Biological Phase Separation Research Articles

Polymer Theory for Sequence-Specific Phase Separation Behaviors of Charged Intrinsically Disordered Proteins

Liquid-liquid phase separation in biology underpins the formation of mesoscopic, liquid-like assemblies of DNA, RNA, and proteins, termed “membraneless organelles” or “biomolecular condensates”. Without a membrane, these assemblies can respond to environmental stimuli rapidly and play a critical role in many cellular regulation processes. Intrinsically disordered proteins (IDPs), proteins that do not fold when isolation because of the depletion of hydrophobicity and the abundance of polar, charged, and aromatic residues, have been observed to form membraneless organelles in vivo with other biomolecules as well as undergo phase separation in vitro by themselves. The phase separation propensity of an IDP is determined by its amino acid sequence, not only the composition but also the sequence pattern of its amino acids. To investigate this “sequence-specific” phenomenon, we develop a polymer physics theory, the “random-phase approximation (RPA)” theory, for the phase separation of charged IDPs. The theory predicts that the charge sequence pattern and the number of aromatic residues are critical for the phase separation propensity of IDP. We have applied this theory to the RNA helicase IDP Ddx4 and its two mutants with scrambled charge sequence and mutations reducing its aromaticity. Our predictions are consistent with experiments. We also applied the theory to 30 model IDP sequences with the same positively and negatively charged residues but different sequence patterns, and discovered a connection between the sequence-specific single-chain compactness and multi-chain phase separation propensity. Aqueous solutions with two IDP species with different charge sequences have also been addressed by our formulation, whereby a “fuzzy” molecular recognition mechanism supported by the similarity/dissimilarity of the two IDP sequences is indicated. Taken together, our RPA theory establishes a framework for systematically investigating and rationalizing sequence-specific phase separation behaviors of charged biomolecules.

The Ins and Outs of Phase Separation in Nucleolar Biology

The Ins and Outs of Phase Separation in Nucleolar Biology

A liquid-like organelle at the root of motile ciliopathy.

Motile ciliopathies are characterized by specific defects in cilia beating that result in chronic airway disease, subfertility, ectopic pregnancy, and hydrocephalus. While many patients harbor mutations in the dynein motors that drive cilia beating, the disease also results from mutations in so-called dynein axonemal assembly factors (DNAAFs) that act in the cytoplasm. The mechanisms of DNAAF action remain poorly defined. Here, we show that DNAAFs concentrate together with axonemal dyneins and chaperones into organelles that form specifically in multiciliated cells, which we term DynAPs, for dynein axonemal particles. These organelles display hallmarks of biomolecular condensates, and remarkably, DynAPs are enriched for the stress granule protein G3bp1, but not for other stress granule proteins or P-body proteins. Finally, we show that both the formation and the liquid-like behaviors of DynAPs are disrupted in a model of motile ciliopathy. These findings provide a unifying cell biological framework for a poorly understood class of human disease genes and add motile ciliopathy to the growing roster of human diseases associated with disrupted biological phase separation.

Liquid-liquid phase separation in artificial cells.

Liquid-liquid phase separation (LLPS) in biology is a recently appreciated means of intracellular compartmentalization. Because the mechanisms driving phase separations are grounded in physical interactions, they can be recreated within less complex systems consisting of only a few simple components, to serve as artificial microcompartments. Within these simple systems, the effect of compartmentalization and microenvironments upon biological reactions and processes can be studied. This review will explore several approaches to incorporating LLPS as artificial cytoplasms and in artificial cells, including both segregative and associative phase separation.

Why does biopolymer condensation matter?

Biomolecular condensation that occurs through phase separation is an emerging topic in cell biology. Ongoing research will continue to provide novel mechanistic insights into the involvement of biological condensates in a variety of normal and pathological cellular processes. In particular, a deeper understanding of the physical basis for biomolecular phase separation and of the properties of biopolymer condensates should emerge in the near future. Accordingly, connections between these processes and the organization of matter in living organisms will undergo fresh scrutiny. Biomolecules can phase separate and form condensates that have roles in diverse cellular processes and contexts. Michnick and Bergeron-Sandoval comment on this rapidly progressing field and envisage that the study of biological phase separation will bring new understanding of cell and developmental biology.

Probing RNA Structure in Liquid-Liquid Phase Separation Using SHAPE-MaP.

RNA is an integral component of many biological condensates. A variety of features of RNAs are linked to their function in biological phase separation. Length and negative charge provide fairly generic chemical inputs that drive condensation while sequence has been shown to influence both the molecular identity and biophysical properties of droplets. mRNA sequence guides the secondary structure of the polymers and RNA secondary structure licenses-specific RNA-RNA interactions and the recruitment of RNA-binding proteins. Here, we describe a method for directly probing the structure of mRNAs in the context of RNP-droplets formed via LLPS.

