Live-cell intracellular immunofluorescence

摘要 免疫荧光 (IF) 是基础生物学研究和临床样本最重要的工具之一 成像由于抗体的广泛可用性以及荧光成像的分辨率和灵敏度。 尽管自最初发明以来 IF 在过去约 60 年里产生了大量数据，但 IF 仍面临着一些问题 来自一个根本的限制：无法询问活细胞中的细胞内靶标（细胞必须是 首先固定，因此仅提供动态小区信令过程的快照）。考虑到 3D 结构 细胞中具有生物学意义的细胞内靶点比细胞膜靶点多得多， 但免疫制剂，如抗体、抗体片段、肽、纳米抗体和 scFv， 高亲水性大分子，不能自发穿过细胞膜。克服障碍 细胞膜和内吞作用对于防止完整生物分子进入细胞非常有效 细胞质中，我们建议开发一种基于独特概念的细胞内蛋白质递送技术：非 共价胆固醇标记。与主要基于 内吞作用（由于捕获的成像剂产生高背景，不适合活细胞成像 在内体中），我们的小分子标签使蛋白质能够渗透细胞膜。更多 重要的是，这是在膜上不产生导致细胞毒性的孔的情况下实现的。如果成功的话，这 平台技术将为分子生物学、药物开发和细胞打开一个全新的维度 工程。

ABSTRACT Immunofluorescence (IF) is one of the most important tools for both basic biology research and clinical sample imaging due to the broad availability of antibodies and the resolution and sensitivity of fluorescence imaging. Despite the tremendous amount of data produced by IF in the past ~60 years since its initial invention, IF suffers from a fundamental limitation: incapability of interrogating intracellular targets in live cells (cells have to be fixed first, thus only providing a snapshot of the dynamic cell signaling process). Considering the 3D structure of cells, there are far more intracellular targets with biological significance than their cell-membrane counterparts, but immunological agents such as antibodies, antibody fragments, peptides, nanobodies, and scFvs, being highly hydrophilic macromolecules, cannot spontaneously cross the cell membranes. To overcome the barriers of cell membrane and endocytosis that are extremely effective in preventing intact biomolecules to enter the cytosol, we propose to develop an intracellular protein delivery technology building on a unique concept: non- covalent cholesterol tagging. In contrast to conventional delivery technologies that are mostly based on endocytosis (inappropriate for live cell imaging due to the high background generated by imaging agents trapped in endosomes), our small-molecule tag enables proteins to permeate through the cell membrane. More importantly, this is achieved without generating pores in the membrane that cause cytotoxicity. If successful, this platform technology should open a whole new dimension for molecular biology, drug development, and cell engineering.