Introduction: Biophysics of biomolecular condensates (2021)

Cells have developed dedicated compartments to perform biochemical reactions. Rapidly emerging evidence shows that many of these compartments are not surrounded by membranes. They are termed membrane-less organelles or biomolecular condensates. Biomolecular condensates form through a process termed liquid–liquid phase separation (LLPS). Key components of biomolecular condensates are intrinsically disordered proteins as well as nucleic acids. Changes in the regulation and material properties of biomolecular condensates are associated with several human diseases. In order to gain insight into the molecular underpinnings of biochemical reactions and disease-associated processes inside biomolecular condensates, it is important to study biophysical mechanisms of LLPS. In this special issue of Protein Science entitled “Biophysics of Biomolecular Condensates,” we present 17 research articles and reviews by incredible protein scientists that use spectroscopy, microscopy, molecular dynamics simulations, and animal model systems of disease to dissect the intriguing biophysical mechanisms of LLPS. The collection of articles and reviews showcases a variety of exciting systems and emerging biophysical approaches, which protein scientists currently use to unravel the interactions and structural as well as dynamic changes in proteins and nucleic acids that are critical for biomolecular condensation. I hope that this special issue of Protein Science will encourage other protein scientists to study LLPS, to develop innovative and powerful biophysical methods to gain quantitative insights into biomolecular condensates and to transform our understanding of biomolecular condensates in human biology and disease. Biomolecular condensates formed through LLPS are highly dynamic and often contain multiple proteins and nucleic acids. Improved biophysical tools are therefore needed to gain insight into the biochemical processes that occur inside biomolecular condensates on a wide range of length- and time-scales. Rezaei-Ghaleh and colleagues (PRO-20-0254) take advantage from the ubiquitous presence of sodium ions in biomolecular samples and show that 23Na NMR spectroscopy provides insight into the internal fluidity of biomolecular condensed phases. 23Na NMR spectroscopy thus is a new tool to probe the influence of physico- and biochemical factors on the material properties of biomolecular condensates. Mittal and coworkers (PRO-21-0123) report on the further improvement of a coarse-grained model for the computational analysis of the sequence dependent phase separation of disordered proteins. To improve computational predictions of LLPS, they introduce the Urry hydropathy scale into the simulations and optimize the overall interaction strength for the new coarse-grained model. The new model is predicted to enable accurate simulations of the LLPS of intrinsically disordered proteins. Computational studies have the potential to provide high-resolution views of the molecular interactions that underlie the formation and stability of biomolecular condensates. This is particularly the case for atomistic molecular dynamics simulations in explicit solvent as shown by Barducci and colleagues (PRO-21-0135). Their atomistic simulations demonstrate that an important factor for RNA-induced complex coacervation of intrinsically disordered peptides and proteins is the propensity of arginine side chains to simultaneously form a high number of specific interactions with oligonucleotides. The multivalency of the guanidinium group of a single arginine residue thus contributes to protein/RNA LLPS. Mazarakos and Zhou (PRO-21-0057) on the other hand, take a step back and ask how interfacial tension of phase-separated biomolecular condensates affects their fusion and multiphase organization. To address this question they perform molecular dynamics simulations of model systems, which consist of binary mixtures of Lennard–Jones particles or chains of such particles. They find that the critical temperature for phase separation is tightly connected to the interfacial tension of phase-separated condensates. Condensation of proteins and nucleic acids inside eukaryotic cells plays an important role in diverse cellular processes including RNA metabolism, ribosome biogenesis, the DNA damage response and transcription. The androgen receptor, which regulates the transcription of multiple genes, can undergo LLPS in solution. Tompa and coworkers (PRO-21-0138) analyze which parts of the androgen receptor enable LLPS. This allows them to suggest that the diminished LLPS ability of a splice variant (AR-v7) of the androgen receptor may contribute to the misregulation of the transcription function of the androgen receptor in prostate cancer. In addition, Salvatella and colleagues (PRO-21-0133) use one part of the androgen receptor, a fragment of the activation domain, to gain insight into physicochemical properties that influence protein LLPS. They make the at first surprising observation that replacing even small fractions of H2O with D2O increases LLPS propensity, but can