A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Constrained dynamics of DNA oligonucleotides in phase-separated droplets.

Understanding the dynamics of biomolecules in complex environments is crucial for elucidating the effect of condensed and heterogeneous environments on their functional properties. A relevant environment-and one that can also be mimicked easily in vitro-is that of phase-separated droplets. While phase-separated droplet systems have been shown to compartmentalize a wide range of functional biomolecules, the effects of internal structuration of droplets on the dynamics and mobility of internalized molecules remain poorly understood. Here, we use fluorescence correlation spectroscopy to measure the dynamics of short oligonucleotides encapsulated within two representative kinds of uncharged and charged phase-separated droplets. We find that the internal structuration controls the oligonucleotide dynamics in these droplets, revealed by measuring physical parameters at high spatiotemporal resolution. By varying oligonucleotide length and salt concentrations (and thereby charge screening), we found that the dynamics are significantly affected in the noncharged droplets compared to the charged system. Our work lays the foundation for unraveling and quantifying the physical parameters governing biomolecular transport in the condensed environment.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

Assembling anisotropic colloids using curvature-mediated lipid sorting.

The use of colloid supported lipid bilayers (CSLBs) for assembling colloidal structures has been of recent interest. Here, we use multi-component lipid bilayer membranes formed around anisotropic colloids and show that the curvature anisotropy of the colloids drives a sorting of the lipids in the membrane along the colloids. We then exploit this curvature-sensitive lipid sorting to create "shape-anisotropic patchy colloids" - specifically, we use colloids with six rods sticking out of a central cubic core, "hexapods", for this purpose and demonstrate that membrane patches self-assemble at the tip of each of the six colloidal rods. The membrane patches are rendered sticky using biotinylated lipids in complement with a biotin-binding streptavidin protein. Finally, using these "shape-anisotropic patchy colloids", we demonstrate the directed assembly of colloidal links, paving the way for the creation of heterogeneous and flexible colloidal structures.

Flow-induced phase separation of active particles is controlled by boundary conditions.

Active particles, including swimming microorganisms, autophoretic colloids, and droplets, are known to self-organize into ordered structures at fluid-solid boundaries. The entrainment of particles in the attractive parts of their spontaneous flows has been postulated as a possible mechanism underlying this phenomenon. Here, combining experiments, theory, and numerical simulations, we demonstrate the validity of this flow-induced ordering mechanism in a suspension of active emulsion droplets. We show that the mechanism can be controlled, with a variety of resultant ordered structures, by simply altering hydrodynamic boundary conditions. Thus, for flow in Hele-Shaw cells, metastable lines or stable traveling bands can be obtained by varying the cell height. Similarly, for flow bounded by a plane, dynamic crystallites are formed. At a no-slip wall, the crystallites are characterized by a continuous out-of-plane flux of particles that circulate and re-enter at the crystallite edges, thereby stabilizing them. At an interface where the tangential stress vanishes, the crystallites are strictly 2D, with no out-of-plane flux. We rationalize these experimental results by calculating, in each case, the slow viscous flow produced by the droplets and the long-ranged, many-body active forces and torques between them. The results of numerical simulations of motion under the action of the active forces and torques are in excellent agreement with experiments. Our work elucidates the mechanism of flow-induced phase separation in active fluids, particularly active colloidal suspensions, and demonstrates its control by boundaries, suggesting routes to geometric and topological phenomena in an active matter.

Farming and public goods production in Caenorhabditis elegans populations.

The ecological and evolutionary dynamics of populations are shaped by the strategies they use to produce and use resources. However, our understanding of the interplay between the genetic, behavioral, and environmental factors driving these strategies is limited. Here, we report on a Caenorhabditis elegans-Escherichia coli (worm-bacteria) experimental system in which the worm-foraging behavior leads to a redistribution of the bacterial food source, resulting in a growth advantage for both organisms, similar to that achieved via farming. We show experimentally and theoretically that the increased resource growth represents a public good that can benefit all other consumers, regardless of whether or not they are producers. Mutant worms that cannot farm bacteria benefit from farming by other worms in direct proportion to the fraction of farmers in the worm population. The farming behavior can therefore be exploited if it is associated with either energetic or survival costs. However, when the individuals compete for resources with their own type, these costs can result in an increased population density. Altogether, our findings reveal a previously unrecognized mechanism of public good production resulting from the foraging behavior of C. elegans, which has important population-level consequences. This powerful system may provide broad insight into exploration-exploitation tradeoffs, the resultant ecoevolutionary dynamics, and the underlying genetic and neurobehavioral driving forces of multispecies interactions.

