DOI: 10.5061/dryad.pc78t
title: Data from: Nucleolar sub-compartments in motion during rRNA synthesis inhibition: contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant

description: 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.
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