Munier Godebert Annie, Weil Dominique, Safieddine Adham
Research Center Saint-Antoine (CRSA), CISA Flow Cytometry Facility, UMRS 938, Sorbonne University, Paris, France.
Sorbonne Université, CNRS, INSERM, Development, Adaptation and Ageing, Dev2A, Paris, France.
Nat Protoc. 2025 Jul 18. doi: 10.1038/s41596-025-01216-x.
Condensates are receiving increasing attention because of their ability to organize subcellular space. In eukaryotes, nuclear condensates include nucleoli and paraspeckles, and cytoplasmic ones include P-bodies (PBs) and stress granules. One approach to investigate condensate biology is through analyzing their protein and RNA content. However, purifying condensates remains a challenge because of their densities being similar to various other organelles, and the absence of protein markers accumulating exclusively in them. These limitations, combined with the generally low number of condensates per cell, necessitate new approaches to tackle their purification. Here, we present a protocol describing fluorescence-activated particle sorting (FAPS) for purifying condensates. In brief, FAPS involves fluorescently labeling condensates to identify and isolate them from other cellular components via sorting. In this Protocol, we focus on PB purification, quality control and downstream characterization of PB protein and RNA contents. Although originally developed to purify PBs from human cell lines, FAPS can be adapted to various condensates across model organisms. The procedure requires knowledge in basic cell culture, molecular biology and flow cytometry and access to a fluorescence-activated cell sorter with sufficient sensitivity. It requires ~25-30 d, including a hands-on period of 15 d, to complete. In summary, FAPS allows the characterization of the content of diverse condensates across cell types and organisms.
由于具有组织亚细胞空间的能力，凝聚物正受到越来越多的关注。在真核生物中，核凝聚物包括核仁和平行斑点，细胞质凝聚物包括P小体（PBs）和应激颗粒。研究凝聚物生物学的一种方法是分析它们的蛋白质和RNA含量。然而，纯化凝聚物仍然是一个挑战，因为它们的密度与各种其他细胞器相似，并且缺乏专门在其中积累的蛋白质标记物。这些限制，再加上每个细胞中凝聚物的数量通常较少，使得需要新的方法来解决它们的纯化问题。在这里，我们提出了一种描述用于纯化凝聚物的荧光激活颗粒分选（FAPS）的方案。简而言之，FAPS包括对凝聚物进行荧光标记，以便通过分选将它们与其他细胞成分识别并分离出来。在本方案中，我们专注于PB的纯化、质量控制以及PB蛋白质和RNA含量的下游表征。尽管FAPS最初是为了从人细胞系中纯化PB而开发的，但它可以适用于跨模式生物的各种凝聚物。该程序需要具备基础细胞培养、分子生物学和流式细胞术方面的知识，并能够使用具有足够灵敏度的荧光激活细胞分选仪。完成该程序大约需要25 - 30天，包括15天的实际操作时间。总之，FAPS能够对不同细胞类型和生物体中各种凝聚物的成分进行表征。
