Data from: Nucleolar sub-compartments in motion during rRNA synthesis inhibition: contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.核仁（nucleolus）可转录生成包含18S、5.8S及28S核糖体RNA（ribosomal RNA, rRNA）序列的大型多顺反子转录本（47S前体），并介导核糖体RNA前体（pre-rRNA）加工的绝大多数核内步骤。其组分丰富多样，包含浓缩染色质与活性核糖体RNA基因，后者呈现更为开放的构象，因此核仁是染色体区域组织（chromosome territory organization）的经典研究模型。活性核糖体RNA基因聚集于多个纤维中心（fibrillar centers, FCs）内，并通过上游结合因子（Upstream Binding Factor, UBF）维持开放构象。本研究借助放线菌素D（Actinomycin D, AMD）抑制核糖体RNA合成所诱导的核仁组分可重复性重构，探究纤维中心与核仁浓缩染色质的时空重构进程。为实现该研究目标，我们采用了两种互补的实验手段：其一，对表达一种或多种绿色荧光蛋白（green fluorescent protein, GFP）标记蛋白（核仁纤维蛋白fibrillarin、UBF、组蛋白H2B）的细胞进行延时共聚焦成像（time-lapse confocal imaging）；其二，对参与重构过程的核仁组分开展超微结构鉴定。通过对延时共聚焦显微镜获取的图像进行精细化三维成像分析，我们证实：放线菌素D处理不会引发单个细胞核内不同核仁的融合，也不会改变其相对位置。与之相对，我们观察到每个核仁内的纤维中心与核仁浓缩染色质均会逐步聚集并融合。为在更高分辨率下解析纤维中心与浓缩染色质的重构过程，我们对同一细胞开展了关联光镜-电镜（correlative light and electron microscopy, CLEM）成像。实验结果表明，核仁内浓缩染色质丝位于复杂的囊泡三维网络中。经放线菌素D处理后，这些结构会发生聚集，随后向核仁周缘浓缩染色质迁移并最终与之融合。在迁移过程中，所有与核仁内浓缩染色质相连的纤维中心会被后者牵拉，最终聚集为分布于核仁周边的帽状结构。