Phase separation in biology

Cells have to organize their complex biochemistry to regulate their metabolism and respond to changes in the environment. Traditionally, intracellular organization has been associated with compartments that are surrounded by lipid membranes. However, in recent years, phase transitions have emerged as a novel form of cellular organization. Phase transition is a physical process whereby a substance changes from one physical state to another. Examples are provided by the freezing of water into ice (liquid to solid) or the heating of water to generate water vapor (liquid to gas).

The Use of Detergents to Purify Membrane Proteins.

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included.

Phase separation in biology; functional organization of a higher order.

Inside eukaryotic cells, macromolecules are partitioned into membrane-bounded compartments and, within these, some are further organized into non-membrane-bounded structures termed membrane-less organelles. The latter structures are comprised of heterogeneous mixtures of proteins and nucleic acids and assemble through a phase separation phenomenon similar to polymer condensation. Membrane-less organelles are dynamic structures maintained through multivalent interactions that mediate diverse biological processes, many involved in RNA metabolism. They rapidly exchange components with the cellular milieu and their properties are readily altered in response to environmental cues, often implicating membrane-less organelles in responses to stress signaling. In this review, we discuss: (1) the functional roles of membrane-less organelles, (2) unifying structural and mechanistic principles that underlie their assembly and disassembly, and (3) established and emerging methods used in structural investigations of membrane-less organelles.

Liquid-liquid phase separation in biology.

Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.

Innovative Integrated Reactor System for Carbon, Sulfur and Nitrogen Removal Based on Biological Phase-Separation

An innovative biological wastewater treatment system for the removal of organic carbon, sulfur and nitrogen was developed based on biological phase-separation principle. This system consists of three reactors integrated together i.e. sulfate reduction and organic matter removal (SR-CR), autotrophic and heterotrophic denitrifying sulfide removal (A&H-DSR) and nitrification (AN) reactors. In this system, the operational parameters for successful bio-phase separation are sulfate and organic loading rate, hydraulic retention time (HRT), COD/SO42- ratio and pH for the SR-CR reactor, and sulfide and nitrate loading rate, HRT, pH, S 2- /NO3- ratio and COD/NO3- for the A&H-DSR reactor. The results from a laboratory scale system demonstrated that for the SR-CR reactor, the optimal operating conditions were HRT≥24 h; sulfate and organic loading rate ≤7.5 kg SO42- /m3•d and ≤10 kgCOD/m3•d; COD/SO42- ≥2; and pH ≥6.5. For A&H-DSR process, the optimal conditions are sulfide loading rate ≤6.0kg S 2- /m3•d; nitrate loading rate ≤3.5 kg NO3-/m3•d; S 2- /NO3-≥1; COD/NO3- ≥1.25:1; and pH≥7.5. Under such conditions, high sulfate, ammonia and organic matter removal of 99%, 90% and 99% were achieved, respectively. In this case, the elemental sulfur (S0) reclamation efficiency reached 6.0 kg S0/m3•d, around 20 times higher than the maximum level as referred in the literatures. DGGE profiling indicated that the predominant functional organisms of Clostridiaceae sp., Desulfomicrobium sp., Methanosaeta sp. dominated in the SR-CR reactor, and Sulfurovum sp., Pseudomonas aeruginosa and Denitratisoma sp. in the A&H-DSR reactor. These species played essential role in metabolic functions in each bio-phase.

The use of detergents to purify membrane proteins.

Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, or biomolecular NMR. Protocols in this unit include the isolation and solubilization of biological membranes, phase separation, and support protocols for detergent removal and detergent exchange using different methods.

Interactions of complement fraction C1q, fibronectin, and immunoglobulin G with polyacrylic microparticles used as solid-phase in immunoassay.

A microparticle-enhanced nephelometric immunoassay was recently described, where polyacrylic, hydrophilic, and polyfunctional microparticles are used as the solid phase. It is a one-step immunoassay based on the nephelometric quantification of microparticle agglutination. In such assays, the measurement of analytes at low concentration may be impaired by the need of using undiluted biological samples. This leads to work with high concentrations of several proteins liable to interfere with the agglutination process. In this paper, we report on a study performed with human serum and purified proteins, which were assayed by classical analytical methods. This work identified three major components of human serum specifically involved in yielding polyacrylic microparticle instability: complement fraction C1q, fibronectin, and immunoglobulins G. In this order of importance, they all showed a marked ability to be adsorbed on the microparticle's surface. Pretreatment of human serum with microparticles decreased the concentrations in C1q (82%), fibronectin (16%), and immunoglobulin G (4%) very unequally. However, it allowed the elimination of microparticle instability, consequently providing the possible use of such polyacrylic microparticles in a one-step nephelometric immunoassay of analytes at low concentration in biological samples, without washes or phase separation.