Bourguignon L Y, Tokuyasu K T, Singer S J
J Cell Physiol. 1978 Jun;95(3):239-57. doi: 10.1002/jcp.1040950302.
Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.
已对小鼠脾T淋巴细胞、B淋巴细胞及其他细胞表面受体的凝集素封帽和抗体封帽进行了实验，其中通过双荧光技术在同一细胞上测定凝集素或抗体的表面分布以及肌球蛋白或肌动蛋白的细胞内分布。为此，开发了一种细胞内染色的通用方法，旨在保存敏感抗原和脆弱的超微结构成分。该方法包括对细胞或组织进行轻度甲醛固定、注入浓缩蔗糖、快速冷冻以及制备厚度小于1微米的冰冻切片。然后将免疫荧光或其他合适的荧光试剂应用于解冻后的切片。在本实验中，使用基于F-肌动蛋白与重酶解肌球蛋白相互作用的荧光染色方法检测细胞内肌动蛋白，而通过间接免疫荧光法检测细胞内肌球蛋白。我们的发现是，每种凝集素或抗体试剂形成封帽时，总是伴随着帽下方肌球蛋白和肌动蛋白的聚集。这些及其他结果表明，封帽是一个活跃过程，其中肌动蛋白和肌球蛋白直接参与所有封帽的形成。这一观点对封帽的分子机制具有重要的新启示。
