Micropipette-based quantification of neuronal protein condensates in live cells

基于微量移液管的活细胞中神经元蛋白凝聚物的定量

• 批准号: 10681474
• 负责人: Zheng Shi
• 金额: $ 18.5万
• 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: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10681474
• 关键词: Affect; Aging; Animals; Autophagocytosis; Binding; Biological; Brain; Bypass; C-terminal; Cell Line; Cell physiology; Cell surface; Cells; Cellular Structures; Communities; Cultured Cells; Cytoplasmic Granules; Cytoskeleton; Data; Disease; Electrophysiology (science); Environment; Fluorescence; Growth; Health; Hela Cells; Human; In Vitro; Individual; Life Cycle Stages; Liquid substance; Measurement; Measures; Mediating; Membrane; Microtubules; Molecular; Monitor; Morphologic artifacts; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurologic Process; Neurons; Neurosciences; Parkinson Disease; Pathologic; Pharmaceutical Preparations; Phase; Phase Transition; Physical condensation; Predisposition; Process; Property; Proteins; Regulation; Reporter; Role; Signal Transduction; Slice; Solid; Source; Surface; Surface Tension; Synapsins; Synaptic Vesicles; System; Techniques; Technology; Testing; Therapeutic; Viscosity; Work; alpha synuclein; aspirate; complex biological systems; disease-causing mutation; experimental study; in vivo; innovation; insight; instrumentation; meter; patch clamp; preservation; prevent; protein aggregation; protein purification; reconstitution; response; seal; success; tool; transmission process; viscoelasticity

Project Summary Biomolecular condensates that arise from liquid-liquid phase separation have emerged as a central player in numerous cellular processes. The material properties of these condensates are associated with various biological roles. For example, the surface tension of liquid condensates governs the interaction between the condensate and other cellular structures, regulating processes such as nucleoli organization, autophagy, microtubule branching, P granule growth, and cell surface signaling. Under abnormal conditions, several types of neuronal protein condensates change from liquid states to solid fibrils that resemble the hallmarks of neurodegeneration. However, current understanding of biomolecular condensates is limited, mainly due to the lack of accurate tools that can perturb and monitor the material properties of these microscale condensates. Established techniques focus on individual aspects of condensate properties and are often susceptible to instrumentation challenges or measurement artifacts. Moreover, quantifications of condensates in live cells are still elusive. Recently, we demonstrated a micropipette-based technique that directly measures both the surface tension and viscosity of purified 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 condensates of several neuronal proteins. This includes not only proteins associated with neurodegeneration, but also synapsin, a highly abundant neuronal protein that regulates synaptic vesicle clustering and transmission. Furthermore, we have tested the compatibility between our micropipette-based technique and patch-clamp recording in live cells. Based on these preliminary data, we hypothesize that micropipette is broadly applicable to measure condensates made of neuronal proteins, allowing mechanistic understanding of the material properties of these condensates in live cells. Our Specific Aims are: (1) Mechanistic understanding of the surface tension and viscoelasticity of neuronal protein condensates through in vitro reconstitutions. (2) Quantification of neuronal protein condensates in live cells. We anticipate the proposed technology can be easily adapted by the broader scientific community to study biomolecule condensates in cells. Data from this project will give direct insights into the roles of condensate material properties in mediating neurological processes and neurodegenerative diseases. The quantification of condensates in cultured cells will also lie the basis for exploring condensate material properties in complex biological systems.

项目摘要 由液-液相分离产生的生物分子冷凝物已经成为生物化学的核心参与者。 许多细胞过程。这些冷凝物的材料性质与各种 生物角色。例如，液体冷凝物的表面张力控制了冷凝物与水之间的相互作用。 凝聚物和其他细胞结构，调节过程，如核仁组织，自噬， 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， 的神经元蛋白质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理解是有限的，主要是由于 缺乏可以扰动和监测这些微尺度冷凝物的材料性质的精确工具。 已建立的技术侧重于凝析油性质的各个方面，并且通常容易受到 仪器挑战或测量伪影。此外，活细胞中的冷凝物的定量是 仍然难以捉摸最近，我们展示了一种基于微量移液器的技术，该技术直接测量了表面 张力和粘度的纯化蛋白质浓缩物，从人工制品的共同来源免费。重要的是我们的 Technique与膜片钳共享其大部分核心硬件，膜片钳是一种由 神经科学家记录活细胞和动物的电信号。 在正在进行的实验中，我们已经将该技术应用于几种神经元蛋白质的浓缩物。这 不仅包括与神经变性相关的蛋白质，还包括突触蛋白，一种高度丰富的神经元蛋白， 调节突触囊泡聚集和传递的蛋白质。此外，我们还测试了兼容性 我们的基于微量移液器的技术和活细胞中的膜片钳记录之间的差异。基于这些初步 数据，我们假设微量移液管广泛适用于测量由神经元蛋白质制成的冷凝物， 允许对活细胞中这些凝聚物的材料性质的机械理解。我们的具体 目的是：（1）对神经元蛋白质的表面张力和粘弹性的机理认识 通过体外重组进行浓缩。(2)活细胞中神经元蛋白质缩合物的定量。 我们预计所提出的技术可以很容易地被更广泛的科学界所适应， 细胞中的生物分子凝聚物。该项目的数据将直接洞察凝析油的作用 在介导神经过程和神经退行性疾病中的材料性质。的量化 培养细胞中的冷凝物也将为探索复杂的冷凝物材料特性奠定基础。 生物系统。