The proteorhodopsins of the dinoflagellate Oxyrrhis marina : ultrastructure and localization by immunofluorescence light
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ORIGINAL ARTICLE
The proteorhodopsins of the dinoflagellate Oxyrrhis marina: ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy Erhard Rhiel 1,2 & Martin Westermann 3 & Frank Steiniger 3 & Christian Hoischen 4 Received: 6 April 2020 / Accepted: 24 June 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles. Keywords Dinoflagellate . Immunofluorescence light microscopy . Freeze-fracture immunolabeling . Oxyrrhis marina . Proteorhodopsin . Transmission electron microscopy
Introduction Proteorhodopsins were first identified in the course of a metagenomic screening of uncultured sea samples from the Monterey Bay in California (SAR86). The name refers to its Handling Editor: Ralph Gräf * Erhard Rhiel [email protected] 1
Planktology, Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzky-Straße 9–11, 26111 Oldenburg, Germany
2
Planktology, ICBM, Carl von Ossietzky University Oldenburg, P. O. B. 2503, 26129 Oldenburg, Germany
3
Electron Microscopy Center at the Jena University Hospital, Friedrich-Schiller-University Jena, Ziegelmühlenweg 1, 07743 Jena, Germany
4
CF Imaging, Leipniz Institute on Aging, Fritz-Lipmann-Institute (FLI), Beutenbergstraße 11, 07745 Jena, Germany
initial identification in the group of γ-proteobacteria, but meanwhile proteorhodopsins were proven for α-, β-, and δproteobacteria, bacteroidetes, chloroflexi, DeinococcusThermus, flavobacteria, firmicutes, planctomycetes, and actinobacteria as well. Proteorhodopsins were also detected in
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