Phase Separation in Biology and Disease; Current Perspectives and Open Questions

Abstract

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in^1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.^4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ∼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.

Keywords: biomolecular condensates; membraneless organelles; phase separation.

Figure 1.

Diverse nuclear processes are compartmentalized within condensates with distinct compositions, locations, and dynamics. Five different nuclear condensates (colored circles) are illustrated to highlight how each engages with specific genomic loci and/or chromatin features (grey line). Investigating differential composition, relationship with genomic loci, and dynamics of formation are crucial to understanding the regulation and function of these high-order assemblies. Reprinted from [118] with permission from Elsevier.

Figure 2.

Different types of biomolecular condensates form in neurons. Left: Neuronal storage and transport granules composed of RNA-binding proteins and RNAs. Right: The synapse is as an example of a multiphase system. Scheme modified from Milicevic, et al. [119].

Figure 3.

Examples of biomolecular condensates in bacteria and protists. A) Rubisco and multivalent scaffold proteins phase separate through complex coacervation. Coacervates fuse to form the pyrenoid in eukaryotic algae. In bacteria, shell proteins may act to restrict coalescence of carboxysomes, controlling their size. Reprinted from [120] with permission from Elsevier. B, C) Transmission electron micrographs of Chlamydomonas reinhardtii and Synechococcus elongatus, respectively. Arrowheads mark the pyrenoid (black) and carboxysomes (white). Reproduced from [53, 71] with permission from Springer Nature.

Figure 4.

Aberrant biomolecular phase separation in cancer. A) Mislocalized PML bodies cluster telomeres to promote homology-directed DNA synthesis for telomere maintenance in ALT cancer cells. B, C) Several fusion oncoproteins that drive oncogenesis in diverse cancers have been shown to undergo phase separation to form aberrant (B) nuclear transcriptional condensates or (C) cytoplasmic signaling condensates. B and C reproduced from [81] with permission; copyright 2023 by the authors.

Figure 5.

High throughput, plate-based screening approaches for identification of condensate modulators. A) In-cell screen, with endogenous levels or overexpression of the marker protein; condensation can be induced by stimuli or by the level of expression of constituents; B) In-cell screen with light-induced condensates; the marker protein is engineered to express a light-inducible oligomerization domain which nucleates condensate formation [115]; C) In vitro screens; the condensates are reconstituted in buffer with a controlled number of recombinant components (selective simplicity), or by seeding cell lysates with the scaffold of choice (controlled complexity).