Sanders S K, Craig S W
J Immunol. 1983 Jul;131(1):370-7.
When viable murine lymphocytes are incubated sequentially with a saturating amount of affinity-purified, rabbit anti-actin and highly conjugated FITC-goat anti-rabbit Ig, about 52% of mesenteric lymph node (MLN) lymphocytes and 36% of thymocytes exhibit a faint, but sharply punctate surface fluorescence. Cell surface actin (CSA) can be distinguished from staining of cytoplasmic actin in permeable cells, which are identified by their uptake of ethidium bromide. Staining of actin in ethidium bromide-permeable cells is 10-fold more intense than staining of actin on ethidium bromide-impermeable cells and is seen as uniformly fluorescent rings or crescents at the periphery of the cell and as dimmer, diffuse fluorescence centrally. Binding of rabbit anti-actin and goat anti-rabbit Ig to the lymphocyte cell surface is not mediated by Fc receptors; F(ab')2 fragments of these antibodies detect the same number of positive cells as do the intact molecules, and affinity-purified anti-KLH does not bind significantly. The cell surface stain, measured by flow cytometry or fluorescence microscopy, can be absorbed by pretreatment of the anti-actin with immobilized actin but not with IgG-Sepharose. Double-label experiments show that about 70% of the non-B cells and 30% of the MLN B cells bear detectable CSA. Although we have not ascertained the origin of CSA, we find that the number and brightness of cells exhibiting CSA cannot be increased by preincubating the cells with exogenous native skeletal muscle actin or with supernatant from dissociated MLN, indicating that there are no free binding sites for exogenous actin. The findings imply that either there is a developmentally expressed binding site(s) for actin, or that at various stages of development lymphocytes express a protein antigenically related to actin on their surface.
当活的小鼠淋巴细胞依次与饱和量的亲和纯化兔抗肌动蛋白和高度偶联的异硫氰酸荧光素（FITC）标记的羊抗兔免疫球蛋白（Ig）一起孵育时，约52%的肠系膜淋巴结（MLN）淋巴细胞和36%的胸腺细胞呈现出微弱但清晰的点状表面荧光。细胞表面肌动蛋白（CSA）可与可渗透细胞中细胞质肌动蛋白的染色区分开来，可渗透细胞通过摄取溴化乙锭来识别。溴化乙锭可渗透细胞中肌动蛋白的染色强度比溴化乙锭不可渗透细胞中肌动蛋白的染色强度高10倍，表现为细胞周边均匀的荧光环或月牙形，以及中央较暗的弥漫性荧光。兔抗肌动蛋白和羊抗兔Ig与淋巴细胞细胞表面的结合不是由Fc受体介导的；这些抗体的F(ab')2片段检测到的阳性细胞数量与完整分子相同，且亲和纯化的抗钥孔血蓝蛋白（KLH）不显著结合。通过流式细胞术或荧光显微镜测量的细胞表面染色可被固定化肌动蛋白预处理抗肌动蛋白所吸收，但不能被IgG - 琼脂糖吸收。双标记实验表明，约70%的非B细胞和30%的MLN B细胞带有可检测到的CSA。尽管我们尚未确定CSA的来源，但我们发现，通过用外源性天然骨骼肌肌动蛋白或解离的MLN上清液预孵育细胞，并不能增加呈现CSA的细胞数量和亮度，这表明不存在外源性肌动蛋白的游离结合位点。这些发现意味着，要么存在一种发育表达的肌动蛋白结合位点，要么在发育的各个阶段淋巴细胞在其表面表达一种与肌动蛋白抗原相关的蛋白质。
