these authors, too. Cryptophytes and some prasinophytes harbour ejectisomes as extrusive organelles. Both types of ejectisomes consist of proteinaceous, coiled ribbons which finally unfurl into long filaments (Kugrens et al. 1994). In cryptophytes, the margins bend along the entire lengths of the filaments so that they finally form hollow tubes (Ammermann et al. 2013; Rhiel and Westermann 2012; Yamagishi et al. 2012). The same is reached in unfurled ejectisomes of prasinophytes by spirally twisting of the coiled ribbons (Manton 1969; Rhiel et al. 2013). R-bodies of the obligate endosymbiotic bacterium C. taeniospiralis unfurl by spirally twisting of the coiled ribbons, too. These observations and the fact that they differ in their polypeptide banding patterns most probably reflect different evolutionary origins. They surely are rather different

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organelles with respect to ultrastructure, mode of extrusion, polypeptide banding pattern (see Fig. 5, lanes 6–10), and evolution in comparison to the trichocysts of dinoflagellates and ciliates which elongated into filaments by decondensation. As (i) the trichocysts of dinoflagellates and ciliates differ in their polypeptide banding patterns too (Fig. 5, lanes 11 and 12) and (ii) are not immunologically related to them (see Fig. 5, lanes 13 and 14), one might assume that the polypeptides which constitute the trichocysts of dinoflagellates are not homolog to the TMPs of ciliates. Further experiments, i.e. amino acid sequence analysis and gene cloning, are needed to elucidate these open questions. Conclusions and outlook For the first time, a method for the isolation of large amounts of trichocysts from a dinophyte was established. The periodicities of the transversal and longitudinal striations and the protein banding patterns of trichocystenriched fractions are compared to what is currently known about the trichocysts of dinophytes and of Paramecium, and about the ejectisomes of cryptophytes and prasinophytes, and about the R-bodies of C. taeniospiralis. Further experiments, i.e. the production of an antiserum and immunogold labelling, will show, if some or all of the polypeptides resolved by SDSPAGE are structural components of the trichocysts of the investigated dinoflagellate. Polypeptide sequence analysis and cloning of the relevant genes are needed to answer the question if the polypeptides which constitute the trichocysts of dinoflagellates are somehow related to the TMPs of ciliates.

Acknowledgments The authors express their gratitude to Silke Ammermann, Ute Friedrich, Edith Kieselhorst, and Susanne Linde for excellent technical assistance, to Dr. Martina Schrallhammer (University of Freiburg) and Dr. Martin Simon (Saarland University) for kindly providing us the recombinant R-body polypeptides, isolated trichocysts of Paramecium and an antiserum directed against them. Conflict of interest The authors declare that they have no conflict of interest.

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Isolation and characterisation of the trichocysts of the dinophyte Prorocentrum micans.

Trichocyst-enriched fractions were isolated from the marine dinophyte Prorocentrum micans. Transmission electron microscopy revealed that most of the ...
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