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Biomolecular condensates are membraneless, intracellular organelles that form via liquid-liquid phase separation (LLPS) and have the ability to concentrate a wide range of molecules in the cellular milieu. These organelles are highly dynamic and play pivotal roles in cellular organization and physiology. Many studies also link the formation and misregulation of condensates to diseases such as neurodegenerative disorders and cancer. Biomolecular condensates represent a special type of colloids that actively interact with their environment to sustain physiological functions, due to which their misregulation may upset cell signaling, resulting in pathological states. In this review, we discuss the mechanisms underlying the formation, dynamics, and evolution of these biological colloids, with a special focus on their surface properties that are critical in their interaction with other components of the cell. We also summarize experimental approaches that enable the detailed characterization of the formation, interactions, and functions of these cellular colloidal organelles.

Liquid-solid and liquid-liquid phase separation (PS) drives the formation of functional and disease-associated biological assemblies. Principles of phase equilibrium are here employed to derive a general kinetic solution that predicts the evolution of the mass and size of biological assemblies. Thermodynamically, protein PS is determined by two measurable concentration limits: the saturation concentration and the critical solubility. Due to surface tension effects, the critical solubility can be higher than the saturation concentration for small, curved nuclei. Kinetically, PS is characterized by the primary nucleation rate constant and a combined rate constant accounting for growth and secondary nucleation. It is demonstrated that the formation of a few number of large condensates is possible without active mechanisms of size control and in the absence of coalescence phenomena. Our exact analytical solution can be used to interrogate how the elementary steps of PS are affected by candidate drugs.

Motivation Proteins able to undergo liquid–liquid phase separation (LLPS) in vivo and in vitro are drawing a lot of interest, due to their functional relevance for cell life. Nevertheless, the proteome-scale experimental screening of these proteins seems unfeasible, because besides being expensive and time-consuming, LLPS is heavily influenced by multiple environmental conditions such as concentration, pH and temperature, thus requiring a combinatorial number of experiments for each protein. Results To overcome this problem, we propose a neural network model able to predict the LLPS behavior of proteins given specified experimental conditions, effectively predicting the outcome of in vitro experiments. Our model can be used to rapidly screen proteins and experimental conditions searching for LLPS, thus reducing the search space that needs to be covered experimentally. We experimentally validate Droppler’s prediction on the TAR DNA-binding protein in different experimental conditions, showing the consistency of its predictions. Availability and implementation A python implementation of Droppler is available at https://bitbucket.org/grogdrinker/droppler Supplementary information Supplementary data are available at Bioinformatics online.

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid-liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a "reentrant" fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics. ispartof: BIOMOLECULES vol:11 issue:4 ispartof: location:Switzerland status: published

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins., Van Lindt, Bratek-Skicki et al. show that at carefully selected pH values, proteins can be kept in solution and their LLPS can then be induced by a jump to native pH. This presents a generic method to study the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behaviour of diverse proteins.

Protein adsorption on solid surfaces provides either beneficial or adverse outcomes, depending on the application. Therefore, the desire to predict, control, and regulate protein adsorption on different surfaces is a major concern in the field of biomaterials. The most widely used surface modification approach to prevent or limit protein adsorption is based on the use of poly (ethylene oxide) (PEO). On the other hand, the amount of protein adsorbed on poly(2-(dimethylamine)ethyl methacrylate) (PDMAEMA) coatings can be regulated by the pH and ionic strength of the medium. In this work, ultra-thin PEO/PDMAEMA coatings were designed from solutions with different ratios of PEO to PDMAEMA, and different molar masses of PEO, to reversibly adsorb and desorb human serum albumin (HSA), human fibrinogen (Fb), lysozyme (Lys), and avidine (Av), four very different proteins in terms of size, shape, and isoelectric points. X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM), and atomic force microscopy (AFM) were used to characterize the mixed polymer coatings, revealing the presence of both polymers in the layers, in variable proportions according to the chosen parameters. Protein adsorption at pH 7.4 and salt concentrations of 10&minus, 3 M was monitored by QCM. Lys and Av did not adsorb on the homo-coatings and the mixed coatings. The amount of HSA and Fb adsorbed decreased with increasing the PEO ratio or its molar mass in a grafting solution. It was demonstrated that HSA and Fb, which were adsorbed at pH 7.4 and at an ionic strength of 10&minus, 3 M, can be fully desorbed by rinsing with a sodium chloride solution at pH 9.0 and ionic strength 0.15 M from the mixed PEO5/PDMAEMA coatings with PEO/PDMAEMA mass ratios of 70/30, and 50/50, respectively. The results demonstrate that mixed PEO/PDMAEMA coatings allow protein adsorption to be finely tuned on solid surfaces.

