液-液相分离在细胞信号调控过程中的作用机制

近年来,液-液相分离(简称相分离)因其独特的功能与组织性,在细胞生物学研究中发展迅速。细胞内部分蛋白质及核酸(多为RNA)等生物大分子通过由多种弱多价相互作用及构象熵共同介导的相变(phase transition)形成无膜细胞器(membraneless organelles, MLOs)。这些无膜结构具有明显的流体性质,包括其圆形外表、可浸润、滴落和彼此融合,并具有动态的内部成分交换。在体内形成的无膜细胞器,广泛参与到包括细胞膜信号传导、膜结合蛋白质组装、染色质重塑、RNA代谢、突触传递、活跃转录中心形成、有丝分裂结构形成,以及蛋白质病理性转变等多种重要的细胞内信号调控过程。本文从相分离的研究背景,相分离发生的分子机制,正常相分离过程参与的多种细胞生理活动,异常相分离与神经性疾病及癌症发生的关系等方面,阐述了生物大分子的相分离在细胞信号调控过程中的普遍性及重要作用,并对研究相分离的实验技术和常用的相分离数据库进行了介绍。生物大分子相分离行为的发现,为重新理解众多结构及细胞生物学现象提供了全新的角度,生物大分子相分离可能作为一种新的生物学过程,帮助重新认识众多信号通路的调控方式,也有望为相关疾病的治疗提供新的方向。     

生物大分子相分离在疾病中的作用

细胞为了维持正常的生理活动进化出膜系统,使各种各样的活动能在特定的空间、时间上高效有序的发生。膜系统参与物质运输、信号传递、能量代谢等过程已被广泛了解,但与无膜区室组装和功能相关的分子细节尚未研究透彻。生物大分子通过相分离在细胞内形成多种无膜区室,如核仁、中心体、应激颗粒等,这些无膜区室被统称为生物分子凝聚体。作为一种细胞生化反应的聚集分离机制,相分离在自然界中普遍存在,并广泛参与信号转导、基因转录调控等多种重要的生理过程。而异常的相分离与许多人类疾病密切相关,如神经退行性疾病、癌症及传染性疾病等。通过介绍相分离形成的细胞结构及功能、相分离发生的机制,进一步阐述相分离在疾病发生发展中的作用。     

无膜颗粒与生物大分子液-液相分离:形成机制、生物功能及数据资源

为了保证细胞内各种生化反应和调控过程的有序进行,细胞内存在一系列隔室将不同的生物分子分隔开来。这其中除了有膜细胞器,还存在一类无膜细胞器或无膜颗粒,使得具有特定功能的蛋白质和核酸在不同无膜颗粒中聚集,保证相应生化过程在特定时空条件下高效进行。大量的研究证据表明,液-液相分离(liquid-liquid phase separation, LLPS)是介导胞内无膜颗粒凝聚形成的重要机制。本文首先围绕相分离介绍了胞内无膜颗粒的形成机制;进一步总结部分胞内无膜颗粒的功能,以及相分离在其行使生理功能时发挥的重要作用;最后总结相分离数据库及其所常采信的实验方法,期望通过对胞内无膜颗粒形成机制、生物功能及相分离数据资源的总结,为初入领域的科研工作者提供参考,并推进高通量方法在相关领域研究的应用与发展。     

生物大分子“液–液相分离”调控染色质三维空间结构和功能

生物大分子的相分离聚集(简称相分离)是驱动细胞内无膜细胞器形成的主要机制,参与众多生物学过程并和多种人类疾病密切相关,如神经退行性疾病等。近年来,研究人员围绕相分离现象的分子机制和生物学功能,发现了相分离与信号传导、染色质结构、基因表达、转录调控等一系列生物学过程存在紧密关联,为理解细胞命运决定和疾病发生提供了新的视角,为疾病治疗和新药研发开辟了新的可能途径。本文在回顾了相分离研究的发展过程、相分离现象在生物学中的应用,以及相分离与疾病的关系的基础上,重点分析了近年来相分离与染色质结构关联方面的研究突破,包括相分离如何感知并重塑染色质结构、超级增强子如何通过相分离调节基因表达、共转录激活因子如何通过相分离参与基因表达调控等,以期为进一步理解相分离与染色质空间结构的关系提供参考。     

相分离与RNA的相互调控

细胞内生物大分子通过相分离形成凝聚体对复杂精细的生物化学反应进行调控,从而保证细胞生命活动高效有序地进行. RNA是细胞内丰度极高的生物大分子,在大部分凝聚体的形成和调控中起着关键作用. RNA自身可以发生相分离,也可以通过其电荷和结构等特征影响蛋白质的相分离.反之,蛋白质的相分离可以调节RNA的生成、代谢和功能等.因此,相分离为蛋白质-RNA生物大分子机器发挥功能提供新维度.本文对RNA与相分离的相互调控进行总结,并对未来相分离研究在RNA生物学中的作用提出展望.     

