Biophysics Reports

2022 Vol. 8, No. 1

Cover Story

Liquid–liquid phase separation (LLPS) is an emerging phenomenon involved in various biological processes. The formation of phase separated condensates is crucial for many intrinsically disordered proteins to fulfill their biological functions. Using the recombinant protein to reconstitute the formation of condensates in vitro has become the standard method to investigate the behavior and function of LLPS. Meanwhile, there is an urgent need to characterize the LLPS in living cells. Importantly, condensates formed through LLPS at physical relevant concentrations are often smaller than the optical diffraction limit, which makes precise characterization and quantification inaccurate due to the scatter of light. The booming devel opment of super resolution optical microscopy enables the visualization of multiple obscured subcellular components and processes, which is also suitable for the LLPS research. In this protocol, the authors provide step-by-step instructions to help users take advantage of super resolution imaging to depict the morphology and quantify the molecule number of endogenous condensates in living cells using RNA Pol II as an example. This streamlined workflow offers exceptional robustness, sensitivity, and precision, which could be easily implemented in any laboratory with an inverted total internal reflection microscope. They expect that super-resolution microscopy will contribute to the investigation of both large and tiny condensates under physiological and pathological conditions and lead our understanding of the mechanism of LLPS to a higher and deeper layer.

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PREFACE
PROTOCOL

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PREFACE

Preface to the special issue of Phase Separation: methodologies tailored for studying biomolecular condensates

Pilong Li

2022, 8(1): 1-1. doi: 10.52601/bpr.2022.210901

Abstract(1221)

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Abstract:

PROTOCOL

Characterization of liquid–liquid phase separation using super-resolution and single-molecule imaging

Hongchen Zhang, Shipeng Shao, Yujie Sun

2022, 8(1): 2-13. doi: 10.52601/bpr.2022.210043

Abstract(2671)

PDF(221)

Abstract:
Liquid – liquid phase separation (LLPS) is an emerging phenomenon involved in various biological processes. The formation of phase-separated condensates is crucial for many intrinsically disordered proteins to fulfill their biological functions. Using the recombinant protein to reconstitute the formation of condensates in vitro has become the standard method to investigate the behavior and function of LLPS. Meanwhile, there is an urgent need to characterize the LLPS in living cells. Importantly, condensates formed through LLPS at physical relevant concentrations are often smaller than the optical diffraction limit, which makes precise characterization and quantification inaccurate due to the scatter of light. The booming development of super-resolution optical microscopy enables the visualization of multiple obscured subcellular components and processes, which is also suitable for the LLPS research. In this protocol, we provide step-by-step instructions to help users take advantage of super-resolution imaging to depict the morphology and quantify the molecule number of endogenous condensates in living cells using RNA Pol II as an example. This streamlined workflow offers exceptional robustness, sensitivity, and precision, which could be easily implemented in any laboratory with an inverted total internal reflection microscope. We expect that super-resolution microscopy will contribute to the investigation of both large and tiny condensates under physiological and pathological conditions and lead our understanding of the mechanism of LLPS to a higher and deeper layer.

Cellular and animal models to investigate pathogenesis of amyloid aggregation in neurodegenerative diseases

Houfang Long, Shuyi Zeng, Dan Li

2022, 8(1): 14-28. doi: 10.52601/bpr.2022.210033

Abstract(1470)

PDF(119)

Abstract:
Abnormal aggregation of amyloid proteins, e.g. amyloid β (Aβ), Tau and α-synuclein (α-syn), is closely associated with a variety of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Cellular and animal models are useful to explore the neuropathology of amyloid aggregates in disease initiation and progression. In this protocol, we describe detailed procedures for how to establish neuronal and PD mouse models to evaluate amyloid pathologies including self-propagation, cell-to-cell transmission, neurotoxicity, and impact on mouse motor and cognitive functions. We use α-syn, a key pathogenic protein in PD, as an example to demonstrate the application of the protocol, while it can be used to investigate the pathologies of other amyloid proteins as well. The established disease models are also useful to assess the activities of drug candidates for therapeutics of neurodegenerative diseases.

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

Yirong Yao, Wenjuan Wang, Chunlai Chen

2022, 8(1): 29-41. doi: 10.52601/bpr.2022.210026

Abstract(886)

PDF(82)

Abstract:
Liquid–liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.

Biochemical and biophysical characterization of pathological aggregation of amyloid proteins

Houfang Long, Shuyi Zeng, Yunpeng Sun, Cong Liu

2022, 8(1): 42-54. doi: 10.52601/bpr.2022.210032

Abstract(1739)

PDF(132)

Abstract:
Protein amyloid fibrillation, a process of liquid to solid phase transition, is involved in the pathogenesis of a variety of human diseases. Several amyloid proteins including α-synuclein (α-syn), Tau, amyloid β (Aβ) protein, and TAR DNA-binding protein 43 kDa (TDP-43) form pathological fibrils and deposit in patient brains of different neurodegenerative diseases (NDs) such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and Amyotrophic lateral sclerosis (ALS). Preparation and characterization of amyloid fibrils in vitro are essential for studying the molecular mechanism underlying the dynamic amyloid aggregation and its pathogenesis in diseases. In this protocol, we take PD-associated α-syn as an example, and describe amyloid protein purification and fibrillation approaches. We then introduce biochemical and biophysical characterization of amyloid fibrils by Thioflavin-T (ThT) fluorescence kinetics assay, transmission electron microscopy (TEM), atomic force microscopy (AFM) and multiple fibril stability measurement assays. The approaches described here are applicable to different amyloid proteins, and are of importance for further study on the structure determination of amyloid fibrils and their pathological function in cells and animal models.