Cellular organelles that lack a surrounding lipid bilayer, such as the nucleolus and stress granule, represent a newly recognized, general paradigm of cellular organization. The formation of such biomolecular condensates which include "membraneless organelles" (MLOs) by liquid-liquid phase separation (LLPS) has been in the focus of a surge of recent studies. Through a combination of in vitro and in vivo approaches, thousands of potential phase-separating proteins have been identified, and it was found that different cellular MLOs share many common components. These perplexing observations raise the question of how cells regulate the timing and specificity of LLPS, and ensure that different MLOs form and disperse at the right moment and cellular location and can preserve their identity and physical separation. This guide gives an overview of basic regulatory mechanisms, which manifest through the action of intrinsic regulatory elements, alternative splicing, post-translational modifications and a broad range of phase-separating partners. We also elaborate on the cellular integration of these different mechanisms and highlight how complex regulation can orchestrate the parallel functioning of a dozen or so different MLOs in the cell.

Proteins at interfaces are a key for many applications in the biomedical field, in biotechnologies, in biocatalysis, in food industry, etc. The development of surface layers that allow to control and manipulate proteins is thus highly desired. In previous works, we have shown that mixed polymer brushes combining the protein-repellent properties of poly(ethylene oxide) (PEO) and the stimuli-responsive adsorption behavior of poly(acrylic acid) (PAA) could be synthesized and used to achieve switchable protein adsorption. With the present work, we bring more insight into the rational design of such smart thin films by unravelling the role of PEO on the adsorption/desorption of proteins. The PEO content of the mixed PEO/PAA brushes was regulated, on the one hand, by using PEO with different molar masses and, on the other hand, by varying the ratio of PEO and PAA in the solutions used to synthesize the brushes. The influence of ionic strength on the protein adsorption behavior was also further examined. The behavior of three proteins-human serum albumin, lysozyme, and human fibrinogen, which have very different size, shape, and isoelectric point-was investigated. X-ray photoelectron spectroscopy, quartz crystal microbalance, atomic force microscopy, and streaming potential measurements were used to characterize the mixed polymer brushes and, in particular, to estimate the fraction of each polymer within the brushes. Protein adsorption and desorption conditions were selected based on previous studies. While brushes with a lower PEO content allowed the higher protein adsorption to occur, fully reversible adsorption could only be achieved when the PEO surface density was at least 25 PEO units per nm

A standard method of protein immobilization is proposed, based on the use of protein-polyelectrolyte complexes (PPCs) as building blocks for layer-by-layer assembly. Thicker multilayers, with a higher polyelectrolyte fraction, are obtained with PPCs compared to single protein molecules. Biological activity is not only maintained, but specific activity is also higher, as demonstrated for lysozyme-poly(styrene sulfonate) complexes. This is attributed to the more hydrated state of the assemblies. This new method of protein immobilization opens up perspectives for biotechnology and biomedical applications.

Cellular organelles that lack a surrounding lipid bilayer, such as the nucleolus and stress granule, represent a newly recognized, general paradigm of cellular organization. The formation of such biomolecular condensates that include 'membraneless organelles' (MLOs) by liquid-liquid phase separation (LLPS) has been in the focus of a surge of recent studies. Through a combination of in vitro and in vivo approaches, thousands of potential phase-separating proteins have been identified, and it was found that different cellular MLOs share many common components. These perplexing observations raise the question of how cells regulate the timing and specificity of LLPS, and ensure that different MLOs form and disperse at the right moment and cellular location and can preserve their identity and physical separation. This guide gives an overview of basic regulatory mechanisms, which manifest through the action of intrinsic regulatory elements, alternative splicing, post-translational modifications, and a broad range of phase-separating partners. We also elaborate on the cellular integration of these different mechanisms and highlight how complex regulation can orchestrate the parallel functioning of a dozen or so different MLOs in the cell.

