Single-molecule investigation of pathological protein condensates by combining microfluidics with fluorescence microscopy

Abstract

Biomolecular condensates, formed via liquid-liquid phase separation (LLPS), are integral to cellular function. Yet, LLPS-driven phase separation of pathological proteins (e.g., tau and TDP43) under physiological conditions suggests a central role for condensate formation in pathological aggregation. This highlights the importance of investigating the microenvironment and molecular interactions within these condensates. However, probing the conformational changes and interactions of single molecules in condensates remains challenging. In this study, flow-based microfluidic technology and fluorescence microscopy are combined to investigate protein aggregation within a controlled droplet environment. Microfluidic devices offer the dual advantage of high-throughput measurements on small sample volumes and precise control over protein self-assembly parameters (e.g., salt concentration). Moreover, a controlled flow rate allows the one-by-one passage of droplets containing trace amounts of fluorescently labeled protein. Combining this method with fluorescence microscopy enables quantitative analysis of diffusion, single-molecule conformational dynamics, and molecular interactions within droplets. The data obtained from this research will provide valuable insights into the relationship between pathological protein aggregation and protein misfolding disorders.