ORCID Profile
0000-0002-0456-3850
Current Organisation
University of Sydney
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Biochemistry and cell biology | Microfluidics and nanofluidics | Structural biology (incl. macromolecular modelling) |
Publisher: Proceedings of the National Academy of Sciences
Date: 21-06-2022
Abstract: Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multicomponent mixtures of biomolecules (e.g., proteins and nucleic acids) when the different components present sufficient physicochemical ersity (e.g., in intermolecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such ersity is highly coupled to the solution conditions (e.g., temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli or emerge progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the progressive intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of aging. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically aging condensates with nonconservative intermolecular forces. Our nonequilibrium simulations of condensate aging predict that single-component condensates that are initially homogeneous and liquid like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age due to gradual and irreversible enhancement of interprotein interactions. The type of multiphase architecture is determined by the aging mechanism, the molecular organization of the gel and liquid phases, and the chemical makeup of the protein. Notably, we predict that interprotein disorder to order transitions within the prion-like domains of intracellular proteins can lead to the required nonconservative enhancement of intermolecular interactions. Our study, therefore, predicts a potential mechanism by which the nonequilibrium process of aging results in single-component multiphase condensates.
Publisher: Wiley
Date: 08-07-2021
Abstract: Liquid–liquid phase‐separated biomolecular systems are increasingly recognized as key components in the intracellular milieu where they provide spatial organization to the cytoplasm and the nucleoplasm. The widespread use of phase‐separated systems by nature has given rise to the inspiration of engineering such functional systems in the laboratory. In particular, reversible gelation of liquid–liquid phase‐separated systems could confer functional advantages to the generation of new soft materials. Such gelation processes of biomolecular condensates have been extensively studied due to their links with disease. However, the inverse process, the gel–sol transition, has been less explored. Here, a thermoresponsive gel–sol transition of an extracellular protein in microgel form is explored, resulting in an all‐aqueous liquid–liquid phase‐separated system with high homogeneity. During this gel–sol transition, elongated gelatin microgels are demonstrated to be converted to a spherical geometry due to interfacial tension becoming the dominant energetic contribution as elasticity diminishes. The phase‐separated system is further explored with respect to the diffusion of small particles for drug‐release scenarios. Together, this all‐aqueous system opens up a route toward size‐tunable and monodisperse synthetic biomolecular condensates and controlled liquid–liquid interfaces, offering possibilities for applications in bioengineering and biomedicine.
Publisher: Wiley
Date: 05-07-2022
Abstract: The emerging applications of hydrogel materials at different length scales, in areas ranging from sustainability to health, have driven the progress in the design and manufacturing of microgels. Microgels can provide miniaturized, monodisperse, and regulatable compartments, which can be spatially separated or interconnected. These microscopic materials provide novel opportunities for generating biomimetic cell culture environments and are thus key to the advances of modern biomedical research. The evolution of the physical and chemical properties has, furthermore, highlighted the potentials of microgels in the context of materials science and bioengineering. This review describes the recent research progress in the fabrication, characterization, and applications of microgels generated from biomolecular building blocks. A key enabling technology allowing the tailoring of the properties of microgels is their synthesis through microfluidic technologies, and this paper highlights recent advances in these areas and their impact on expanding the physicochemical parameter space accessible using microgels. This review finally discusses the emerging roles that microgels play in liquid–liquid phase separation, micromechanics, biosensors, and regenerative medicine.
Publisher: Elsevier BV
Date: 05-2015
Publisher: Cold Spring Harbor Laboratory
Date: 15-08-2022
DOI: 10.1101/2022.08.15.503964
Abstract: A wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.
