Haidar Ali, Simonelig Martine, Ramat Anne
Institute of Human Genetics, Université de Montpellier, CNRS, Montpellier, France.
Bio Protoc. 2025 Mar 20;15(6):e5242. doi: 10.21769/BioProtoc.5242.
Super-resolution imaging of RNA-protein (RNP) condensates has shown that most are composed of different immiscible phases reflected by a heterogenous distribution of their main components. Linking RNA-protein condensate's inner organization with their different functions in mRNA regulation remains a challenge, particularly in multicellular organisms. germ granules are a model of RNA-protein condensates known for their role in mRNA storage and localized protein production in the early embryo. Present at the posterior pole of the embryo within a specialized cytoplasm called germplasm, they are composed of maternal mRNAs as well as four main proteins that play a key role in germ granule formation, maintenance, and function. Germ granules are necessary and sufficient to drive germ cell formation through translational regulation of maternal mRNAs such as . Due to their localization at the posterior tip of the ovoid embryo and small size, the classical imaging setup does not provide enough resolution to reach their inner organization. Here, we present a specific mounting design that reduces the distance between the germ granule and the objectives. This method provides optimal resolution for the imaging of germ granules by super-resolution microscopy, allowing us to demonstrate their biphasic organization characterized by the enrichment of the four main proteins in the outermost part of the granule. Furthermore, combined with the direct visualization of mRNA translation using the Suntag approach, this method enables the localization of translation events within the germ granule's inner organization and thus reveals the spatial organization of its functions. This approach reveals how germ granules serve simultaneously as mRNA storage hubs and sites of translation activation during development. This work also highlights the importance of considering condensates' inner organization when investigating their functions. Key features • Method for super-resolution imaging of germ granules in early embryo. • Analysis of RNP condensate functional organization. • Simultaneous recording of RNP condensate function and organization.
RNA-蛋白质（RNP）凝聚物的超分辨率成像表明，大多数凝聚物由不同的不混溶相组成，这通过其主要成分的异质分布得以体现。将RNA-蛋白质凝聚物的内部组织与其在mRNA调控中的不同功能联系起来仍然是一项挑战，尤其是在多细胞生物中。生殖颗粒是RNA-蛋白质凝聚物的一种模型，因其在早期胚胎中的mRNA储存和局部蛋白质产生中的作用而闻名。它们存在于胚胎后极的一种称为种质的特殊细胞质中，由母体mRNA以及四种在生殖颗粒形成、维持和功能中起关键作用的主要蛋白质组成。生殖颗粒对于通过对母体mRNA（如 ）的翻译调控来驱动生殖细胞形成是必要且充分的。由于它们位于卵形胚胎的后尖端且尺寸较小，传统的成像设置无法提供足够的分辨率来观察其内部组织。在此，我们提出了一种特定的固定设计，可减少生殖颗粒与物镜之间的距离。这种方法通过超分辨率显微镜为生殖颗粒成像提供了最佳分辨率，使我们能够证明其双相组织，其特征是四种主要蛋白质在颗粒的最外层富集。此外，结合使用SunTag方法对mRNA翻译的直接可视化，这种方法能够在生殖颗粒的内部组织中定位翻译事件，从而揭示其功能的空间组织。这种方法揭示了生殖颗粒在发育过程中如何同时作为mRNA储存中心和翻译激活位点。这项工作还强调了在研究凝聚物功能时考虑其内部组织的重要性。关键特性 • 早期胚胎中生殖颗粒的超分辨率成像方法。 • RNP凝聚物功能组织的分析。 • RNP凝聚物功能与组织的同步记录。
