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Isolation and characterisation of the trichocysts of the dinophyte Prorocentrum micans

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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 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.

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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.

Author information

Correspondence to Erhard Rhiel.

Additional information

This paper is dedicated to Renate Kort on the occasion of her 65th birthday.

Handling Editor: Reimer Stick

Electronic supplementary material

Below is the link to the electronic supplementary material.

MPEG 1-formated movie of negative-stained trichocysts of Prorocentrum micans. The tilt series was obtained using the TEMography software packages Recorder, Composer and Visualizer kai (System in Frontier Inc., Tokyo, Japan). The specimen was tilted from −69.01° to 58.05° and the micrographs were taken at 1° intervals (total of 129 micrographs) (MPG 1158 kb)

Online Resource 1

Micrographs showing thecal pores of Prorocentrum micans either by scanning electron microscopy (A, B) or freeze-etching (C, D). Trichocysts in the process of being protruded are visible in B and C. The trichocyst in C withstood the replica cleaning process with proteinase K and hot nitric acid and its residue is visible as black structure. The scale bars are indicated (TIFF 9094 kb)

Online Resource 2

A Cryo-TEM micrograph of an isolated discharged trichocyst of Prorocentrum micans. After FFT analysis (B), a longitudinal frequency at 3.8 nm and a transversal frequency at 16.0 nm were detected (encircled spots in B). The encircled FFT spots at 16 and 3.8 nm were filtered out and an inverse FFT was calculated for the two frequencies. The transversal periodicity is visualized in C by an overlay of A and the inverse FFT of the 16.0 nm spots. The longitudinal periodicity is visualized in D by an overlay of A and the inverse FFT of the 3.8 nm spots. The scale bar represents 100 nm (TIFF 11779 kb)

Online Resource 3

MPEG 1-formated movie of negative-stained trichocysts of Prorocentrum micans. The tilt series was obtained using the TEMography software packages Recorder, Composer and Visualizer kai (System in Frontier Inc., Tokyo, Japan). The specimen was tilted from −69.01° to 58.05° and the micrographs were taken at 1° intervals (total of 129 micrographs) (MPG 1158 kb)

Online Resource 4

Pictures of 3-D reconstructed trichocysts obtained using the TEMography software packages Recorder, Composer and Visualizer kai (System in Frontier Inc., Tokyo, Japan). The micrographs shown in A and C were turned and tilted in order to get front views of the square-shaped profiles of trichocyst filaments (marked with arrows in B and D) (TIFF 3963 kb)

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Westermann, M., Steiniger, F., Gülzow, N. et al. Isolation and characterisation of the trichocysts of the dinophyte Prorocentrum micans . Protoplasma 252, 271–281 (2015) doi:10.1007/s00709-014-0675-3

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Keywords

  • Trichocyst
  • Dinophyte
  • Dinoflagellate
  • Prorocentrum micans
  • Transmission electron microscopy