Publisher: Wiley
Date: 20-11-2022
Abstract: Multicellular cancer spheroids (MCSs) have emerged as a promising in vitro model that recaptures many features of solid tumours in vivo. To generate cancer spheroids, cells are encapsulated in microgels with high throughput. While the biophysical properties of the cancer spheroid and biomaterial influence the function and behavior of the cells, characterization of these properties remains largely unexplored. In addition, many existing techniques lack control over cell positioning, resulting in the formation of spheroids with large variability. Herein, a versatile, microfluidic platform for generating biocompatible core–shell microgels with uniform cancer spheroids is reported. In addition, an in situ micromechanics measuring device to determine the stiffness of in idual artificial cancer niches is used. The power of the microfluidics‐based method by making MCS‐laden microgels to model the interactions of stromal cancer cells is demonstrated. Together with in vivo data, it is shown that tumor cell proliferation is promoted by cocultured fibroblasts.
Publisher: Springer Science and Business Media LLC
Date: 02-2022
DOI: 10.1557/S43577-022-00279-5
Abstract: Micron-scale soft materials are finding a wide range of applications in bioengineering and molecular medicine, while also increasingly emerging as useful components for consumer products. The mechanical characterization of such microscale soft objects is conventionally performed with techniques such as atomic force microscopy or micropipette aspiration that measure the local properties of micron scale objects in a serial manner. To permit scalable characterization of the global mechanical properties of soft microscale objects, we developed and describe here a microfluidic platform that can be used for performing parallelized integrated measurements of the shear modulus of in idual microscale particles. We demonstrate the effectiveness of this approach by characterizing the mechanical properties of multiple protein microgels in parallel, and show that the obtained values are in good agreement with conventional serial measurements. This platform allows parallelized in situ measurements of the mechanical properties of soft deformable micron-scale particles, and builds on scalable single-layer soft-photolithography fabrication, making the measurement system readily adaptable for a range of potential applications.
Publisher: Elsevier BV
Date: 11-2018
DOI: 10.1016/J.CELL.2018.11.020
Abstract: The use of optogenetic approaches has revealed new roles for intracellular protein condensates described in two papers in this issue of Cell (Bracha et. al., 2018 Shin et al., 2018). These results show that growing condensates are able to exert mechanical forces resulting in chromatin rearrangement, establishing a new role for liquid-liquid phase separation in the mechanobiology of the cell.
Publisher: Proceedings of the National Academy of Sciences
Date: 07-08-2023
Abstract: A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.
Publisher: Wiley
Date: 24-07-2017
Publisher: Springer Science and Business Media LLC
Date: 10-06-2021
DOI: 10.1038/S41467-021-23813-6
Abstract: The abundance of plant-derived proteins, as well as their biodegradability and low environmental impact make them attractive polymeric feedstocks for next-generation functional materials to replace current petroleum-based systems. However, efforts to generate functional materials from plant-based proteins in a scalable manner have been h ered by the lack of efficient methods to induce and control their micro and nanoscale structure, key requirements for achieving advantageous material properties and tailoring their functionality. Here, we demonstrate a scalable approach for generating mechanically robust plant-based films on a metre-scale through controlled nanometre-scale self-assembly of water-insoluble plant proteins. The films produced using this method exhibit high optical transmittance, as well as robust mechanical properties comparable to engineering plastics. Furthermore, we demonstrate the ability to impart nano- and microscale patterning into such films through templating, leading to the formation of hydrophobic surfaces as well as structural colour by controlling the size of the patterned features.
Publisher: Wiley
Date: 14-11-2022
Abstract: Living cells comprise erse subcellular structures, such as cytoskeletal networks, which can regulate essential cellular activities through dynamic assembly and synergistic interactions with biomolecular condensates. Despite extensive efforts, reproducing viscoelastic networks for modulating biomolecular condensates in synthetic systems remains challenging. Here, a new aqueous two‐phase system (ATPS) is proposed, which consists of poly( N ‐isopropylacrylamide) (PNIPAM) and dextran (DEX), to construct viscoelastic networks capable of being assembled and dissociated dynamically to regulate the self‐assembly of condensates on‐demand. Viscoelastic networks are generated using liquid–liquid phase‐separated DEX droplets as templates and the following liquid‐to‐solid transition of the PNIPAM‐rich phase. The resulting networks can dissolve liquid fused in sarcoma (FUS) condensates within 5 min. This work demonstrates rich phase‐separation behaviors in a single ATPS through incorporating stimuli‐responsive polymers. The concept can potentially be applied to other macromolecules through other stimuli to develop materials with rich phase behaviors and hierarchical structures.
