Liao WY and Fugmann SD (2017), Lectins identify distinct populations of coelom...

ECB-ART-45862

PLoS One 2017 Nov 10;1211:e0187987. doi: 10.1371/journal.pone.0187987.

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Lectins identify distinct populations of coelomocytes in Strongylocentrotus purpuratus.

Liao WY , Fugmann SD .

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Coelomocytes represent the immune cells of echinoderms, but detailed knowledge about their roles during immune responses is very limited. One major challenge for studying coelomocyte biology is the lack of reagents to identify and purify distinct populations defined by objective molecular markers rather than by morphology-based classifications that are subjective at times. Glycosylation patterns are known to differ significantly between cell types in vertebrates, and furthermore they can vary depending on the developmental stage and activation states within a given lineage. Thus fluorescently labeled lectins that recognize distinct glycan structures on cell surface proteins are routinely used to identify discrete cell populations in the vertebrate immune system. Here we now employed a panel of fifteen fluorescently-labeled lectins to determine differences in the glycosylation features on the surface of Strongylocentrotus purpuratus coelomocytes by fluorescence microscopy and flow cytometry. Eight of the lectins (succinylated wheat germ agglutinin, Len culinaris lectin, Pisum sativum agglutinin, Saphora japonica agglutinin, Solanum tuberosum lectin, Lycopersicon esculentum lectin, Datura stramonium lectin, Vicia villosa lectin) showed distinct binding patterns to fixed and live cells of three major coelomocyte classes: phagocytic cells, red spherule cells, and vibratile cells. Importantly, almost all lectins bound only to a subgroup of cells within each cell type. Lastly, we established fluorescently-labeled lectin-based fluorescence activated cell sorting as a strategy to purify distinct S. purpuratus coelomocyte (sub-)populations based on molecular markers. We anticipate that this will become a routine approach in future studies focused on dissecting the roles of different coelomocytes in echinoderm immunity.

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Species referenced: Echinodermata
Genes referenced: egr3 foxj1 LOC100887844 LOC115919910 LOC588806 LOC591536 LOC762504 tefl

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	Fig 1. Lectin staining of fixed coelomocytes.Total coelomocytes were separated over a density gradient to obtain cell fractions enriched for phagocytes (ph), vibratile cells (v), and red spherule cells (rs). Cells were settled on glass slides, fixed with paraformaldehyde, and stained with DAPI and the indicated lectins that were labeled with (A-D) rhodamine or (E-H) fluorescein. Note that only staining patterns consistent across three individual are shown. Representative images were taken on a Zeiss Axioimager.Z2 microscope with an Apotome.2 structured illumination accessory using a Plan-Apochromat 40x objective and a cooled CCD camera. Respective phase contrast images were taken (without the Apotome.2 feature) to confirm the identity of each cell. The images for the fluorescent channels are shown individually and merged.
	Fig 2. Overview of the lectin binding profiles of live and fixed coelomocytes using fluorescence microscopy.The staining profile for each lectin for each cell type obtained by analyzing at least three individual S. purpuratus is shown. For each lectin/cell type pair the upper half of the rectangle corresponds to live cell conditions and the lower half the fixed cell data. Solid bars indicate that all cells of a given type were either bound by the lectin (red or green) or not (grey). Diagonal red or green stripes indicate that only some but not all cells were stained. The small solid bars for PSA and LCA for fixed cells indicated that the respective lectin stained this cell type only in some but not all of the sea urchins we tested (for representative images see S1 Fig).
	Fig 3. Lectin staining of live settled coelomocytes.Total coelomocytes were stained with the indicated fluorescently labeled lectins (A-D: rhodamine; F-J: fluorescein), and then settled onto glass slides. Representative fluorescence images are shown that were taken on a Zeiss Axioimager.Z2 microscope with an Apotome.2 structured illumination accessory using a Plan-Apochromat 40x objective and a cooled CCD camera. The corresponding phase contrast images were taken using a color SLR camera. The identity of each cell is indicated in the phase contrast image (ph: phagocyte; rs: red spherule cell; v: vibratile cell). Live vibratile cells were not bound by any of the lectins under these conditions, and a phase contrast image of one such cell is shown for comparison (E).
	Fig 4. Lectin staining of live coelomocytes.Total coelomocytes were settled onto glass slides, and subsequently stained with the indicated fluorescently labeled lectins (A-D: rhodamine; E-J: fluorescein). Representative fluorescence images are shown that were taken on a Zeiss Axioimager.Z2 microscope with an Apotome.2 structured illumination accessory using a Plan-Apochromat 40x objective and a cooled CCD camera. The corresponding phase contrast images were taken using a color SLR camera. The identity of each cell is indicated in the phase contrast image (ph: phagocyte; rs: red spherule cell).
	Fig 5. Flow cytometry analysis of lectin stained coelomocytes.(A) Forward/side scatter profile of total coelomocytes from sea urchin B. The gating of distinct populations is shown as red, blue, and yellow ovals, and the percentage of live cells in each of these gates is provided. (B) Histogram plots of coelomocytes that were either unstained or stained with the indicated fluorescently labelled lectins from sea urchin B. The data from each of the three gates (red, blue, and yellow) is shown as an overlay and the percentage of positive cells is provided. (C) Total coelomocytes from sea urchin A were stained with the indicated combinations of fluorescently labeled lectins, and analyzed by flow cytometry. The forward/side scatter profiles of each gated population are show. The gates of the three distinct populations (red, blue, and green ovals) from Fig 5A and the percentage of events in each of the gates are provided.
	Fig 6. Flow cytometry based cell sorting of lectin-labeled coelomocytes.Total coelomocytes from sea urchin B were stained with DSL-fluorescein and LCA-rhodamine. Live cells (A) were gated based on their forward/side scatter profile, and four different populations (B) were sorted based on their distinct fluorescence profiles. (C) The forward/side scatter profiles of each indicated population (red dots) was overlaid on that of all cells in the sample (gray dots). (D) The expression levels of the phagocyte specific genes Sp-B7L3 and Sp-Egr, the genes unique for a mix of vibratile and colorless spherule cells Sp-P2rx4 and Sp-FoxJ1, and the red spherule cell specific genes Sp-Pks1 and Sp-Giant were quantified in each of the indicated sorted cell populations from sea urchin B (blue) and sea urchin C (red) by qRT-PCR. Values are normalized to the levels of Sp-RPL39 in each sample. The means of two technical replicates are shown and the error bars indicate the standard error of the mean.

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Allison, The effects of various fixatives on subsequent lectin binding to tissue sections. 1987, Pubmed