Over the last two decades, developments in single-molecule microscopy (SMM) have enabled imaging and tracking of individual, fluorescently labelled proteins in biological systems, and most of these studies have focused on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied the protein dynamics of the membrane protein H-Ras, mutants of this protein, and its membrane-anchoring domain, C10H-Ras, in epidermal cells of living two-day-old embryos, using a total internal reflection fluorescence microscopy (TIRFM) setup. For all proteins studied, our results consistently confirm the presence of a fast- and a slow-diffusing subpopulations of molecules, which both confine to microdomains within the plasma membrane. Although the mobility patterns of H-Ras, mutants of this proteins and its membrane-anchoring domain were remarkably similar, the constitutively active H-Ras mutant, H-Ras^V12, exhibited significantly higher diffusion rates than the wild-type H-Ras and its inactive mutant, H-Ras^N17. Ultimately, we characterized variability in our data obtained using the zebrafish embryo model and demonstrated that differences between cells within the same embryo are the largest source of variation in our data. Our findings are in line with a model in which the cellular architecture of individual cells within a tissue determine the mobility of H-Ras proteins anchored in the plasma membrane cytoplasmic leaflet. Thereby, our results underline the growing importance of performing SMM imaging in vivo in order to better understand factors influencing the protein dynamics in an intact living organism.

中文翻译：

---

体内 H-Ras 迁移模式的分析显示表皮组织内的细胞异质性

在过去的二十年中，单分子显微镜 (SMM) 的发展使生物系统中单个荧光标记蛋白质的成像和跟踪成为可能，其中大部分研究都集中在培养细胞内蛋白质迁移模式的分析上。在本研究中，SMM 应用于体内，使用斑马鱼胚胎模型。我们使用全内反射荧光显微镜 (TIRFM) 研究了膜蛋白 H-Ras、该蛋白质的突变体及其膜锚定域 C10H-Ras 在活两天大胚胎的表皮细胞中的蛋白质动力学） 设置。对于所有研究的蛋白质，我们的结果一致证实存在快速和缓慢扩散的分子亚群，它们都局限于质膜内的微域。尽管 H-Ras、该蛋白质的突变体及其膜锚定域的迁移模式非常相似，但组成型活性的 H-Ras 突变体 H-Ras ^V12表现出比野生型 H-Ras 和它的非活性突变体 H-Ras ^N17. 最终，我们对使用斑马鱼胚胎模型获得的数据的变异性进行了表征，并证明同一胚胎内细胞之间的差异是我们数据中最大的变异来源。我们的发现与一个模型一致，在该模型中，组织内单个细胞的细胞结构决定了锚定在质膜细胞质小叶中的 H-Ras 蛋白的移动性。因此，我们的结果强调了在体内进行 SMM 成像的重要性，以便更好地了解影响完整生物体中蛋白质动力学的因素。