Publisher: Wiley
Date: 24-11-2023
Abstract: Thrombotic diseases have a high rate of mortality and disability, and pose a serious threat to global public health. Currently, most thrombolytic drugs especially protein drugs have a short blood‐circulation time, resulting in low thrombolytic efficiency. Therefore, a platelet membrane (Pm) cloaked nanotube (NT‐RGD/Pm) biomimetic delivery system with enhanced thrombolytic efficiency is designed. Nanotubes (NT) with an excellent clot‐penetration properties are used to load a protein thrombolytic drug urokinase (Uk). Platelet‐targeting arginine glycine‐aspartic peptide (RGD) is grafted onto the surface of the nanotubes (NT‐RGD) prior to cloaking. Multiple particle tracking (MPT) technique and confocal laser scanning microscope (CLSM) analysis are applied and the results show that the nanotubes possess a strong penetration and diffusion capacity in thrombus clots. After the Pm cloaking on NT‐RGD/Uk, it shows a thrombus microenvironmental responsive release property and the half‐life of Uk is six times longer than that of free Uk. Most importantly, NT‐RGD‐Uk/Pm exhibits a 60% thrombolytic efficiency in the FeCl 3 ‐induced thrombosis mouse model, and it is able to significantly reduce the bleeding side effects of Uk. This Pm‐cloaked nanotube system is an effective and promising platform for the controlled and targeted delivery of drugs for the thrombus treatment.
Publisher: Springer Science and Business Media LLC
Date: 13-07-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 11-02-2013
Abstract: Biofilms are antibiotic-resistant, sessile bacterial communities that occupy most moist surfaces on Earth and cause chronic and medical device-associated infections. Despite their importance, basic information about biofilm dynamics in common ecological environments is lacking. Here, we demonstrate that flow through soil-like porous materials, industrial filters, and medical stents dramatically modifies the morphology of Pseudomonas aeruginosa biofilms to form 3D streamers, which, over time, bridge the spaces between obstacles and corners in nonuniform environments. We discovered that accumulation of surface-attached biofilm has little effect on flow through such environments, whereas biofilm streamers cause sudden and rapid clogging. We demonstrate that flow-induced shedding of extracellular matrix from surface-attached biofilms generates a sieve-like network that captures cells and other biomass, which add to the existing network, causing exponentially fast clogging independent of growth. These results suggest that biofilm streamers are ubiquitous in nature and strongly affect flow through porous materials in environmental, industrial, and medical systems.
Publisher: Wiley
Date: 09-10-2019
Abstract: Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.
Publisher: Wiley
Date: 11-06-2020
Publisher: American Chemical Society (ACS)
Date: 24-03-2021
Publisher: Cold Spring Harbor Laboratory
Date: 28-01-2020
DOI: 10.1101/2020.01.21.912857
Abstract: A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies 1 , germ granules 2 , stress granules 3 and Cajal bodies 4 . The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis 1–4 . RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases 5–7 . It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation 6 . Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning 8,9 , which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.
Publisher: Elsevier BV
Date: 2011
Publisher: Springer Science and Business Media LLC
Date: 24-04-2017
Abstract: Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from β-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.
