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

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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.