Understanding the kinetics and underlying physicochemical forces of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for the analysis of often severely aggregation-prone proteins. Frequently applied approaches, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, however, often lead to very different kinetic behaviors. Here we suggest that at extreme pH values even proteins such as the low-complexity domain (LCD) of hnRNPA2, TDP-43, and NUP-98 can be kept in solution, and then their LLPS can be induced by a jump to native pH, resulting in a system that can be easily controlled. This approach represents a generic method for studying LLPS under near native conditions, providing a platform for studying the phase-separation behavior of diverse proteins.

Manipulation of high-density materials, such as crystals and liquid condensates, is of great importance for many applications, including serial crystallography, structural and molecular biology, chemistry, and medicine. In this work, we describe an acoustic technique to focus and harvest flowing crystals and liquid condensates. Moreover, we show, based on numerical simulations, that the acoustic waves can be used for size-based particle (crystals, droplets, etc.) separation. This is an essential technological step in biological research, medical applications, and industrial processes. The presented technology offers high precision, biocompatibility, ease of use and additionally, is non-invasive and inexpensive. With the recent advent of X-ray Free Electron Laser (XFEL) technology and the associated enormous importance of a thin jet of crystals, this technology might pave the way to a novel type of XFEL injector.

Selective protein adsorption is a key challenge for the development of biosensors, separation technologies, and smart materials for medicine and biotechnologies. In this work, a strategy was developed for selective protein adsorption, based on the use of mixed polymer brushes composed of poly(ethylene oxide) (PEO), a protein-repellent polymer, and poly(acrylic acid) (PAA), a weak polyacid whose conformation changes according to the pH and ionic strength of the surrounding medium. A mixture of lysozyme (Lyz), human serum albumin (HSA), and human fibrinogen (Fb) was used to demonstrate the success of this strategy. Polymer brush formation and protein adsorption were monitored by quartz crystal microbalance, whereas protein identification after adsorption from the mixture was performed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principal component analysis and gel electrophoresis with silver staining. For the ToF-SIMS measurements, adsorption was first performed from single-protein solutions in order to identify characteristic peaks of each protein. Next, adsorption was performed from the mixture of the three proteins. Proteins were also desorbed from the brushes and analyzed by gel electrophoresis with silver staining for further identification. Selective adsorption of Lyz from a mixture of Lyz/HSA/Fb was successfully achieved at pH 9.0 and ionic strength of 10 M, while Lyz and HSA, but not Fb, were adsorbed at ionic strength 10 M and pH 9.0. The results demonstrate that by controlling the ionic strength, selective adsorption can be achieved from protein mixtures on PEO/PAA mixed brushes, predominantly because of the resulting control on electrostatic interactions. In well-chosen conditions, the selectively adsorbed proteins can also be fully recovered from the brushes by a simple ionic strength stimulus. The developed systems will find applications as responsive biointerfaces in the fields of separation technologies, biosensing, drug delivery, and nanomedicine.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

Controlled deposition of nanoparticles and bioparticles is necessary for their separation and purification by chromatography, filtration, food emulsion and foam stabilization, etc. Compared to numerous experimental techniques used to quantify bioparticle deposition kinetics, the quartz crystal microbalance (QCM) method is advantageous because it enables real time measurements under different transport conditions with high precision. Because of its versatility and the deceptive simplicity of measurements, this technique is used in a plethora of investigations involving nanoparticles, macroions, proteins, viruses, bacteria and cells. However, in contrast to the robustness of the measurements, theoretical interpretations of QCM measurements for a particle-like load is complicated because the primary signals (the oscillation frequency and the band width shifts) depend on the force exerted on the sensor rather than on the particle mass. Therefore, it is postulated that a proper interpretation of the QCM data requires a reliable theoretical framework furnishing reference results for well-defined systems. Providing such results is a primary motivation of this work where the kinetics of particle deposition under diffusion and flow conditions is discussed. Expressions for calculating the deposition rates and the maximum coverage are presented. Theoretical results describing the QCM response to a heterogeneous load are discussed, which enables a quantitative interpretation of experimental data obtained for nanoparticles and bioparticles comprising viruses and protein molecules.