Biophysics Reports

2022 Vol. 8, No. 2

Cover Story

Liquid–liquid phase separation (LLPS) has proved to be ubiquitous in living cells, forming membraneless organelles (MLOs) and dynamic condensations essential in physiological processes. However, some underlying mechanisms remain challenging to unravel experimentally, making theoretical modeling an indispensable aspect. Here the authors present a protocol for understanding LLPS from fundamental physics to detailed modeling procedures. The protocol involves a comprehensive physical picture on selecting suitable theoretical approaches, as well as how and what to interpret and re-solve from the results. On the particle based level, they elaborate on coarse grained simulation procedures from building up models, identifying crucial interactions to running simulations to obtain phase diagrams and other concerned properties. They also outline field based theories which give the system’s density profile to determine phase diagrams and provide dynamic properties by studying the time evolution of density field, enabling us to characterize LLPS systems with larger time and length scales and to further include other nonequilibrium factors such as chemical reactions.

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PROTOCOL

Theoretical modelling of liquid–liquid phase separation: from particle-based to field-based simulation

Lin-ge Li, Zhonghuai Hou

2022, 8(2): 55-67. doi: 10.52601/bpr.2022.210029

Abstract(3359)

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Abstract:
Liquid–liquid phase separation (LLPS) has proved to be ubiquitous in living cells, forming membraneless organelles (MLOs) and dynamic condensations essential in physiological processes. However, some underlying mechanisms remain challenging to unravel experimentally, making theoretical modeling an indispensable aspect. Here we present a protocol for understanding LLPS from fundamental physics to detailed modeling procedures. The protocol involves a comprehensive physical picture on selecting suitable theoretical approaches, as well as how and what to interpret and resolve from the results. On the particle-based level, we elaborate on coarse-grained simulation procedures from building up models, identifying crucial interactions to running simulations to obtain phase diagrams and other concerned properties. We also outline field-based theories which give the system's density profile to determine phase diagrams and provide dynamic properties by studying the time evolution of density field, enabling us to characterize LLPS systems with larger time and length scales and to further include other nonequilibrium factors such as chemical reactions.

Measuring the elasticity of liquid–liquid phase separation droplets with biomembrane force probe

Min Sun, Hui Chen, Qinghua Ji, Jianhui Xiao, Yanzhe Hou, Jizhong Lou

2022, 8(2): 68-79. doi: 10.52601/bpr.2022.210038

Abstract(1639)

PDF(101)

Abstract:
Numerous biomacromolecules undergo liquid–liquid phase separation (LLPS) inside living cells and LLPS plays important roles in their functions. The droplets formed by LLPS molecules are complex fluids and their behavior follows fluid mechanics, thus studies on rheological and material properties are required to gain full insight into the biophysical mechanism of these droplets. Biophysical force spectroscopy techniques are particularly useful in this aspect. Indeed, atomic force microscopy and optical tweezers have been used to quantify the elasticity and the viscoelasticity of LLPS droplets. The Biomembrane Force Probe (BFP) is a single-molecule technique designed to investigate liquid-like objects and is more suitable to quantify the material properties of LLPS droplets, but its usage on LLPS droplets is not yet described. Here we present an experimental protocol to measure the Young’s modulus of LLPS droplets using BFP, we believe that the application of BFP on phase separation studies can be expanded and will be very helpful in deciphering the underlying principles of LLPS.

Visualizing carboxyl-terminal domain of RNA polymerase II recruitment by FET fusion protein condensates with DNA curtains

Linyu Zuo, Jiawei Ding, Zhi Qi

2022, 8(2): 80-89. doi: 10.52601/bpr.2022.210027

Abstract(1248)

PDF(62)

Abstract:
Many recent references show that living cells can form some membrane-less organelles by liquid–liquid phase separation (LLPS) of biomolecules, like proteins and nucleic acids. LLPS has been confirmed to link with many important biological functions in living cells, and one of the most important functions of biomolecular condensates is in the field of RNA transcription. Many studies confirm that mammalian RNA polymerase II (Pol II) molecules containing the CTD with different phosphorylation level are purposed to shuttle between initiation condensates and elongation condensates of RNA transcription. Traditional ensemble assays often experience difficulties in quantitively and directly recording the transient recruitment of Pol II CTD. Novel single-molecule approach — DNA curtains can be used to directly visualize biomolecular condensates formation and also recruitment of RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) at the target sites in solution and in real time. This method can offer the potential for new insights into the mechanism of gene transcription. Here, we highlight the detailed protocol of DNA curtains method for studying LLPS.

Driving force of biomolecular liquid–liquid phase separation probed by nuclear magnetic resonance spectroscopy

Hanyu Zhang, Weiwei Fan, Gilbert Nshogoza, Yaqian Liu, Jia Gao, Jihui Wu, Yunyu Shi, Xiaoming Tu, Jiahai Zhang, Ke Ruan

2022, 8(2): 90-99. doi: 10.52601/bpr.2022.210034

Abstract(1143)

PDF(80)

Abstract:
The assembly of biomolecular condensates is driven by liquid–liquid phase separation. To understand the structure and functions of these condensates, it is essential to characterize the underlying driving forces, e.g., protein–protein and protein–RNA interactions. As both structured and low-complexity domains are involved in the phase separation process, NMR is probably the only technique that can be used to depict the binding topology and interaction modes for the structured and nonstructured domains simultaneously. Atomic-resolution analysis for the intramolecular and intermolecular interactions between any pair of components sheds light on the mechanism for phase separation and biomolecular condensate assembly and disassembly. Herein, we describe the procedures used for the most extensively employed NMR techniques to characterize key interactions for biomolecular phase separation.

REVIEW

Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid–liquid phase separation

Zhulou Wang, Huizhi Zhang, Lin Jian, Bo Ding, Keying Huang, Wolun Zhang, Qian Xiao, Shaohui Huang

2022, 8(2): 100-118. doi: 10.52601/bpr.2022.210047

Abstract(2157)

PDF(192)

Abstract:
Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.