Kam Z, Volberg T, Geiger B
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
J Cell Sci. 1995 Mar;108 ( Pt 3):1051-62. doi: 10.1242/jcs.108.3.1051.
Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.
共振能量转移（RET）的定量显微成像技术被用于对细胞黏附中几种细胞骨架成分进行免疫高分辨率邻近图谱分析。为进行此项分析，开发了一种显微系统，该系统由一个高度稳定的场照明器、用于快速多色成像的计算机控制滤光轮以及一个灵敏的高分辨率电荷耦合器件（CCD）相机组成，能够进行定量数据记录和处理。利用该系统，我们研究了四种与黏附相关的蛋白质，即纽蛋白、踝蛋白、α - 辅肌动蛋白和肌动蛋白的空间相互关系及组织情况。使用荧光素和罗丹明偶联抗体或鬼笔环肽对培养的鸡晶状体细胞中的每种连接分子进行双重标记。通过荧光素激发和罗丹明发射滤光设置获取RET图像，对荧光素和罗丹明荧光进行校正，并将其归一化到荧光素图像。结果表明，在粘着斑中纽蛋白、踝蛋白和F - 肌动蛋白的局部密度较高，其平均RET值分别为15%、12%和10%。另一方面，对同一细胞进行α - 辅肌动蛋白的双重免疫荧光标记后，观察到相对较低的值（小于1%）。对这四种蛋白质中的两两进行双重间接标记（使用荧光团偶联抗体或鬼笔环肽），除α - 辅肌动蛋白和肌动蛋白这一对产生显著较高的值（13 - 15%）外，其他对的RET值均为5%或更低。这些结果表明，尽管它们的染色模式重叠，但在光学显微镜分辨率水平上，斑块蛋白纽蛋白和踝蛋白在分子水平上并非均匀散布，而是形成分离的簇。另一方面，α - 辅肌动蛋白似乎不形成这样的簇，而是与肌动蛋白紧密相互作用。我们在此讨论RET测量的概念和应用方面以及结果对黏附型连接的亚细胞分子组织的影响。
