Lee G M, Johnstone B, Jacobson K, Caterson B
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599.
J Cell Biol. 1993 Dec;123(6 Pt 2):1899-907. doi: 10.1083/jcb.123.6.1899.
Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b-HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan-aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.
尽管细胞周基质厚度可达数微米，但由于其在相差显微镜或微分干涉差显微镜下不可见，所以是一种难以捉摸的结构。这种基质通过排除诸如固定红细胞等大颗粒而易于可视化，在胚胎发育和软骨维持中很重要。虽然已知围绕软骨细胞和多种其他细胞的细胞周基质主要由蛋白聚糖和透明质酸组成，后者与细胞表面受体结合，但其大分子组织仍然是推测性的。由于传统固定和脱水技术会导致细胞被膜塌陷，以前无法确定大分子组织。直到现在，还没有办法研究透明质酸与其聚集的蛋白聚糖在活细胞上的动态排列。在这项研究中，直接观察了从牛关节软骨分离的活细胞的细胞周基质中透明质酸 - 聚集蛋白聚糖复合物的排列和流动性。这些复合物用与硫酸角质素抗体5 - D - 4偶联的30至40纳米胶体金标记，并用视频增强光学显微镜观察。根据我们对细胞周基质大分子运动的观察，我们报告软骨细胞的细胞周基质是一种动态结构，由从细胞表面向外突出的单个拴系分子复合物组成。这些复合物经历有限的旋转或摆动。当细胞用促进基质成分产生的抗坏血酸培养时，细胞被膜的大小和金探针相对于质膜的位置没有改变。然而，拴系运动的速度和程度降低了。用链霉菌透明质酸酶处理去除了表现出拴系运动的分子。向经透明质酸酶处理过的细胞中添加透明质酸和聚集蛋白聚糖，产生了与天然细胞被膜观察到的相同标记模式和拴系运动。为了确定聚集蛋白聚糖是否负责复合物的延伸构型，仅向经透明质酸酶处理过的细胞中添加透明质酸。使用生物素化透明质酸结合区域（b - HABR）和金链霉抗生物素蛋白检测透明质酸的位置和流动性。发现金标记的b - HABR仅在细胞表面附近。基于这些观察结果，组成细胞被膜的透明质酸 - 聚集蛋白聚糖复合物被认为是以类似于先前描述的在良溶剂中的高密度接枝聚合物的方式以刷状构型延伸。
