Stolpen A H, Pober J S, Brown C S, Golan D E
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1988 Mar;85(6):1844-8. doi: 10.1073/pnas.85.6.1844.
Interferon gamma induces striking phenotypic alterations in confluent cultures of human vascular endothelial cells (HEC), including cell shape change from polygonal to elongated and cytoskeletal actin rearrangement from dense peripheral bands to longitudinal bundles of stress fibers. Since many transmembrane proteins, including class I major histocompatibility complex (MHC) proteins, interact with cytoskeletal actin, an interferon-gamma-induced anisotropic arrangement of stress fibers might cause anisotropic lateral diffusion of HEC class I MHC proteins. To test this hypothesis, we adapted the fluorescence photobleaching recovery technique to allow measurement of anisotropic diffusion of fluorescently labeled molecules on two-dimensional surfaces. A highly eccentric elliptical Gaussian laser beam was used to photobleach the sample and to monitor fluorescence recovery. In this technique, named "line fluorescence photobleaching recovery," lateral diffusion is measured along that axis of the sample that is perpendicular to the major axis of the elliptical beam. The lateral diffusion coefficient and fractional mobility are obtained by fitting the experimental data to a theoretical recovery curve, the form of which is determined by the solution to a modified version of the diffusion equation in which a tensor is used to describe diffusion in two orthogonal directions. Fluorescein-conjugated murine monoclonal antibodies were used to label class I MHC proteins on interferon-gamma-treated HEC and human dermal fibroblasts. These two cultured human cell types were found to be similar in their elongated shape and anisotropic stress fiber organization. Class I MHC protein lateral mobility was compared to that of fluorescein-labeled phosphatidyl-ethanolamine, a membrane phospholipid probe. Class I MHC proteins diffused anisotropically on human dermal fibroblasts, whereas fluorescein-labeled phosphatidylethanolamine diffused isotropically on this cell type. In contrast, both class I MHC proteins and fluorescein-labeled phosphatidylethanolamine diffused isotropically on interferon-gamma-treated HEC. These data suggest that neither elongated shape nor anisotropic stress fiber arrangement is sufficient to induce anisotropic diffusion of proteins on the HEC plasma membrane.
干扰素γ可诱导人血管内皮细胞（HEC）汇合培养物发生显著的表型改变，包括细胞形状从多边形变为细长形，以及细胞骨架肌动蛋白从密集的周边带重新排列为纵向的应力纤维束。由于许多跨膜蛋白，包括I类主要组织相容性复合体（MHC）蛋白，都与细胞骨架肌动蛋白相互作用，干扰素γ诱导的应力纤维各向异性排列可能会导致HEC I类MHC蛋白的各向异性侧向扩散。为了验证这一假设，我们采用了荧光光漂白恢复技术，以测量荧光标记分子在二维表面上的各向异性扩散。使用高度偏心的椭圆形高斯激光束对样品进行光漂白并监测荧光恢复。在这项名为“线荧光光漂白恢复”的技术中，侧向扩散是沿着样品中垂直于椭圆光束主轴的轴进行测量的。通过将实验数据拟合到理论恢复曲线来获得侧向扩散系数和分数迁移率，该曲线的形式由扩散方程的修正版本的解确定，其中使用张量来描述在两个正交方向上的扩散。用荧光素偶联的鼠单克隆抗体标记经干扰素γ处理的HEC和人皮肤成纤维细胞上的I类MHC蛋白。发现这两种培养的人类细胞类型在其细长形状和各向异性应力纤维组织方面相似。将I类MHC蛋白的侧向迁移率与荧光素标记的磷脂酰乙醇胺（一种膜磷脂探针）的侧向迁移率进行比较。I类MHC蛋白在人皮肤成纤维细胞上各向异性扩散，而荧光素标记的磷脂酰乙醇胺在这种细胞类型上各向同性扩散。相比之下，I类MHC蛋白和荧光素标记的磷脂酰乙醇胺在经干扰素γ处理的HEC上均各向同性扩散。这些数据表明，细长的形状和各向异性的应力纤维排列都不足以诱导HEC质膜上蛋白质的各向异性扩散。
