Schaaf Marcel J M, Koopmans Wiepke J A, Meckel Tobias, van Noort John, Snaar-Jagalska B Ewa, Schmidt Thomas S, Spaink Herman P
Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands.
Biophys J. 2009 Aug 19;97(4):1206-14. doi: 10.1016/j.bpj.2009.05.044.
It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.
多年来，通过单分子显微镜研究荧光标记蛋白的动力学已成为可能，但直到现在，这项技术仅应用于培养中的单个细胞。在本研究中，它被扩展到干细胞和活体脊椎动物生物体。作为感兴趣的分子，我们使用了与人H-Ras膜锚定蛋白融合的黄色荧光蛋白，该蛋白已被证明可作为质膜锚定蛋白的模型。我们使用宽场荧光显微镜装置来观察斑马鱼细胞系（ZF4）和原代胚胎干细胞中的单个分子。全内反射显微镜装置用于活体成像，特别是在2天大的斑马鱼胚胎皮肤的表皮细胞中。我们的结果表明，在活生物体中存在膜微区，其中膜蛋白的扩散受到限制。这种膜组织与在培养细胞中观察到的有显著差异，说明了在活生物体中进行单分子显微镜研究的相关性。