Self-Driven Phase Transitions Drive Myxococcus xanthus Fruiting Body Formation.

Combining high-resolution single cell tracking experiments with numerical simulations, we show that starvation-induced fruiting body formation in Myxococcus xanthus is a phase separation driven by cells that tune their motility over time. The phase separation can be understood in terms of cell density and a dimensionless Péclet number that captures cell motility through speed and reversal frequency. Our work suggests that M. xanthus takes advantage of a self-driven nonequilibrium phase transition that can be controlled at the single cell level.

A versatile class of cell surface directional motors gives rise to gliding motility and sporulation in Myxococcus xanthus.

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.

Chimera states in mechanical oscillator networks.

The synchronization of coupled oscillators is a fascinating manifestation of self-organization that nature uses to orchestrate essential processes of life, such as the beating of the heart. Although it was long thought that synchrony and disorder were mutually exclusive steady states for a network of identical oscillators, numerous theoretical studies in recent years have revealed the intriguing possibility of "chimera states," in which the symmetry of the oscillator population is broken into a synchronous part and an asynchronous part. However, a striking lack of empirical evidence raises the question of whether chimeras are indeed characteristic of natural systems. This calls for a palpable realization of chimera states without any fine-tuning, from which physical mechanisms underlying their emergence can be uncovered. Here, we devise a simple experiment with mechanical oscillators coupled in a hierarchical network to show that chimeras emerge naturally from a competition between two antagonistic synchronization patterns. We identify a wide spectrum of complex states, encompassing and extending the set of previously described chimeras. Our mathematical model shows that the self-organization observed in our experiments is controlled by elementary dynamical equations from mechanics that are ubiquitous in many natural and technological systems. The symmetry-breaking mechanism revealed by our experiments may thus be prevalent in systems exhibiting collective behavior, such as power grids, optomechanical crystals, or cells communicating via quorum sensing in microbial populations.

Interfacial mechanisms in active emulsions.

Active emulsions, i.e., emulsions whose droplets perform self-propelled motion, are of tremendous interest for mimicking collective phenomena in biological populations such as phytoplankton and bacterial colonies, but also for experimentally studying rheology, pattern formation, and phase transitions in systems far from thermal equilibrium. For fuelling such systems, molecular processes involving the surfactants which stabilize the emulsions are a straightforward concept. We outline and compare two different types of reactions, one which chemically modifies the surfactant molecules, the other which transfers them into a different colloidal state. While in the first case symmetry breaking follows a standard linear instability, the second case turns out to be more complex. Depending on the dissolution pathway, there is either an intrinsically nonlinear instability, or no symmetry breaking at all (and hence no locomotion).

Emergent dynamics due to chemo-hydrodynamic self-interactions in active polymers.

The field of synthetic active matter has, thus far, been led by efforts to create point-like, isolated (yet interacting) self-propelled objects (e.g. colloids, droplets, microrobots) and understanding their collective dynamics. The design of flexible, freely jointed active assemblies from autonomously powered sub-components remains a challenge. Here, we report freely-jointed active polymers created using self-propelled droplets as monomeric units. Our experiments reveal that the self-shaping chemo-hydrodynamic interactions between the monomeric droplets give rise to an emergent rigidity (the acquisition of a stereotypical asymmetric C-shape) and associated ballistic propulsion of the active polymers. The rigidity and propulsion of the chains vary systematically with their lengths. Using simulations of a minimal model, we establish that the emergent polymer dynamics are a generic consequence of quasi two-dimensional confinement and auto-repulsive trail-mediated chemical interactions between the freely jointed active droplets. Finally, we tune the interplay between the chemical and hydrodynamic fields to experimentally demonstrate oscillatory dynamics of the rigid polymer propulsion. Altogether, our work highlights the possible first steps towards synthetic self-morphic active matter.

Metabolically-driven flows enable exponential growth in macroscopic multicellular yeast.

The ecological and evolutionary success of multicellular lineages is due in no small part to their increased size relative to unicellular ancestors. However, large size also poses biophysical challenges, especially regarding the transport of nutrients to all cells; these constraints are typically overcome through multicellular innovations (e.g., a circulatory system). Here we show that an emergent biophysical mechanism - spontaneous fluid flows arising from metabolically-generated density gradients - can alleviate constraints on nutrient transport, enabling exponential growth in nascent multicellular clusters of yeast lacking any multicellular adaptations for nutrient transport or fluid flow. Surprisingly, beyond a threshold size, the metabolic activity of experimentally-evolved snowflake yeast clusters drives large-scale fluid flows that transport nutrients throughout the cluster at speeds comparable to those generated by the cilia of extant multicellular organisms. These flows support exponential growth at macroscopic sizes that theory predicts should be diffusion limited. This work demonstrates how simple physical mechanisms can act as a 'biophysical scaffold' to support the evolution of multicellularity by opening up phenotypic possibilities prior to genetically-encoded innovations. More broadly, our findings highlight how co-option of conserved physical processes is a crucial but underappreciated facet of evolutionary innovation across scales.

