Physicochemical properties driving membraneless organelle assembly in bacteria

驱动细菌无膜细胞器组装的物理化学特性

• 批准号: 10274445
• 负责人: Julie Biteen
• 金额: $ 59.65万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10274445
• 关键词: Affect; Automobile Driving; Bacteria; Bacterial Chromosomes; Bacterial DNA; Bacterial Physiology; Behavior; Biochemical; Biochemistry; Biogenesis; Cell Separation; Cell Survival; Cell physiology; Cells; Chromosome Condensation; Chromosome Structures; Chromosomes; Complement; Complex; Cytoplasm; DNA; DNA Binding; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Diffuse; Diffusion; Engineering; Environment; Eukaryotic Cell; Feedback; Genetic; Goals; Growth; Image; In Vitro; Kinetics; Life; Light; Liquid substance; Maps; Measures; Mediating; Membrane; Microscopy; Modeling; Molecular; Motion; Nucleic Acids; Organelles; Organism; Phase; Phase Transition; Physical condensation; Physiological; Process; Property; Proteins; Proteomics; Research; Resolution; Ribosomes; Role; Spatial Distribution; Spectrum Analysis; Stress; Structure; System; Testing; Time; Trees; Work; antibiotic design; cell injury; chemical property; chromatin immunoprecipitation; chromosome conformation capture; crystallinity; defined contribution; environmental change; experimental study; genome-wide; genomic locus; in silico; in vivo; innovation; liquid crystal; macromolecule; mechanical behavior; mechanical properties; multidisciplinary; novel; organizational structure; physical property; response; single molecule; small molecule; theories; tool; viscoelasticity

Project Summary Recently, breakthrough work has led to a wave of discoveries of biomolecular condensates. Such membraneless organelles that cluster specific biomolecules away from the surrounding cellular milieu have long been theorized and are now experimentally tractable. These dynamic structures contain a wide range of proteins and nucleic acids and assemble through the process of phase separation. While many proteins are prone to phase separation (either by themselves or via complexation with other proteins, nucleic acids, or small molecules), these condensates have primarily been found in eukaryotic cells. Since bacteria do not typically contain membrane-enclosed organelles, we hypothesize that bacteria instead use phase-separated membraneless organelles as novel organizers of their cytoplasm to regulate biochemical activity while they respond to changing environmental conditions. In this proposal, our multidisciplinary team combines state-of-the-art in vitro approaches, in vivo experiments, and in silico modeling and theory to explore the structural organization of the bacterial cytoplasm and characterize phase-separated membraneless organelles in bacteria. We will focus on a candidate protein system, the DNA-binding protein from starved cells (Dps), that drives the organization of the bacterial chromosome and leads DNA to form a separate subcellular compartment within bacterial cells upon stress. We will first study this system’s chemical and mechanical properties, map the phase space for condensate formation, ascertain whether it occurs through spinodal decomposition or nucleation and condensate droplet growth, and determine its kinetics in vitro. Next, we will elucidate how phase separation controls the access of cytoplasmic and nucleoid-associated biomolecules to the bacterial chromosome and image the structure of membraneless DNA-organizing organelles in living bacteria to measure the effect of condensation on chromosome structure and dynamics in vivo. Finally, we will characterize the impact of chromosome phase separation on the mobility of cytoplasmic and DNA-binding proteins in vivo and determine the role of chromosomal condensation in bacterial physiology and survival. Together, our results will define the contributions of the unique physicochemical properties of the bacterial cytoplasm to compartmentalization within these cells. Phase separation provides an alternate mechanism for spatial and functional organization in the bacterial domain of life. Indeed, phase separation is emerging as a universal organizing principle across the tree of life, and our work will ultimately shed light on the origin of life and provide new targets for rationally designed antibiotics.

项目摘要 最近，突破性的工作导致了一波生物分子凝聚物的发现。等 无膜细胞器聚集特定的生物分子远离周围的细胞环境， 长期以来一直是理论化的，现在已经可以在实验中使用了。这些动态结构包含广泛的 蛋白质和核酸通过相分离过程组装。虽然许多蛋白质 倾向于相分离（通过自身或通过与其他蛋白质、核酸或小分子复合）， 分子），这些缩合物主要在真核细胞中发现。因为细菌通常 含有膜封闭的细胞器，我们假设细菌，而不是使用相分离 无膜细胞器作为其细胞质的新组织者来调节生化活性， 应对不断变化的环境条件。 在这项提案中，我们的多学科团队结合了最先进的体外方法，体内实验， 以及计算机模拟和理论来探索细菌细胞质的结构组织， 表征细菌中相分离的无膜细胞器。我们将重点研究一种候选蛋白质 系统，来自饥饿细胞的DNA结合蛋白（Dps），驱动细菌的组织 染色体，并导致DNA在压力下在细菌细胞内形成单独的亚细胞区室。我们 首先研究了该体系的化学和力学性质，绘制了凝析油的相空间图 形成，确定它是通过亚稳分解还是通过成核和冷凝液滴发生的 生长，并测定其体外动力学。接下来，我们将阐明相分离如何控制 细胞质和核相关的生物分子的细菌染色体和图像的结构 活细菌中的无膜DNA组织细胞器，以测量冷凝对 体内染色体结构和动力学。最后，我们将描述染色体相位的影响 分离细胞质和DNA结合蛋白在体内的迁移率，并确定 细菌生理学和存活中的染色体浓缩。我们的结果将共同定义 细菌细胞质独特的物理化学性质对区室化的贡献 在这些细胞中。相分离为空间和功能组织提供了一种替代机制， 生命的细菌领域。事实上，相分离正在成为一个普遍的组织原则， 生命之树，我们的工作将最终揭示生命的起源，并为合理的研究提供新的目标。 设计的抗生素