生物大分子“液-液”相分离:现状与展望

生物大分子"液-液"相分离是近年来在生命科学领域迅速发展起来的新概念。相分离概念的提出,为我们深入理解细胞内生物大分子的组织模式和功能调控提供了新的观点和研究工具,因此迅速成为生命科学领域的研究前沿。但是围绕生物大分子相分离的生物物理学特性及其在细胞中扮演的角色仍有很多未解之谜。该文对近年来生物大分子相分离的相关研究进行了综述,对其在细胞中的功能和未来的发展趋势进行了初步探讨和展望。     

植物细胞质外体多肽与蛋白研究

细胞质膜以外的质外体是植物细胞的重要组成部分, 质外体是植物细胞的重要信号源和细胞器。当植物遭受生物或非生物环境刺激时,可能首先引起质外体信号系统的变化; 同时质外体作为植物细胞之间最方便的通道, 在细胞间信号传递和信息交流上起重要作用, 从而成为协调植物细胞分化、器官形成和整体生长发育的决定性因素之一。本文概括地介绍了我室在此领域的一些研究进展。     

植物细胞质外体多肽与蛋白研究

细胞质膜以外的质外体是植物细胞的重要组成部分,质外体是植物细胞的重要信号源和细胞器。当植物遭受生物或非生物环境刺激时,可能首先引起质外体信号系统的变化;同时质外体作为植物细胞之间最方便的通道,在细胞间信号传递和信息交流上起重要作用,从而成为协调植物细胞分化、器官形成和整体生长发育的决定性因素之一。本文概括地介绍了我室在此领域的一些研究进展。     

试析世纪之交植物生理学研究的动向

简要介绍了当今植物生理学研究中值得注意的四个动向,它们是从研究生物大分子到阐明复杂生命活动——基因组学、基因结构与功能的研究;实现生命整体性的重要环节——信号传递的研究;生命活动的能量和物质基础——代谢及其调节的研究;植物与环境(非生物和生物环境)的相互关系——生物的协同进化和适应的研究。     

Phase separation in plants: New insights into cellular compartmentalization

A fundamental challenge for cells is how to coordinate various biochemical reactions in space and time. To achieve spatiotemporal control, cells have developed organelles that are surrounded by lipid bilayer membranes. Further, membraneless compartmentalization, a process induced by dynamic physical association of biomolecules through phase transition offers another efficient mechanism for intracellular organization. While our understanding of phase separation was predominantly dependent on yeast and animal models, recent findings have provided compelling evidence for emerging roles of phase separation in plants. In this review, we first provide an overview of the current knowledge of phase separation, including its definition, biophysical principles, molecular features and regulatory mechanisms. Then we summarize plant-specific phase separation phenomena and describe their functions in plant biological processes in great detail. Moreover, we propose that phase separation is an evolutionarily conserved and efficient mechanism for cellular compartmentalization which allows for distinct metabolic processes and signaling pathways, and is especially beneficial for the sessile lifestyle of plants to quickly and efficiently respond to the changing environment.     

The emergence of phase separation as an organizing principle in bacteria

Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This article assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria.     

Get closer and make hotspots: liquid–liquid phase separation in plants

Liquid–liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus‐responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.     

The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43

The TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43′s intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43′s functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43′s physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320–340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.     

Liquid-liquid phase separation in biology: mechanisms,physiological functions and human diseases

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation(LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization,gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin-and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre-and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.     

Membraneless organelles: P granules in Caenorhabditis elegans

Membraneless organelles are distinct compartments within a cell that are not enclosed by a traditional lipid membrane and instead form through a process called liquid‐liquid phase separation. Examples of these non‐membrane‐bound organelles include nucleoli, stress granules, P bodies, pericentriolar material and germ granules. Many recent studies have used Caenorhabditis elegans germ granules, known as P granules, to expand our understanding of the formation of these unique cellular compartments. From this work, we know that proteins with intrinsically disordered regions (IDRs) play a critical role in the process of phase separation. IDR phase separation is further tuned through their interactions with RNA and through protein modifications such as phosphorylation and methylation. These findings from C elegans, combined with work done in other model organisms, continue to provide insight into the formation of membraneless organelles and the important role they play in compartmentalizing cellular processes.     

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells

In cells at steady state, two forms of cell compartmentalization coexist: membrane‐bound organelles and phase‐separated membraneless organelles that are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments referred to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA‐based processing‐bodies (P‐bodies) and stress granules that form in response to oxidative, endoplasmic reticulum (ER), osmotic and nutrient stress as well as many others. Interestingly, P‐bodies and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Furthermore, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, such as metabolic enzymes foci, proteasome storage granules, EIF2B bodies, U‐bodies and Sec bodies, some of which are not RNA‐based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.     

Quality Control of Membraneless Organelles

The formation of membraneless organelles (MLOs) by phase separation has emerged as a new way of organizing the cytoplasm and nucleoplasm of cells. Examples of MLOs forming via phase separation are nucleoli in the nucleus and stress granules in the cytoplasm. The main components of these MLOs are macromolecules such as RNAs and proteins. In order to assemble by phase separation, these proteins and RNAs have to undergo many cooperative interactions. These cooperative interactions are supported by specific molecular features within phase-separating proteins, such as multivalency and the presence of disordered domains that promote weak and transient interactions. However, these features also predispose phase-separating proteins to aberrant behavior. Indeed, evidence is emerging for a strong link between phase-separating proteins, MLOs, and age-related diseases. In this review, we discuss recent progress in understanding the formation, properties, and functions of MLOs. We pay special attention to the emerging link between MLOs and age-related diseases, and we explain how changes in the composition and physical properties of MLOs promote their conversion into an aberrant state. Furthermore, we discuss the key role of the protein quality control machinery in regulating the properties and functions of MLOs and thus in preventing age-related diseases.     

Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring?

Liquid–liquid phase separation of RNA-binding proteins underlies the formation of membraneless organelles, whose composition is dynamic and whose existence may be transient. These organelles are involved in regulation of RNA processing and translation and, if they behave abnormally, in pathologies. Because disorder phenomena are essential in their formation and dynamics, established methodology is insufficient for characterizing their structure. In this review, we consider the current and potential contribution of NMR and EPR spectroscopy to the understanding of structure and dynamics of phase-separating RNA-binding proteins in, both, their dispersed and condensed state in vitro. We discuss which experiments are applicable under what conditions and which information can be obtained from them. Because for these phenomena, the accessible information depends crucially on metastable phase equilibria, we also consider aspects of sample preparation for NMR and EPR experiments.