京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21778号 / 工博第4595号 / 新制||工||1716(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 木村 俊作, 教授 瀧川 敏算, 教授 大内 誠 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Orderliness, speed, and rhythm in biochemistry are vital for cellular function. In order to achieve this, cells implement compartmentalization via several methods, one of which is the formation of membrane-less compartments. These compartments, often referred to as “biomolecular condensates”, are understood to be formed by separation of proteins and other biomolecules into dense and dilute phases. While the formation of the resulting protein-rich condensates is fundamental for spatiotemporal organization of biochemistry within the cell, a vast majority of proteins found to phase separate in vitro do so at a concentration an order of magnitude above their endogenous expression levels. Recently, a theoretical study has shown that membrane binding of phase separating proteins can result in phase separation spatially occurring at the membrane well below bulk saturation concentrations. However, much remains unknown about the formation mechanism and function of these condensates. To that end, for my doctoral project, I used a synthetic system composed of supported lipid bilayers decorated with lipid-bound NTA(Ni) to allow for coordination and thus membrane binding of the well-characterized protein FUS via a C-terminal His-tag. Through this model system I found that 2D phase separation of FUS could occur an order of magnitude below the experimentally determined bulk saturation concentration. FUS was able to form dense and dilute phases in 2D and the transition point to form these phases could be controlled by modulating buffer conditions. Additionally, membrane-bound FUS condensates were able to further recruit FUS from the bulk to form a multilayer of protein through a prewetting transition. With this characterization of 2D phase separation of FUS, I then explored a physiologically relevant protein in the form of PAR-3, a fundamental protein of the PAR polarity system, which is necessary for the establishment of polarity in the C. elegans zygote. I found that full-length PAR-3 was able to phase separate under physiological salt conditions with a Csat of 100nM. Further, I identified a C-terminal predicted prion-like domain to act as a driver for phase separation. Additionally, I determined PAR-3’s affinity and specificity for PI(4,5)P2 and found that it could form 2D condensates upon binding to the membrane at physiological concentrations. Furthermore, these condensates were able to recruit PAR-6 alone and PAR-6 in complex with PKC-3 to the membrane, ultimately resulting the reconstitution of the anterior PAR complex which is known to exist in a condensed clustered form in vivo. Taken together, this work provides insight into a mechanism where phase separation can be locally triggered by membrane binding under sub-saturation concentration, offering a robust and potentially universal mechanism by which cells can spatially control phase separation and pattern cellular membranes.

Interactions among the multitude of macromolecules populating the cytoplasm can lead to the emergence of coexisting phases formed via phase separation. This phenomenon plays a crucial role in the spatial organization of cells and the regulation of their functions. Many of the molecules that drive phase separation can undergo transitions among different states. Proteins, for example, can go through conformational transitions and switch among different phosphorylation states. In addition, proteins that are relevant for phase separation can assemble into oligomers of different sizes. Both molecular transitions and oligomerization can be described as chemical reactions in the context of theories that account for phase separation in multicomponent mixtures. In this work, we discuss how chemical reactions can be used to control coexisting phase composition and shape. In particular, focusing on molecular transitions among two states of a protein, we find a discontinuous thermodynamic phase transition in the composition of the protein-dense phase, as a function of temperature. Breaking detailed balance of the molecular transition by continuous fuel addition can also be used to control the number of distinct coexisting phases and their composition. Additionally, fuel turnover can lead to the emergence of novel patterns as the system approaches a non-equilibrium stationary state. We focus on the mechanism that leads to the formation of ring-like patterns, motivated by the observation of similar shapes in experiments with chemical reaction cycles coupled to a fuel reservoir. We propose that, due to chemical reactions, the composition at the centre of the dense phase can be altered, leading to an instability that drives the formation of a new interface. Controlling the composition of coexisting phases becomes crucial when the number of components and the number of reactions among them rises. This is the case in mixtures containing proteins that can be found in a monomeric state but also form aggregates of arbitrary size. We characterise the equilibrium of such systems in the limit of maximum aggregate size going to infinity. For systems that phase separate, we show that the aggregate size distribution can be different in each of the coexisting phases and is determined by the temperature and the energy of bonds between monomers. Mixtures composed of disk-like or spherical aggregates can undergo a gelation transition. Gelation can be considered as a special case of phase coexistence between a dilute phase (the 'sol') containing aggregates of finite size, and a 'gel' phase, corresponding to an aggregate of infinite size. Lowering the temperature leads to a transition from two coexisting ''sol' phases to the coexistence of a 'sol' phase and a ''gel'' phase. In summary, this work provides a theoretical framework to study phase-separating systems composed of many components that undergo chemical reactions. Furthermore, we discuss how to exploit such reactions to control the composition of coexisting phases.