Publisher: Cold Spring Harbor Laboratory
Date: 09-10-2021
DOI: 10.1101/2021.10.09.463670
Abstract: Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multi-component mixtures of biomolecules (e.g. proteins and nucleic acids) when the different components present sufficient physicochemical ersity (e.g. in inter-molecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such ersity is highly coupled to the solution conditions (e.g. temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli, or progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of ageing. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically ageing condensates with non-conservative inter-molecular forces. Our nonequilibrium simulations of condensate ageing predict that single-component condensates that are initially homogeneous and liquid-like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age, due to gradual and irreversible enhancement of inter-protein interactions. The type of multiphase architecture is determined by the ageing mechanism, the molecular organization of the gel and liquid phases, and the chemical make up of the protein. Notably, we predict that inter-protein disorder-to-order transitions within the prion-like domains of intracellular proteins could lead to the required non-conservative enhancement of inter-molecular interactions. Our study, therefore, predicts a potential mechanism Biomolecular condensates are highly erse systems spanning not only homogeneous liquid droplets, but also gels, glasses, and even multiphase architectures that contain various coexisting liquid-like and/or gel-like inner phases. Multiphase architectures form when the different biomolecular components in a multi-component condensate establish sufficiently imbalanced inter-molecular forces to sustain different coexisting phases. While such a requirement seems, at first glance, impossible to fulfil for a condensate formed exclusively of chemically-identical proteins (i.e., single-component), our simulations predict conditions under which this may be possible. During condensate ageing, a sufficiently large imbalance in inter-molecular interactions can emerge intrinsically from the accumulation of protein structural transitions—driving even single-component condensates into nonequilibrium liquid-core/gel-shell or gel-core/liquid-shell multiphase architectures.
Publisher: Elsevier BV
Date: 07-2012
Publisher: Wiley
Date: 13-09-2021
Abstract: Microcapsules are a key class of microscale materials with applications in areas ranging from personal care to biomedicine, and with increasing potential to act as extracellular matrix (ECM) models of hollow organs, tissues, or biomolecular condensates. Such capsules are conventionally generated from non‐ECM materials including synthetic polymers. Here, robust microcapsules with controllable shell thickness from physically‐ and enzymatically‐crosslinked gelatin are fabricated, and a core–shell architecture is achieved by exploiting a liquid–liquid phase‐separated aqueous system in a one‐step microfluidic process. Microfluidic mechanical testing reveals that the mechanical robustness of thicker‐shell capsules could be controlled through modulation of the shell thickness. Furthermore, the microcapsules demonstrate environmentally‐responsive deformation, including buckling driven by osmosis and external mechanical forces. A sequential release of cargo species is obtained through the degradation of the capsules. Stability measurements show the capsules are stable at 37 °C for more than 2 weeks. Finally, through gel–sol transition, microgels function as precursors for the formation of all‐aqueous liquid–liquid phase‐separated systems that are two‐phase or multiphase. These smart capsules that can undergo phase transition are promising models of hollow biostructures, microscale drug carriers, and building blocks or compartments for active soft materials and robots.
Publisher: Elsevier BV
Date: 10-2023
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4CP01086H
Abstract: The integration of Microbial Fuel Cells (MFCs) in a microfluidic geometry can significantly enhance the power density of these cells, which would have more active bacteria per unit volume. Moreover, microfluidic MFCs can be operated in a continuous mode as opposed to the traditional batch-fed mode. Here we investigate the effect of fluid flow on the performance of microfluidic MFCs. The growth and the structure of the bacterial biofilm depend to a large extent on the shear stress of the flow. We report the existence of a range of flow rates for which MFCs can achieve maximum voltage output. When operated under these optimal conditions, the power density of our microfluidic MFC is about 15 times that of a similar-size batch MFC. Furthermore, this optimum suggests a correlation between the behaviour of bacteria and fluid flow.
Publisher: Cold Spring Harbor Laboratory
Date: 24-03-2023
DOI: 10.1101/2023.03.22.533832
Abstract: Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other ex les across the cell whereby biomolecular condensates closely juxtapose cell membranes.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 10-2023
End Date: 10-2026
Amount: $441,454.00
Funder: Australian Research Council
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