Selection and Regulation of Compartments by Fuel-driven Phase Separation

Project Information

FuelledLife

Grant agreement ID: 949021

DOI

10.3030/949021

EC signature date 17 August 2020

Start date 1 September 2021

End date 31 August 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 498 852,00

EU contribution

€ 1 498 852,00

Coordinated by

UNIVERSITAET AUGSBURG
Germany

Periodic Reporting for period 3 - FuelledLife (Selection and Regulation of Compartments by Fuel-driven Phase Separation)

Reporting period: 2024-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Living cells rely on the compartmentalization of thousands of different molecules and their chemical reactions. Remarkably, many of such compartments form by phase separation of heteropolymers controlled by sequence-specific interactions and fuel that drives reactions away from equilibrium. If we knew how such polymers with sequence-specific interactions evolve and compartmentalize in fuel-driven multi-component mixtures, we would better understand the role of phase separation in living cells and how synthetic or prebiotic cells emerge.

The overall objective of this proposal is to study how fuel-driven phase separation can drive the selection and replication of hetero-polymers with sequence-specific interactions, the control of their chemical reactions, and the emergence and selection of different compartments. My team and I will develop a theory for phase separation and chemical reactions in multi-component mixtures driven away from equilibrium by irreversible, fuel-driven reactions. This theory will provide a link between phenomena on the compartment scale and coarse-grained properties of sequences. First, we will use this theory to study how compartments control biochemical reactions and how this control is determined by sequence. Second, we will investigate how sequences are selected, replicated, and evolve under cyclic, non-equilibrium conditions. Third, we will use our theory to unravel how fuel-driven chemical reactions regulate the formation and division of compartments and affect the selection of different compartments within a population. Our theoretical studies will elucidate the physical mechanisms and conditions, which will be experimentally scrutinized by our collaborators.

Our results will help us understand how living cells regulate phase separation, such as the formation of stress granules, by selecting RNA. Moreover, our results will elucidate the role of phase separation in the emergence of life by determining the prerequisites for a protocell to divide, replicate, and undergo selection.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Within the first funding round of this proposal, we have derived a theory for multi-component phase separation and fuel-driven chemical reactions (Bauermann, AdP 2022; Michaels, PRR 2022, Pönisch, Biophys. J. 2023, Laha, Arxiv 2024, Bartolucci, Bioarxiv 2024).
Recently, we even accounted for hydrodynamic flows and developed numerical techniques enabling efficient computations.
We also applied this theoretical framework to various experimental systems of our experimental collaborators. By comparing theory and experiments,
we could quantitatively extract chemical reaction rates - a prerequisite to deciphering the functionalities of chemical reaction cycles in system chemistry and biology.

We also developed theoretical frameworks to numerically calculate the growth and shape dynamics of millions of compartments of different compositions, where the components are subject
to fuel-driven, irreversible chemical reactions. In several recent publications, we set the groundwork for this goal (Bauermann, AdP 2022, Laha, Arxiv 2024). Currently, we are applying this framework to study
the selection of compartments.

We also studied the selection, replication, and evolution of molecules with sequence-specific interactions. In particular, we investigated which
types of sequences get selected under which physical condition. In collaboration with the Braun group (Bartolucci et al, PNAS, 2023), we could show that when subjecting phase separating system by a cycling sequence pool
gives rise to the selection of specific sequences. This selection is based on specific sequence-sequences interaction triggering phase separation. In simple words, the cycles fish for better sequences in each cycle while expelling less
well-interacting sequences. In the next step, we start accounting for polymerization processes (Bartolucci, Bioarxiv, 2023).
Moreover, for the last funding round, we will continue with detailed studies on systems where compartments can undergo replication and selection of compartments.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

A key achievement beyond the state of the art is the derivation of the mass action law for systems with coexisting phases (Bauermann, Laha et al., JACS 2022). The key challenge was to find a self-consistent theoretical framework for non-dilute conditions and where phases are at phase equilibrium. Our results are simple and coupled ordinary differential equations that extend the laws of chemical kinetics, enabling us to account for the chemical kinetics of non-dilute mixtures in the presence of coexisting phases. It is not only key to deciphering the functions of biomolecular condensates in living cells but also represents a framework to guide recent developments in synthetic life through compartmentalization.

Another key achievement is the first comparison between in vivo phase separation data (in C. elegans) and a theoretical model from polymer physics (Fritsch et al., PNAS, 2021). Our work suggests that biomolecular condensates in vivo can establish local thermodynamic equilibria despite the non-equilibrium conditions in living cells. Moreover, due to the thorough quantification and analysis of microscope data, this work also constitutes an essential contribution to the flourishing research field of intracellular phase separation.

We developed several fundamental dynamic theories for phase separation in the presence of chemical reactions maintained away from equilibrium (Bauermann, AdP 2022; Michaels, PRR 2022, Pönisch, Biophys. J. 2023, Laha, Arxiv 2024, Bartolucci, Bioarxiv 2024). These theories can serve a large community of scientists to understand their observations of coacervate regulation by chemical processes, or biomolecular condensate formation and the emergence of biological functionality in living cells.

In collaboration with the chemist's group of J. Boekhoven, we found new non-equilibrium steady states for liquid phase separated states with chemical reaction: liquid shells that are stable due to persistent non-equilibrium fluxes. We worked out the underlying physical-chemical mechanisms in several theoretical (Bartulucci, Biophys. J. 2021; Bauermann, PRR, 2023) and interdisciplinary (experiment+theory) studies (Bergmann, Nat Com. 2023).