Biophysics of liquid droplets in bacteria

简要描述研究背景，包括潜在影响：生物细胞的细胞质被组织成称为细胞器的室室，通常是膜结合的，例如细胞核，由单或双磷脂双层膜组成，实现不同分子的时空分离。这种区隔化可以控制细胞中同时发生的大量生化反应，例如，通过将特定分子集中在一起并将分子与其他成分隔离开来。然而，液-液相分离（LLPS）最近成为驱动生物分子凝聚物或“无膜细胞器”形成的关键现象，其作用类似于膜结合室，但没有将内容物与细胞质分离的周围膜。生物分子相分离的热力学驱动力是大分子-溶剂相互作用的交换，包括大分子-大分子相互作用和溶剂-溶剂相互作用。除了环境条件外，LLPS还受到细胞内活性过程的调节，包括转录、翻译后修饰和分子机器的atp依赖性活性。然而，一些尚未解决的问题仍然存在，特别是生物分子LLPS液滴的物理特性的作用以及它如何影响生物功能，解决这些问题可能会通过制药开发产生工业影响。最近，我们在开发一种称为“aggresome”的细菌LLPS系统方面取得了重要进展，它显示出解决这些问题的明显潜力。宗旨和目标：主要通过一系列先进的生物物理实验和模拟，更详细地了解细菌聚集体的物理特性。2. 确定集合组装和拆卸的“物理规则”。确定聚合体的物理化学微环境如何影响其功能。使用这些规则来确定如何“调优”总体组成和物理属性。研究方法，包括将研究的工程和物理科学的新知识或新技术：聚合体将使用免疫沉淀法提取，用于液滴的“离体”研究。聚合体的粘弹性特性将通过结合光学捕获和高分辨率单分子荧光成像来测量。为了评估哪些蛋白质是聚集体形成所必需的，以及哪些蛋白质-蛋白质或蛋白质-RNA相互作用稳定了聚集体结构，将在体外重建一个最小的聚集体，从而确定不同蛋白质或RNA成分对液滴生物物理特性的影响。在提取聚合体后，将研究聚合体分解的精确条件，并利用聚合物物理理论模拟形成条件。与以前用荧光显微镜研究过的不同成分将被标记，以更详细地询问聚合体的内部结构，不同成分的流动性，以及生物物理性质如何随着时间和环境的变化而变化。与EPSRC的战略和研究领域保持一致：生命物理学；医疗保健技术；老龄——终身健康和福利方案；合作者：范白教授（中国北京大学）和蒲莹莹教授（中国武汉大学）；公司参与可能涉及富士胶片在后期阶段。

Brief description of the context of the research including potential impact:The cytoplasm of biological cells is organised into compartments called organelles which are typically membrane-bound, for example the nucleus, consisting of either a single or double phospholipid bilayer membrane to achieve spatiotemporal separation of different molecules. This compartmentalisation allows the plethora of biochemical reactions that take place simultaneously in the cell to be controlled, for example, by concentrating particular molecules together and sequestering molecules from other components. However, liquid-liquid phase separation (LLPS) has recently emerged as a key phenomenon driving the formation of biomolecular condensates, or "membraneless organelles", which perform a similar role to membrane-bound compartment but have no surrounding membrane separating the contents from the cytoplasm. The thermodynamic driving force for the phase separation of biomolecules is the exchange of macromolecule-solvent interactions, with macromolecule-macromolecule and solvent-solvent interactions. In addition to environmental conditions, LLPS is regulated by active processes in the cell, including transcription, post-translational modification, and the ATP-dependent activity of molecular machines. However, several unresolved questions remain, especially the role of the physical properties of biomolecular LLPS droplets and how this influences biological function, and addressing this may have industrial impact through Pharma development. Recently, we made important progress in developing a bacterial LLPS system called the "aggresome" which shows clear potential to address these questions.Aims and objectives:1. Understand the bacterial aggresome in more far greater detail in terms of its physical properties, primarily through a range of advanced biophysical experimentation and simulations. 2. Determine the "physical rules" for aggresome assembly and disassembly.3. Establish how the physicochemical microenvironment of the aggresome affects is function4. Use these rules to determine how to "tune" the aggresome composion and physical properties. The research methodology, including new knowledge or techniques in engineering and physical sciences that will be investigated:Aggresomes will be extracted using immunoprecipitation for "ex vivo" investigations of the liquid droplets. The viscoelastic properties of the aggresome will be measured by combining optical trapping with high-resolution, single-molecule fluorescence imaging. To assess which proteins are essential for aggresome formation, and which protein-protein or protein-RNA interactions stabilise the aggresome structure, a minimal aggresome will be reconstituted in vitro, enabling the effects of different protein or RNA components on the biophysical properties of the droplet to be determined. Having extracted aggresomes, the precise conditions for disaggregation of the assembly will be investigated and formation conditions simulated using polymer physics theory. Different components to those which have been previously investigated with fluorescence microscopy will be tagged to interrogate in more detail the internal structure of the aggresome, the mobility of different components, and how the biophysical properties change over time and with changing environments. Alignment to EPSRC's strategies and research areas:Physics of Life; Healthcare Technologies ; Ageing - lifelong health and wellbeing programme; Tackling Infections, Any companies or collaborators involved:Collaborators - Prof Bai Fan (Peking University, China) and Prof Yingying Pu (Wuhan University, China); company engagement may involve Fujifilm during later stages.