Filled hydrogel particles consisting of emulsified oil droplets encapsulated within a hydrogel matrix were fabricated based on the phase separation of proteins and polysaccharides through aggregative and segregative mechanisms. A 3% (wt/wt) pectin and 3% (wt/wt) caseinate mixture at pH 7 separated into an upper pectin-rich phase and a lower casein-rich phase. Casein-coated lipid droplets added to this mixture partitioned into the lower casein-rich phase. When shear was applied, an oil-in-water-in-water (O/W1/W2) emulsion consisting of oil droplets (O) contained within a casein-rich dispersed phase (W1) suspended in a pectin-rich continuous phase (W2) was formed. Acidification from pH 7 to 5 promoted adsorption of pectin onto casein-rich W1 droplets, forming filled hydrogel particles. Particles were then cross-linked using transglutaminase. Particles were assessed for stability to changes in pH, increasing levels of salts (sodium chloride and calcium chloride), and susceptibility to lipid oxidation. Both cross-linked and not cross-linked particles were stable at low pH (pH 2-5). At high pH, cross-linked particles maintained their integrity while not cross-linked particles disintegrated. Particles were stable to sodium chloride (0-500 mM). Calcium chloride levels above 4 mM resulted in system gelation. The rate of lipid oxidation for 1% (vol/vol) fish oil encapsulated within filled hydrogel particles was compared to that of oil-in-water emulsions stabilized by either Tween 20 or casein. Emulsions stabilized by Tween 20 oxidized faster than either filled hydrogel particles or casein stabilized emulsions, while filled hydrogel particles and casein stabilized emulsions showed similar oxidation rates. Using an in-vitro digestion model, the digestion of lipid encapsulated within filled hydrogel particles was compared to that of a casein stabilized oil-in-water emulsion. Results showed similar rates of digestion for both hydrogel and emulsion samples. Attempts to fabricate particles using free oil (rather than emulsified oil) were unsuccessful and resulted in the formation of large non-encapsulated oil droplets (d ~10 μm). By controlling particle concentrations of biopolymer, water, and oil, it was possible to fabricate particles that were highly resistant to gravitational separation which was attributed to the equivalent density of the continuous and particle phases. Results highlight the potential applications and versatility of this delivery system.

Liquid-liquid phase separation (LLPS) is an important phenomenon in soft condensed matter that explains many properties of membraneless organelles in living cells. The research on this topic is, therefore, a field with a wide range of applications such as biopharmacy and biomaterials. In this project, we investigate the LLPS of the antibody protein Immunoglobulin G (IgG) by analyzing the liquid dynamics of IgG solutions at a wide range of temperatures with dynamic light scattering (DLS). It was found that the slow component of the autocorrelation function increases with decreasing temperature below 0 °C. This can be attributed to either the number of protein clusters increasing as the sample approaches phase separation or LLPS droplets forming in the solution. LLPS was detected through optical microscopy, visualising the droplet formation in the IgG solution. This work confirms that LLPS can be detected for bovine IgG solutions without the presence of cosolvents and without water freezing in the sample.

