Micropipette-based quantification of neuronal protein condensates in live cells

点击翻译按钮获取中文摘要

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.