Conrad Mara, DeNobile JoAnna, Chaikhoutdinov Irina, Escribano Douglas, Lee Kyeng-Gea, Cohen William D
Department of Biological Sciences, Hunter College, New York, NY 10021, USA.
Biol Bull. 2004 Aug;207(1):56-66. doi: 10.2307/1543628.
One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.
循环中的鲎血细胞的主要功能之一是在接收到适当信号后实现血液凝固。然而，在先前关于其细胞骨架组织的研究中，鲎血细胞与脊椎动物血小板和血栓细胞在本质上相似的假设尚未得到充分验证。早期数据来自对有限数量细胞的薄切片进行的透射电子显微镜（TEM）观察，而现在，能够保留细胞形态的改进荧光标记方法使我们能够在完整的单个血细胞以及激活前后的大量血细胞群体中研究F-肌动蛋白和微管组织。抗微管蛋白免疫荧光显示，在大多数未激活的细胞中，微管的边缘带（MB）呈椭圆形，基本上不存在其他微管。然而，也观察到了少数呈盘状或尖状的细胞亚群，其微管的相应排列暗示了形态发生中间体。德克萨斯红鬼笔环肽标记了未激活血细胞中富含F-肌动蛋白的皮质，这解释了在TEM薄切片中观察到的MB和颗粒与质膜的分离，并支持了早期关于MB通过对皮质层施加压力来维持扁平血细胞形态的模型。细菌脂多糖激活后的形状转变主要归因于细胞外区域中呈刺状和扩散状的F-肌动蛋白，MB转变为扭曲的、与细胞核相关的形式，最终变得无法辨认。这些激活前后主要的细胞骨架特征类似于血小板和非哺乳动物脊椎动物血栓细胞的特征，支持将鲎血细胞视为更特化的凝血细胞类型的代表性进化前体。
