Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.

Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.

A framework for understanding the functions of biomolecular condensates across scales.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

Partitioning of Cancer Therapeutics in Nuclear Condensates

Over the past two decades, there has been a growing interest in the use of plasmonic nanoparticles as sources of heat remotely controlled by light, giving rise to the field of thermoplasmonics. The ability to release heat on the nanoscale has already impacted a broad range of research activities, from biomedicine to imaging and catalysis. Thermoplasmonics is now entering an important phase: some applications have engaged in an industrial stage, while others, originally full of promise, experience some difficulty in reaching their potential. Meanwhile, innovative fundamental areas of research are being developed. In this Review, we scrutinize the current research landscape in thermoplasmonics, with a specific focus on its applications and main challenges in many different fields of science, including nanomedicine, cell biology, photothermal and hot-electron chemistry, solar light harvesting, soft matter and nanofluidics. Thermoplasmonics is based on the use of plasmonic nanoparticles as sources of heat remotely controlled by light. This Review discusses its current applications and challenges in a broad range of scientific fields, from nanomedicine to hot-electron chemistry and nanofluidics.

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Applications and challenges of thermoplasmonics.

Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo. HDAC6 modulates acetylation at multiple lysine residues in the N-terminal intrinsically disordered region of RNA helicase DDX3X to regulate liquid–liquid phase separation and stress granule maturation.

Acetylation of intrinsically disordered regions regulates phase separation.

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.

Non-invasive perturbations of intracellular flow reveal physical principles of cell organization

A finite element approach for vector- and tensor-valued surface PDEs

We consider the numerical investigation of surface bound orientational order using unit tangential vector fields by means of a gradient flow equation of a weak surface Frank–Oseen energy. The energy is composed of intrinsic and extrinsic contributions, as well as a penalization term to enforce the unity of the vector field. Four different numerical discretizations, namely a discrete exterior calculus approach, a method based on vector spherical harmonics, a surface finite element method, and an approach utilizing an implicit surface description, the diffuse interface method, are described and compared with each other for surfaces with Euler characteristic 2. We demonstrate the influence of geometric properties on realizations of the Poincare–Hopf theorem and show examples where the energy is decreased by introducing additional orientational defects.

Orientational Order on Surfaces: The Coupling of Topology, Geometry, and Dynamics

Orientational order on surfaces - the coupling of topology, geometry and dynamics

We consider a thin film limit of a Landau-de Gennes Q-tensor model. In the limiting process, we observe a continuous transition where the normal and tangential parts of the Q-tensor decouple and various intrinsic and extrinsic contributions emerge. The main properties of the thin film model, like uniaxiality and parameter phase space, are preserved in the limiting process. For the derived surface Landau-de Gennes model, we consider an L2-gradient flow. The resulting tensor-valued surface partial differential equation is numerically solved to demonstrate realizations of the tight coupling of elastic and bulk free energy with geometric properties.

https://royalsocietypublishing.org/doi/pdf/10.1098/rspa.2017.0686

Michael Nestler

Papers

Non-invasive perturbations of intracellular flow reveal physical principles of cell organization

A finite element approach for vector- and tensor-valued surface PDEs

Orientational Order on Surfaces: The Coupling of Topology, Geometry, and Dynamics

Orientational order on surfaces - the coupling of topology, geometry and dynamics

Nematic liquid crystals on curved surfaces: a thin film limit.