School of Life Sciences, SUSTech University.
Shenzhen PKU-HKUST Medical Center; Division of Life Science, Hong Kong University of Science and Technology.
J Vis Exp. 2024 Jan 19(203). doi: 10.3791/66080.
Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.
利用 GFP（绿色荧光蛋白）和其他荧光标签对活细胞中的蛋白质进行可视化，极大地提高了我们对蛋白质定位、动态和功能的理解。与免疫荧光相比，活细胞成像更准确地反映了蛋白质的定位，而不会出现组织固定带来的潜在假象。重要的是，活细胞成像能够对蛋白质水平和定位进行定量和时间特征分析，这对于理解细胞运动或分裂等动态生物学过程至关重要。然而，荧光标记方法的一个主要限制是需要足够高的蛋白质表达水平才能成功进行可视化。因此，许多表达水平相对较低的内源性荧光蛋白无法被检测到。另一方面，使用病毒启动子的异位表达有时会导致蛋白质在生理环境中的错误定位或功能改变。为了解决这些限制，提出了一种利用活胚胎中高度敏感的抗体介导的蛋白质检测方法，实质上是在无需组织固定的情况下进行免疫荧光。作为原理验证，原本在活胚胎中几乎无法检测到的内源性 GFP 标记 Notch 受体，在注射抗体后可以成功可视化。此外，该方法还被改编用于可视化活胚胎中的翻译后修饰（PTM），从而可以检测到早期胚胎发生过程中酪氨酸磷酸化模式的时间变化，并揭示出顶端膜下一种新的磷酸酪氨酸（p-Tyr）亚群。该方法可以进行修改以适应其他蛋白质特异性、标签特异性或 PTM 特异性抗体，并且应该与其他可注射模型生物或细胞系兼容。该方案为使用传统荧光标记方法以前难以检测的低丰度蛋白质或 PTM 的活细胞成像开辟了新的可能性。
