Vogel G, Thilo L, Schwarz H, Steinhart R
J Cell Biol. 1980 Aug;86(2):456-65. doi: 10.1083/jcb.86.2.456.
The recognition step in the phagocytotic process of the unicellular amoeba dictyostelium discoideum was examined by analysis of mutants defective in phagocytosis, Reliable and simple assays were developed to measure endocytotic uptake. For pinocytosis, FITC-dextran was found to be a suitable fluid-phase marker; FITC-bacteria, latex beads, and erythrocytes were used as phagocytotic substrates. Ingested material was isolated in one step by centrifuging through highly viscous poly(ethyleneglycol) solutions and was analyzed optically. A selection procedure for isolating mutants defective in phagocytosis was devised using tungsten beads as particulate prey. Nonphagocytosing cells were isolated on the basis of their lower density. Three mutant strains were found exhibiting a clear-cut phenotype directly related to the phagocytotic event. In contrast to the situation in wild-type cells, uptake of E. coli B/r by mutant cells is specifically and competitively inhibited by glucose. Mutant amoeba phagocytose latex beads normally but not protein-coated latex, nonglucosylated bacteria, or erythrocytes. Cohesive properties of mutant cells are altered: they do not form EDTA-sensitive aggregates, and adhesiveness to glass or plastic surfaces is greatly reduced. Based upon these findings, a model for recognition in phagocytosis is proposed: (a) A lectin-type receptor specifically mediates binding of particles containing terminal glucose (E. coli B/r). (b) A second class of "nonspecific" receptors mediate binding of a variety of particles by hydrophobic interaction. Nonspecific binding is affected by mutation in such a way that only strongly hydrophobic (latex) but not more hydrophilic particles (e.g., protein-coated latex, bacteria, erythrocytes) can be phagocytosed by mutant amoebae.
通过对吞噬作用有缺陷的突变体进行分析，研究了单细胞变形虫盘基网柄菌吞噬过程中的识别步骤。开发了可靠且简单的测定方法来测量内吞摄取。对于胞饮作用，发现异硫氰酸荧光素标记的葡聚糖是一种合适的液相标记物；异硫氰酸荧光素标记的细菌、乳胶珠和红细胞用作吞噬底物。通过在高粘性聚乙二醇溶液中离心一步分离摄取的物质，并进行光学分析。设计了一种使用钨珠作为颗粒猎物来分离吞噬作用有缺陷的突变体的筛选程序。非吞噬细胞根据其较低的密度进行分离。发现三个突变株表现出与吞噬事件直接相关的明确表型。与野生型细胞的情况相反，突变细胞对大肠杆菌B/r的摄取受到葡萄糖的特异性和竞争性抑制。突变变形虫正常吞噬乳胶珠，但不吞噬蛋白包被的乳胶、非糖基化细菌或红细胞。突变细胞的凝聚特性发生改变：它们不形成对乙二胺四乙酸敏感的聚集体，并且对玻璃或塑料表面的粘附性大大降低。基于这些发现，提出了一种吞噬作用中的识别模型：(a) 一种凝集素型受体特异性介导含有末端葡萄糖的颗粒（大肠杆菌B/r）的结合。(b) 第二类“非特异性”受体通过疏水相互作用介导各种颗粒的结合。非特异性结合受到突变的影响，使得只有强疏水性（乳胶）而不是亲水性更强的颗粒（例如蛋白包被的乳胶、细菌、红细胞）能够被突变变形虫吞噬。
