Investigating Ageing of Neurodegenerative Disease Relevant Protein Condensates

Change log
Zhou, Alexandra 

The phenomenon of liquid-liquid separation (LLPS) has been a popular topic within soft matter physics. There is now a substantial body of literature on biological macromolecules such as proteins that can undergo the LLPS process and form biomolecular condensates. It has been demonstrated repeatedly that these condensates can play a wide range of cellular roles, from transcription to metabolism. In addition, an increasing amount of research is devoted to investigating aberrant protein condensates. The ongoing effort to find the link between dysfunctional proteins condensates and neurodegenerative disease pathogenesis complements the current research.

Protein condensates often become pathological when their physical properties change, from a liquid state to a more gel-like or solid-like state. This process is referred to as a solution-to-gel (sol-gel) transition. There are many external factors leading to this property change, such as temperature and stress. There has been as yet no systematic examination of the physical property changes as a function of time, in other words, the ageing of biomolecular condensates. Therefore, this thesis focuses on the ageing behaviour of several neurodegenerative disease-related proteins with a biophysical approach. More specifically, condensate properties including viscosity, complex modulus, and fusion behaviour are investigated and revealed with innovative tools and techniques.

At present, little is known about the absolute viscosity of biomolecular condensates both in vivo and in vitro, especially as a function of time. An initial study using micro-rheology was conducted to acquire viscosity measurements of fresh and aged protein condensates. Moreover, an analysis of the complex modulus of the condensates supplemented our understanding of condensate properties. A combined platform integrating micro-rheology and fluorescence lifetime imaging (FLIM) was then established to take the findings one step further. One difficulty in studying biomolecular condensate properties is the lack of accessibility to condensates in in vivo systems. The combined platform was developed to measure both viscosity and aggregation state in in vitro environments and provide a relationship between the two quantities. The relationship can then be applied to infer the viscosity or aggregation state of in vivo condensates in a situation where only one of the measurements can be obtained easily. Such a platform provides better insight into the ageing of condensates in a biologically relevant context.

To further improve the understanding of biomolecular condensates, this thesis also encompasses an investigation of the physical interaction between condensates. Leveraging a dual-trap optical tweezer setup with bright-field and fluorescence imaging and force response probes, the fusion behaviour of protein condensates was studied in detail. Bright-field microscope allowed protein fusion driven by surface tension to be observed and recorded, and force measurements reinforced the findings with highly accurate readouts on the characteristic fusion time. Two-channel confocal imaging highlighted new and interesting observations on how fresh and aged condensates could influence each other.

This thesis may contribute towards a better understanding of biomolecular condensate ageing, especially the ones that are accounted for prominent neurodegenerative diseases when becoming aberrant. Building upon the tools and techniques developed within this thesis, further studies on a broader range of biomolecular condensates can reveal additional implications of condensate ageing in biology and medicine.

Knowles, Tuomas
Biomolecular condensates, Liquid-liquid phase separation, Protein physics, Solution-to-gel transition
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge