DOI: 10.5061/dryad.vx0k6dk59
title: Data from: Hierarchical interactions between nucleolar and heterochromatin condensates are mediated by a dual-affinity protein

description: Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH),
 reflecting a conserved spatial association between the two largest
 biomolecular condensates in eukaryotic nuclei. Nucleoli are the sites of
 ribosome synthesis, while the repeat-rich PCH is essential for chromosome
 segregation, genome stability, and transcriptional silencing, yet the
 mechanisms for their co-assembly are unclear. Here, we use high-resolution
 live imaging during Drosophila embryogenesis and reveal that de novo
 establishment of PCH-nucleolar associations is highly dynamic, as PCH
 transitions from extending along the nuclear edge to surrounding the
 nucleolus. Eliminating the nucleolus by removing the ribosomal RNA genes
 (rDNA) disrupted this process, causing increased PCH compaction followed
 by its reorganization into a toroidal structure. Further, in embryos
 lacking rDNA, nucleolar proteins were redistributed into new bodies or
 ‘neocondensates’, including enrichment in the PCH toroidal hole. Combining
 these in vivo observations with molecular dynamics simulations based on
 multiphase wetting theory revealed that nucleolar-PCH associations could
 be mediated by a hierarchy of interaction strengths between PCH, nucleoli,
 and proteins with dual affinities for both compartments. We validated this
 model by identifying such a protein, a DEAD-Box RNA Helicase called
 Pitchoune, and showed that modulating its affinity for either nucleolar or
 PCH components alters nucleolar-PCH organization. Together, this study
 unveils a dynamic program for establishing nucleolar-PCH associations
 during animal development and demonstrates how interaction hierarchies and
 dual-affinity molecular linkers co-organize compositionally distinct
 condensates.
description: # Hierarchical interactions between nucleolar and heterochromatin
 condensates are mediated by a dual-affinity protein Dataset DOI:
 [10.5061/dryad.vx0k6dk59](https://doi.org/10.5061/dryad.vx0k6dk59) ##
 Description of the data and file structure **Dataset Title**\ Microscopy
 source data for “Hierarchical interactions between nucleolar and
 heterochromatin condensates are mediated by a dual-affinity protein.”
 **Authors**\ Srivarsha Rajshekar, Omar Adame-Arana, Gaurav Bajpai, Serafin
 U. Colmenares, Hannah Papoi, Lucy D. Brennan, Shingo Tsukamoto, Samuel
 Safran, Gary H. Karpen **Corresponding Author Contact**\
 [gkarpen@berkeley.edu](mailto:gkarpen@berkeley.edu) **First Author
 Contact**\ [srajshekar@berkeley.edu](mailto:srajshekar@berkeley.edu)
 **Dataset Overview**\ This dataset contains the source data for all the
 microscopy images presented in the figures of the associated publication.
 Data are organized by figure type (main figures and extended data
 figures). **Associated Publication**\ Rajshekar S, Adame-Arana O, Bajpai
 G, Colmenares SU, Papoi H, Brennan LD, Tsukamoto S, Safran S, Karpen
 GH. *Hierarchical interactions between nucleolar and heterochromatin
 condensates are mediated by a dual-affinity protein. Nature Cell Biology.*
 2025. ### Files and variables #### File:
 RajshekarS_2025_Dryad_Submission.zip **Description:** This master dataset
 contains all the microscopy data used to generate the figures in Rajshekar
 et al., 2025.  **Sub-folders** Main Figures Raw Data: Contains microscopy
 files corresponding to Figures 1, 2, 4, and 6 in the published article.
 Extended Data Figures Raw Data: Contains microscopy files corresponding to
 Extended Data Figures 1, 2, 3, 5, 6, 7, and 9. **File Nomenclature**\
 Filenames include: (1) Figure and panel number. (2) Experimental condition
 (e.g., developmental stage, cell type, genetic background). (3) Targeted
 molecule and fluorophore (e.g., protein of interest with tagged
 fluorescent protein). **File Format and Access**\ File types include .czi
 (Airyscan-processed) or .tif. All files can be accessed using Fiji/ImageJ.
 **Acquisition Notes**\ Detailed imaging parameters and methods are
 available in the *Methods* section of the associated publication and in
 the Light Microscopy Reporting Table supplement.