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

项目总结 细胞中的相分离会导致生物分子凝聚体的形成，也称为无膜凝聚体。 细胞器。这些凝聚体的物质性质与各种生物学和病理学有关。 角色。例如，液体冷凝液的表面张力决定了它与膜和 无膜细胞器，调节过程，如自噬，囊泡运输，核仁组织， 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 的生物分子凝聚物从液态转变为固体纤维，类似于 神经退行性变。然而，目前对生物分子凝析油材料性质的理解 严重缺乏两个重要方面：1)活细胞内凝结物的定量评估；2)a) 对控制凝析油性质和功能的因素的机械理解。 最近，我们演示了微吸管吸入法的使用，这是一种研究膜的技术， 在量化蛋白质凝聚物的表面张力和粘度时，不含常见的 手工艺品。重要的是，我们的技术与膜片钳共享其核心硬件的很大一部分，膜片钳是一种成熟的 神经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 已经将这项技术应用于几种不同类型的生物分子缩合物。这包括蛋白质 与神经退行性变和突触素有关，突触素是一种高度丰富的神经元蛋白，调节 突触小泡聚集和传递。此外，我们还测试了我们的技术与 细胞膜片钳记录。根据这些初步数据，我们假设微吸管可以 广泛应用于了解活细胞中生物分子凝聚物的材料性质。 在接下来的五年里，我们将首先将基于微吸管的技术发展成一种准确、广泛的 适用且易于获取的工具，用于对普通细胞系和 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，从而直接给出 对凝析油材料性质在调节广泛的生物过程中的作用的洞察。我们 将研究表面张力在调节突触蛋白凝聚体完整性中的作用，以及 凝集液粘度在调节突触囊泡释放和胞吐动力学中的作用。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 在突触和紧密连接处。在病理相关性方面，我们将重点阐明凝析油 神经退变相关蛋白异常相变的物质特性。我们会 利用我们技术的胞质通道直接测试药物分子的效果 目标是细胞内的冷凝物。我们对培养神经元中凝集物的定量研究也将 探索复杂神经系统中生物分子凝聚物的阶段。

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.