Biophysics of liquid droplets in bacteria

Brief description of the context of the research including potential impact:

The cytoplasm of biological cells is organised into compartments called organelles which are typically membrane-bound, for example the nucleus, consisting of either a single or double phospholipid bilayer membrane to achieve spatiotemporal separation of different molecules. This compartmentalisation allows the plethora of biochemical reactions that take place simultaneously in the cell to be controlled, for example, by concentrating particular molecules together and sequestering molecules from other components.

However, liquid-liquid phase separation (LLPS) has recently emerged as a key phenomenon driving the formation of biomolecular condensates, or "membraneless organelles", which perform a similar role to membrane-bound compartment but have no surrounding membrane separating the contents from the cytoplasm. The thermodynamic driving force for the phase separation of biomolecules is the exchange of macromolecule-solvent interactions, with macromolecule-macromolecule and solvent-solvent interactions.

In addition to environmental conditions, LLPS is regulated by active processes in the cell, including transcription, post-translational modification, and the ATP-dependent activity of molecular machines. However, several unresolved questions remain, especially the role of the physical properties of biomolecular LLPS droplets and how this influences biological function, and addressing this may have industrial impact through Pharma development.

Recently, we made important progress in developing a bacterial LLPS system called the "aggresome" which shows clear potential to address these questions. Aims and objectives:

1. Understand the bacterial aggresome in more far greater detail in terms of its physical properties, primarily through a range of advanced biophysical experimentation and simulations. 2. Determine the "physical rules" for aggresome assembly and disassembly. 3. Establish how the physicochemical microenvironment of the aggresome affects is function

4. Use these rules to determine how to "tune" the aggresome composion and physical properties.

The research methodology, including new knowledge or techniques in engineering and physical sciences that will be investigated:

Aggresomes will be extracted using immunoprecipitation for "ex vivo" investigations of the liquid droplets. The viscoelastic properties of the aggresome will be measured by combining optical trapping with high-resolution, single-molecule fluorescence imaging. To assess which proteins are essential for aggresome formation, and which protein-protein or protein-RNA interactions stabilise the aggresome structure, a minimal aggresome will be reconstituted in vitro, enabling the effects of different protein or RNA components on the biophysical properties of the droplet to be determined.

Having extracted aggresomes, the precise conditions for disaggregation of the assembly will be investigated and formation conditions simulated using polymer physics theory. Different components to those which have been previously investigated with fluorescence microscopy will be tagged to interrogate in more detail the internal structure of the aggresome, the mobility of different components, and how the biophysical properties change over time and with changing environments.

Alignment to EPSRC's strategies and research areas: Physics of Life; Healthcare Technologies ; Ageing - lifelong health and wellbeing programme; Tackling Infections, Any companies or collaborators involved:

Collaborators - Prof Bai Fan (Peking University, China) and Prof Yingying Pu (Wuhan University, China); company engagement may involve Fujifilm during later stages.