Department of Cellular Biology, University of Georgia, Athens, GA 30602.
School of Electrical and Computer Engineering, University of Georgia, Athens, GA 30602.
Proc Natl Acad Sci U S A. 2021 Jun 8;118(23). doi: 10.1073/pnas.2024690118.
The impact of the experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in .
实验系统对现代生物学研究的影响不可低估。能够标记内源性表达的蛋白质对于最大限度地利用这种模式生物至关重要。在这里，我们描述了一种在特定细胞类型中特异性标记内源性蛋白质的方法，使用自互补的分裂荧光蛋白（split FPs）。将 FP 编码序列的一小段插入特定的基因组位置，而剩余的 FP 则使用可用的驱动线表达。因此，互补荧光允许检查特定细胞中的蛋白质定位。此外，当在同一基因组位置插入短 FP 片段的串联重复时，我们可以大大增强荧光信号。在亚细胞水平的活细胞成像中，增强的信号非常有价值。我们还可以使用正交分裂 FPs 实现多色标记系统。然而，除了 split GFP 之外，其他正交分裂 FPs 不适用于体内成像。通过蛋白质工程和体内功能研究，我们报告了一种红色分裂 FP，我们可以用于复杂组织中内源性蛋白质的双光子可视化。使用这两个正交的分裂 FP 系统，我们可以同时对位于不同亚突触隔室中的蛋白质进行成像。我们的方法允许我们研究在 中的几乎任何细胞类型中内源性表达的多种蛋白质之间的接近程度和定位。
