INM - Leibniz Institute for New Materials , D-66123 Saarbrücken, Germany.
Department of Biophysics, Saarland University , D-66421 Homburg, Germany.
ACS Nano. 2017 Nov 28;11(11):11108-11117. doi: 10.1021/acsnano.7b05258. Epub 2017 Oct 16.
Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.
膜蛋白通过动态寡聚化形成活性复合物来控制细胞中的许多重要功能。然而，目前用于研究其在细胞结构中的分布和功能状态的分析方法受到限制。在这里，我们介绍了一种用于研究完整质膜中天然存在的单一膜蛋白的技术。SKBR3 乳腺癌细胞在带有薄氮化硅窗口的硅微芯片上生长。将细胞固定，并用量子点 (QD) 纳米颗粒特异性标记表皮生长因子受体 ErbB2。对于相关的荧光和液相电子显微镜，我们通过化学气相沉积 (CVD) 石墨烯膜将液体样品封闭。根据局部细胞厚度，QD 标记以低电子剂量在 2nm 的空间分辨率下成像。在几个不同的细胞位置确定了 ErbB2 受体的分布和化学计量组装，包括隧道纳米管，在连接部位发现了更高水平的同源二聚化。这种实验方法适用于广泛的细胞系和膜蛋白，特别适合于研究蛋白质分布和表达的细胞内和细胞间异质性。
