Liao Wen-Yun, Fugmann Sebastian D
Department of Biomedical Sciences, Chang Gung University, Kwei-Shan District, Tao-Yuan, Taiwan.
Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Kwei-Shan District, Tao-Yuan, Taiwan.
PLoS One. 2017 Nov 10;12(11):e0187987. doi: 10.1371/journal.pone.0187987. eCollection 2017.
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.
体腔细胞代表着棘皮动物的免疫细胞，但关于它们在免疫反应中作用的详细知识非常有限。研究体腔细胞生物学的一个主要挑战是缺乏试剂来识别和纯化由客观分子标记物定义的不同群体，而不是基于有时主观的形态学分类。已知脊椎动物中不同细胞类型之间的糖基化模式存在显著差异，此外，它们还会因发育阶段和特定谱系内的激活状态而有所不同。因此，识别细胞表面蛋白上不同聚糖结构的荧光标记凝集素经常被用于识别脊椎动物免疫系统中的离散细胞群体。在这里，我们使用了一组15种荧光标记的凝集素，通过荧光显微镜和流式细胞术来确定紫海胆体腔细胞表面糖基化特征的差异。其中8种凝集素（琥珀酰化麦胚凝集素、刀豆凝集素、豌豆凝集素、槐凝集素、马铃薯凝集素、番茄凝集素、曼陀罗凝集素、绒毛豌豆凝集素）对三种主要体腔细胞类型（吞噬细胞、红色球状体细胞和颤动细胞）的固定细胞和活细胞表现出不同的结合模式。重要的是，几乎所有凝集素仅与每种细胞类型中的一个细胞亚群结合。最后，我们建立了基于荧光标记凝集素的荧光激活细胞分选方法，作为一种基于分子标记物纯化不同紫海胆体腔细胞（亚）群体的策略。我们预计，这将成为未来研究中剖析不同体腔细胞在棘皮动物免疫中作用的常规方法。
