Phase Separation in the Nucleus and at the Nuclear Periphery during Post-Mitotic Nuclear Envelope Reformation

Abstract

Membrane-enclosed organelle compartmentalization is not the only way by which cell processes are spatially organized. Phase separation is emerging as a new driver in the organization of membrane-less compartments and biological processes. Liquid-liquid phase separation has been indicated as a new way to control the kinetics of molecular reactions and is based on weak multivalent interactions affecting the stoichiometry of the molecules involved. In the nucleus, liquid-liquid phase separation may represent an ancestral means of controlling genomic activity by forming discrete chromatin regions, regulating transcriptional activity, contributing to the assembly of DNA damage response foci, and controlling the organization of chromosomes. Liquid-liquid phase separation also contributes to chromatin function through its role in the reorganization of the nuclear periphery in the post-mitotic phase. Herein, we describe the basic principles regulating liquid-liquid phase separation, analyze examples of phase separation occurring in the nucleus, and dedicate attention to the implication of liquid-liquid phase separation in the reorganization of the nuclear periphery by the endosomal sorting complexes required for transport (ESCRT) machinery. Although some caution is warranted, current scientific knowledge allows for the hypothesis that many factors and processes in the cell are yet to be discovered which are functionally associated with phase separation.

Keywords: chromatin organization; liquid–liquid phase separation; nuclear condensate; post-mitotic nuclear envelope; telomeres.

Figure 1

Features shared by LLPS condensates. (A) LLPS condensates (gray circles) must be round and prone to coalesce. (B) LLPS condensates depend on the concentration of the molecules (blue and yellow circles, different types of molecules) composing them and on environmental factors that influence their formation and dissolution. (C) One system used to study LLPS is FRAP, a technique quantifying fluorescence recovery kinetics after bleaching. The figure schematized the comparison of the recovery time for full photobleaching of an LLPS-dependent and an LLPS-independent organelle. In LLPS, fluorescence recovery has been often suggested to proceed faster. (D) LLPS condensates can be sensitive to the 1,6-hexanediol, which disrupts weak hydrophobic interactions.

Figure 2

Factors contributing to LLPS condensates formation. (A) Scaffold (blue circles) and client (yellow circles) components of LLPS condensates and their role in condensate formation. Clients bind scaffold elements in a regulated way, and their composition varies in response to stimuli (orange lightning). Clients are dispensable for the assembly of the condensate; otherwise, scaffold elements are necessary to assemble a LLPS condensate. (B) Three classes of proteins contribute to LLPS: proteins containing repetitions of modular domains (green boxes); proteins that contain ordered and intrinsically disordered regions (IDRs; green); intrinsically disordered proteins (IDPs). (C) Examples of non-covalent pi-cation interactions between aromatic residues (blue hexagons) and cations (red circles) of positively charged amino acids that could account for LLPS formation by proteins with IDRs.

Figure 3

Examples of LLPS condensates in the nucleus. (A) LLPS based heterochromatin organization acts at chromatin regions enriched in H3K9me3 histone modifications (green) bound by proteins containing chromodomains, such as HP1 (red) and SUVAR39H1 (light blue), increasing the compaction of the chromatin. (B) ALT-associated PML bodies (APBs) are formed by LLPS clustering telomeres (purple), PML protein (blue), and other factors such as BLM (green), DNA damage response proteins (orange), and the long non-coding RNA associated with telomeres, (TERRA; red). (C) At site of DNA damage, foci are promoted by 53BP1 (blue) and by the long non-coding RNAs (dilncRNA; red), which increases the concentration of DNA damage response proteins (orange) such as p53.

Figure 4

LLPS at the reforming nuclear envelope. During nuclear envelope reformation, membranes are reassembled and nuclear holes surrounding remaining microtubules are sealed by ESCRT machinery. The recruitment of CHMP7 (green) and ESCRT III nucleation is promoted by LLPS of LEM2 (pink) that interacts with BAF (red), which in turn interacts with chromatin (purple) and with CHMP7 through its winged-helix domain.