Liquid-liquid phase separation (LLPS) has been proposed as the underlying physical principle leading to the formation of membrane-less organelles in eukaryotic cells, following advancements, in the last two decades, in experimental observations owing to progress in confocal microscopy. These organelles can act as compartments in sequestering molecules and tuning rates of biochemical reactions, among a repertoire of functions they serve. Biochemical reactions are constantly in progress in living cells and are driven out of equilibrium due to fuel consumption in the form of ATP or GTP molecules. Free diffusion of reactive molecules through these compartments leads to their spatiotemporal sequestration and automatically implies an interplay between phase separation and chemical reactions. In this work, we are specifically interested to understand how the two processes closely affect each other and applying the understanding to tune better bottom-up design principles for synthetic life, which involves coupling compartmentalization and chemical reactions. The first part of this work is devoted to studying the interplay between phase separation and chemical reactions. To this end, we developed the theory of mass action kinetics of equilibrium and out-of-equilibrium processes occurring at phase equilibrium in a multicomponent mixture. Phase equilibrium is imposed at all times, thus restricting the chemical kinetics to the binodal manifold. We learn more about circumstances in which reaction rates can differ in coexisting phases. Next, we decouple the phase-forming components (scaffolds) and the dilute reactive components (clients), which means that the reactive dilute components respond to the heterogeneous profile in the system set by the scaffold but do not affect it. This allows us to investigate to what extent compartments can affect chemical reactions in terms of their yield at steady state for a bimolecular reaction or initial reaction rate for a nucleation process compared to the absence of compartments. We use the effective droplet model and mass reaction kinetics at phase equilibrium to address the above questions. We can understand better how the properties of reactions can be optimally tuned by compartment size. Following the theoretical developments in the first part of this work, we proceed to use the theoretical model of mass action kinetics at phase equilibrium to study emergent properties of parasitic behavior in a system composed of multiple fuel-driven reaction cycles, which lead to the formation of so-called 'building blocks' which can phase separate. This study also helps us probe the buffering capacity of phase separation. It further provides insights into how the lifetime of reactive 'building blocks' can be tuned via phase separation. Synthetic cells are generally realized by localizing minimalistic reactions in micron-scale water-filled environments, thus mimicking compartmentalization. Here we apply our model to understand how the localization of an autocatalytic process (PEN-DNA reaction) inside proteinosomes affect the reaction rates compared to the reactions in a homogeneous buffer solution. To summarize, we developed theoretical approaches to study the interplay of chemical reactions with compartmentalization and apply such approaches to systems chemistry and synthetic biology experimental studies to unravel how reactions can be controlled through compartmentalization.:1 Introduction 1 1.1 Phase Separation - A brief overview of the development of the field . . . . . . 1 1.2 Thermodynamics of phase separation in a multi-component mixture . . . . . 4 1.2.1 Mean field free energy . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.2 Other possible free energy considerations: Beyond mean-field . . . . . 7 1.2.3 Exchange chemical potential, chemical activity and osmotic pressure . 8 1.2.4 Thermodynamic instability leads to phase separation . . . . . . . . . 9 1.2.5 Phase equilibrium conditions . . . . . . . . . . . . . . . . . . . . . . . 10 1.2.6 Relaxation dynamics to phase equilibrium . . . . . . . . . . . . . . . . 13 1.3 Thermodynamics of chemical reactions in homogeneous mixtures . . . . . . . 14 1.3.1 Chemical equilibrium conditions . . . . . . . . . . . . . . . . . . . . . 14 1.3.2 Relaxation to chemical equilibrium . . . . . . . . . . . . . . . . . . . . 16 1.4 Thermodynamic equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.5 Scope of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2 Chemical reaction kinetics at phase equilibrium 21 2.1 Kinetics of chemical reactions relaxing to thermodynamic equilibrium . . . . 21 2.1.1 Volume fraction field and phase volume kinetics . . . . . . . . . . . . 22 2.1.2 Diffusive exchange rates between phases . . . . . . . . . . . . . . . . . 22 2.1.3 Reaction rates at phase equilibrium . . . . . . . . . . . . . . . . . . . 23 2.1.4 Properties of chemical reactions at phase equilibrium . . . . . . . . . 24 2.2 Unimolecular chemical reactions in coexisting phases . . . . . . . . . . . . . . 25 2.3 Bimolecular chemical reactions in coexisting phases . . . . . . . . . . . . . . . 27 2.4 Chemical reactions maintained away from chemical equilibrium . . . . . . . . 28 2.4.1 Tie-line selecting curve . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.5 Summary and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3 Chemical reactions of dilute clients in phase-separated compartments 33 3.1 Thermodynamics of a multicomponent mixture of scaffold and clients . . . . 34 3.1.1 Phase equilibrium conditions: Dilute client limit . . . . . . . . . . . . 35 3.1.2 Relaxation dynamics to phase equilibrium: Dilute client limit . . . . . 38 3.1.3 Chemical equilibrium conditions: Dilute client limit . . . . . . . . . . 40 3.1.4 Relaxation dynamics to chemical equilibrium: Dilute client limit . . . 41 3.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.2.1 Two-state transitions controlled by a drop . . . . . . . . . . . . . . . . 43 3.2.2 Bimolecular reaction controlled by a drop . . . . . . . . . . . . . . . . 45 3.2.3 Nucleation reaction controlled by a drop . . . . . . . . . . . . . . . . . 47 3.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4 Fuel-driven chemical reactions in the dilute phase at phase equilibrium 50 4.1 Chemical reaction network and its properties . . . . . . . . . . . . . . . . . . 51 4.1.1 Observations from individual reaction cycles . . . . . . . . . . . . . . 52 4.1.2 Observations from combined reaction cycles . . . . . . . . . . . . . . . 53 4.2 Kinetic equations at phase equilibrium . . . . . . . . . . . . . . . . . . . . . . 55 4.3 Construction of the ternary phase diagram . . . . . . . . . . . . . . . . . . . . 57 4.4 Mechanism of co-phase separation . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4.1 Composition of droplets . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.5 Co-phase separation with periodic fueling . . . . . . . . . . . . . . . . . . . . 61 4.6 Effects of activation rate constants on host-parasite identity . . . . . . . . . . 63 4.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5 Study of enzymatic kinetics in compartmentalized systems 65 5.1 Autocatalytic reactions and their properties . . . . . . . . . . . . . . . . . . . 66 5.2 PEN DNA mass action kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . 67 5.3 Proteinosomes affect the PEN DNA reactions . . . . . . . . . . . . . . . . . . 70 5.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6 Conclusion and Outlook 72 A Free energy calculations for block charged polymers using RPA 76 B Numerical Methods 79 C Linear first order corrections to scaffold equilibrium volume fractions 83 D Dynamic equations in dilute limit 86 E Spatial solutions 88 F Fitting routine and extracted rate coefficients 90 G Experimental methods 95 H Calibration constants and reaction rate coefficients of PEN DNA study 98 List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

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