Bee J A, von der Mark K
Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK.
J Cell Sci. 1990 Jul;96 ( Pt 3):527-36. doi: 10.1242/jcs.96.3.527.
To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5 K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5 K cell adhesion molecule.
为了研究肢体骨骼元件形成过程中细胞间黏附的机制，我们在体外研究了肢芽细胞聚集的过程。肢芽细胞在无钙条件下经胰蛋白酶：胶原酶解离后立即具有聚集能力。这种聚集在很大程度上不依赖Ca2+(CI)，并被环己酰亚胺完全且可逆地抑制。相比之下，当肢芽细胞首先从无Ca2+的胰蛋白酶：胶原酶解离中恢复时，存活细胞群体的聚集完全依赖Ca2+(CD)，并被环己酰亚胺完全且可逆地抑制。初始细胞解离过程中存在外源钙会保留功能性的CD聚集机制。然而，用乙二醇双四乙酸(EGTA)孵育这些细胞会释放CD成分，并将细胞转化为主要的CI聚集。用表现出恢复的CD聚集机制的肢芽细胞免疫兔子，并筛选所得免疫血清对细胞聚集的影响。相对于免疫前血清，完整的免疫IgG凝集解离的肢芽细胞，而免疫Fab片段抑制其聚集。抑制聚集的抗血清识别五种主要的肢芽细胞表面成分，其表观分子量分别为72K、50K、23K、14.5K和8.5K(K = 10(3) Mr)。通过蔗糖梯度密度离心分离肢芽细胞表面质膜，并用溴化氰将去污剂溶解的蛋白质偶联到琼脂糖4B上。将等量的细胞表面质膜蛋白用125I碘化并应用于亲和柱。在外源钙存在下进行肢芽细胞表面蛋白亲和层析，得到一种表观分子量约为8.5K的单一蛋白质。这种蛋白质分子在0.6M NaCl处洗脱，表明具有高亲和力，被抑制聚集的抗血清识别，并且其本身能够抑制CD肢芽细胞聚集。从专门针对亲和纯化物质的兔抗血清制备的Fab片段也抑制CD肢芽细胞聚集，并且这种抑制被8.5K蛋白质中和。因此，我们的数据表明CD肢芽细胞聚集不是由纤连蛋白和/或I型胶原介导的，并表明该过程由一种新的8.5K细胞黏附分子控制。