rationalize it by the importance of hydrophobic interactions for phase separation. Banerjee and colleagues (PRO-21-0137) investigate the importance of LLPS of fusion transcription factors for different types of cancer. In particular, they focus on the oncofusion protein of fused in sarcoma (FUS) fused to the DNA-binding domain DDIT3. Using an integrative approach including single-molecule correlative force-fluorescence microscopy they show that the fusion protein forms condensates on DNA surfaces to which essential components of the transcriptional machinery are recruited. Protein condensation and LLPS are also connected to protein quality control. For example, the shuttle protein UBQLN2, which binds to ubiquitinated substrates, undergoes LLPS in vitro and forms condensates in cells. Castañeda and coworkers (PRO-21-0060) find that LLPS of UBQLN2 does not exclusively depend on its intrinsically disordered regions, but is modulated by its folded domains. Using NMR spectroscopy they then show that the influence of the folded domains of UBQLN2 on its phase separation originates from previously uncharacterized interactions between its folded and intrinsically disordered domains. LLPS is highly sensitive to environmental stimuli and the presence of co-solutes. Recently, it was demonstrated that ATP - the “energy currency” of the cell - behaves as a biological hydrophobe to inhibit protein LLPS at mM concentrations. Jianxing Song (PRO-21-0022) reviews the current knowledge about the influence of ATP on LLPS of intrinsically disordered proteins and regions. In his review, he summarizes the results from biophysical studies and suggests a potential link between cellular ATP concentrations and protein-aggregation diseases. ATP is also at the center of the research article of Madl and colleagues (PRO-21-0136). They show that in contrast to most of the highly abundant cellular metabolites, ATP and other nucleotides bind to the folded RNA-recognition motif as well as the disordered RG/RGG regions of the model protein CIRBP. Consistent with a competitive mechanism, ATP-binding inhibits RNA-induced LLPS of CIRBP. A lot of interest arises from the potential connection between protein LLPS and the misfolding and aggregation of proteins in neurodegenerative diseases. In 2017 it was shown by three independent studies that the protein tau, which plays an important role in Alzheimer's disease and several other neurodegenerative diseases, has high LLPS propensity (Figure 1). Since then several biophysical studies have investigated the molecular factors that influence tau LLPS. Together with Samrat Mukhopadhyay and his coworkers (PRO-21-0053), we reviewed the rapidly developing field of tau LLPS and its importance for tau physiology and pathology. Even in this special issue of Protein Science, however, there are already three new studies investigating the molecular properties and regulation of tau condensates. Uversky and colleagues (PRO-21-0012) demonstrate that the small heat shock protein 22 kDa (Hsp22) can modulate both LLPS and aggregation of tau in vitro. Han and coworkers (PRO-21-0108) show that subtle changes in solution conditions can lead to the formation of different types of tau condensates with distinct micro-viscosity. My lab (PRO-20-0281) investigated the interplay between LLPS of tau and the intrinsically disordered protein α-synuclein, because tau-based pathologies may act synergistically with α-synuclein pathology to confer worse prognosis in Parkinson's disease with dementia. We report that α-synuclein is recruited to tau droplets in a phosphorylation-dependent manner providing a mechanism for the formation of synergistic α-synuclein/tau pathologies. Strong evidence also suggests that aggregation of the RNA-binding protein FUS within phase-separated compartments is an important pathogenic mechanism in amyotrophic lateral sclerosis. Fawzi and colleagues (PRO-20-0294) focus in their study on the consequences of N-terminal acetylation of FUS on LLPS and aggregation. They show that FUS is N-terminally acetylated in cells. N-terminal acetylation of the low complexity domain of FUS promotes LLPS of this domain, while at the same time decreasing its aggregation propensity. The Ferreon lab (PRO-21-0042) investigates another protein associated with amyotrophic lateral sclerosis, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). They show that conditions that favor LLPS of the disordered C-terminal domain of hnRNPA1 promote fibrillization of this domain. Elbaum-Garfinkle and coworkers (PRO-21-0081) even go a step further and study the properties of polyglutamine aggregates in the aging Caenorhabditis elegans neuron using confocal and fluorescence lifetime imaging microscopy. The results from these experiments are best in agreement with a core/shell architecture of polyglutamine aggregates, which might contribute to neuronal dysfunction in Huntington's disease. In summary, I hope that the articles and reviews in this special issue of Protein Science provide an exciting opportunity for many protein scientists to dive into the miraculous biophysical world of biomolecular condensates.