Micropipette-based quantification of neuronal protein condensates in live cells

项目摘要 由液-液相分离产生的生物分子凝析油已经成为 无数的细胞过程。这些凝析油的物质性质与各种 生物角色。例如，液体冷凝物的表面张力控制着 凝结物和其他细胞结构，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转导。在异常情况下，有几种类型 神经元蛋白凝聚物从液态转变为固体纤维，类似于 神经退行性变。然而，目前对生物分子凝聚物的理解是有限的，主要是由于 缺乏准确的工具来干扰和监控这些微尺度凝析油的材料性质。 已有的技术侧重于凝析油性质的各个方面，而且往往容易受到 仪表化挑战或测量构件。此外，活细胞中冷凝物的量化是 仍然难以捉摸。最近，我们展示了一种基于微吸管的技术，它可以直接测量两个表面 纯化蛋白质凝聚物的张力和粘度，不含常见人工制品来源。重要的是，我们的 Technology与膜片钳共享其核心硬件的很大一部分，后者是一种成熟的工具，用于 神经学家记录活细胞和动物的电信号。 在正在进行的实验中，我们已经将这项技术应用于几种神经元蛋白质的凝聚体。这 不仅包括与神经退行性变有关的蛋白质，还包括突触素，一种高度丰富的神经元 调节突触小泡聚集和传递的蛋白质。此外，我们还对兼容性进行了测试 我们基于微吸管的技术和活细胞中的膜片钳记录之间的区别。基于这些初步的 数据，我们假设微吸管广泛适用于测量由神经元蛋白质组成的凝集物， 从而能够从机械上了解活细胞中这些冷凝物的材料性质。我们的特定 目的是：(1)从力学角度理解神经元蛋白质的表面张力和粘弹性 通过体外重组进行浓缩。(2)活细胞内神经元蛋白凝集物的定量。 我们预计，拟议的技术可以很容易地被更广泛的科学界采用来研究 生物分子在细胞中凝聚。来自该项目的数据将使人们直接了解凝析油的作用 调节神经过程和神经退行性疾病的材料特性。的定量化 培养细胞中的冷凝物也将为探索复合体中冷凝物材料的性质奠定基础 生物系统。

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.