Understanding the viscoelasticity, surface tension, and membrane interactions of biomolecular condensates in live cells

了解活细胞中生物分子凝聚物的粘弹性、表面张力和膜相互作用

• 批准号: 10707259
• 负责人: Zheng Shi
• 金额: $ 22.58万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707259
• 关键词: Animals; Autophagocytosis; Biological; Biological Process; Cell Line; Cell membrane; Cell surface; Cells; Cellular Structures; Complex; Cytoplasmic Granules; Data; Exocytosis; Growth; Liquid substance; Mechanics; Mediating; Membrane; Microtubules; Molecular; Morphologic artifacts; Mutation; Nerve Degeneration; Nervous System; Neurodegenerative Disorders; Neurons; Organelles; Pathologic; Pharmaceutical Preparations; Phase; Phase Transition; Physical condensation; Process; Property; Proteins; Role; Signal Transduction; Solid; Source; Surface Tension; Synapses; Synapsins; Synaptic Vesicles; Techniques; Testing; Therapeutic; Tight Junctions; Vesicle; Viscosity; Work; aspirate; experimental study; insight; patch clamp; tool; trafficking; transmission process; vesicular release; viscoelasticity

PROJECT SUMMARY Phase separation in cells can lead to the formation of biomolecular condensates, also known as membraneless organelles. The material properties of these condensates are associated with various biological and pathological roles. For example, the surface tension of a liquid condensate governs its interaction with both membranous and membraneless organelles, regulating processes such as autophagy, vesicle trafficking, nucleoli organization, microtubule branching, P granule growth, and cell surface signaling. Under abnormal conditions, several types of biomolecular condensates change from liquid states to solid fibrils that resemble the hallmarks of neurodegeneration. However, current understanding of the material properties of biomolecular condensates severely lacks in two important aspects: 1) quantitative assessments of condensates in live cells; 2) a mechanistic understanding of factors that control the properties and functions of condensates. Recently, we demonstrated the use of micropipette aspiration, a technique known for studying membranes, in quantifying both the surface tension and viscosity of protein condensates, free from common sources of artifacts. Importantly, our technique shares a large part of its core hardware with patch-clamp, a well-established tool used by neuroscientists to record electrical signals in live cells and animals. In ongoing experiments, we have applied the technique to several different types of biomolecular condensates. This includes proteins associated with neurodegeneration as well as synapsin, a highly abundant neuronal protein that regulates synaptic vesicle clustering and transmission. Furthermore, we have tested the compatibility of our technique with cellular patch-clamp recording. Based on these preliminary data, we hypothesize that micropipettes can be broadly applied to understand the material properties of biomolecular condensates in live cells. In the next five years, we will first develop the micropipette-based technique into an accurate, broadly applicable, and easily accessible tool for quantifications of biomolecular condensates in common cell lines and primary neurons. This new tool will allow us to collect the much-needed quantitative data that can give direct insights into the roles of condensate material properties in mediating a wide range of biological processes. We will study the role of surface tension in governing the integrity of synapsin condensates, and the role of condensate viscosity in modulating the dynamics of synaptic vesicle release and exocytosis. We will also investigate the interplays between cell membrane mechanics and membrane-wetting condensates such as those at synapses and tight junctions. On the front of pathological relevance, we will focus on elucidating condensate material properties that underlie the aberrant phase transition of neurodegeneration-associated proteins. We will take advantage of the cytosolic access of our technique to directly test the effect of drug molecules that are targeted to intracellular condensates. Our quantitative studies of condensates in cultured neurons will also set the stage for exploring biomolecular condensates in complex nervous systems.

项目摘要 细胞中的相分离可导致生物分子凝聚物的形成，也称为无膜 细胞器这些冷凝物的材料性质与各种生物和病理学相关。 角色例如，液体冷凝物的表面张力控制其与膜和膜之间的相互作用。 无膜细胞器，调节自噬、囊泡运输、核仁组织等过程， 微管分支，P颗粒生长和细胞表面信号传导。在非正常情况下， 的生物分子凝聚物从液态变成固体纤维， 神经变性然而，目前对生物分子凝聚物的材料性质的理解 严重缺乏两个重要方面：1）活细胞中冷凝物的定量评估; 2） 对控制冷凝物性质和功能的因素的机械理解。 最近，我们展示了微管抽吸的使用，这是一种用于研究细胞膜的技术， 在定量蛋白质缩合物的表面张力和粘度时，不含常见的 藏物重要的是，我们的技术与膜片钳技术共享其核心硬件的很大一部分，膜片钳技术是一种成熟的技术。 神经科学家用来记录活细胞和动物电信号的工具。在正在进行的实验中，我们 已经将该技术应用于几种不同类型的生物分子凝聚物。其中包括蛋白质 与神经变性以及突触蛋白相关，突触蛋白是一种高度丰富的神经元蛋白， 突触囊泡聚集和传递。此外，我们还测试了我们的技术与 细胞膜片钳记录。基于这些初步数据，我们假设微量移液器可以 广泛应用于了解活细胞中生物分子凝聚物的材料特性。 在接下来的五年里，我们将首先将基于微量移液器的技术发展成为一种准确，广泛的 用于定量常见细胞系中的生物分子缩合物的适用且容易获得的工具， 初级神经元这一新工具将使我们能够收集急需的定量数据， 深入了解冷凝物材料特性在介导广泛的生物过程中的作用。我们 将研究表面张力在控制突触蛋白凝聚物的完整性中的作用，以及 凝聚物粘度在调节突触囊泡释放和胞吐的动力学。我们还将 研究细胞膜力学和膜润湿冷凝物之间的相互作用， 突触和紧密连接处在病理相关性方面，我们将重点阐述凝聚 神经变性相关蛋白质的异常相变的基础材料性质。我们将 利用我们的技术的胞质访问，直接测试药物分子的作用， 靶向细胞内冷凝物。我们对培养神经元中凝聚物的定量研究也将为 探索复杂神经系统中生物分子凝聚物的阶段。