Lasing and waveguiding in smectic A liquid crystal optical fibers.

We demonstrate a new class of soft matter optical fibers, which are self-assembled in a form of smectic-A liquid crystal microtubes grown in an aqueous surfactant dispersion of a smectic-A liquid crystal. The diameter of the fibers is highly uniform and the fibers are highly birefringent. They are characterized by a line topological defect in the core of the fiber with an optical axis pointing from the defect core towards the surface. We demonstrate guiding of light along the fiber and Whispering Gallery Mode (WGM) lasing in a plane perpendicular to the fiber. The light guiding as well as the lasing threshold are significantly dependent on the polarization of the excitation beam. The observed threshold for WGM lasing is very low (≈ 75µJ/cm(2)) when the pump beam polarization is perpendicular to the direction of the laser dye alignment and is similar to the lasing threshold in nematic droplets. The smectic-A fibers are soft and flexible and can be manipulated with laser tweezers demonstrating a promising approach for realization of soft photonic circuits.

Myelin structures formed by thermotropic smectic liquid crystals.

We report on transient structures, formed by thermotropic smectic-A liquid crystals, resembling the myelin figures of lyotropic lamellar liquid crystals. The thermotropic myelin structures form during the solubilization of a smectic-A droplet in an aqueous phase containing a cationic surfactant at concentrations above the critical micelle concentration. Similar to the lyotropic myelin figures, the thermotropic myelins appear in an optical microscope as flexible tubelike structures growing at the smectic/aqueous interface. Polarizing microscopy and confocal fluorescence microscopy show that the smectic layers are parallel to the tube surface and form a cylindrically bent arrangement around a central line defect in the tube. We study the growth behavior of this new type of myelins and discuss similarities to and differences from the classical lyotropic myelin figures.

Tuning active emulsion dynamics via surfactants and topology.

We study water-in-oil emulsion droplets, running the Belousov-Zhabotinsky reaction, that form a new type of synthetic active matter unit. These droplets, stabilised by surfactants dispersed in the oil medium, are capable of internal chemical oscillations and self-propulsion. Here we present studies of networks of such self-propelled chemical oscillators and show that the resulting dynamics depend strongly on the topology of the active matter units and their connections. The chemical oscillations can couple via the exchange of promoter and inhibitor type of reaction intermediates across the droplets under precise conditions of surfactant bilayer formation between the droplets. The self-emerging synchronization dynamics are then characterized by the topology of the oscillator networks. Further, we show that the chemical oscillations inside the droplets cause oscillatory speed variations in the motion of individual droplets, extending our previous studies on such swimmers. Finally, we demonstrate that qualitatively new types of self-propelled motion can occur when simple droplet networks, for example two droplets connected by a bilayer, are set into motion. Altogether, these results lead to exciting possibilities in future studies of autonomous active matter.

Multispecies autocatalytic RNA reaction networks in coacervates.

Robust localization of self-reproducing autocatalytic chemistries is a key step in the realization of heritable and evolvable chemical systems. While autocatalytic chemical reaction networks already possess attributes such as heritable self-reproduction and evolvability, localizing functional multispecies networks within complex primitive phases, such as coacervates, has remained unexplored. Here, we show the self-reproduction of the Azoarcus ribozyme system within charge-rich coacervates where catalytic ribozymes are produced by the autocatalytic assembly of constituent smaller RNA fragments. We systematically demonstrate the catalytic assembly of active ribozymes within phase-separated coacervates-both in micron-sized droplets as well as in a coalesced macrophase, underscoring the facility of the complex, charge-rich phase to support these reactions in multiple configurations. By constructing multispecies reaction networks, we show that these newly assembled molecules are active, participating both in self- and cross-catalysis within the coacervates. Finally, due to differential molecular transport, these phase-separated compartments endow robustness to the composition of the collectively autocatalytic networks against external perturbations. Altogether, our results establish the formation of multispecies self-reproducing reaction networks in phase-separated compartments which in turn render transient robustness to the network composition.

Solubilization of thermotropic liquid crystal compounds in aqueous surfactant solutions.

We study the micellar solubilization of three thermotropic liquid crystal compounds by immersing single drops in aqueous solutions of the ionic surfactant tetradecyltrimethylammonium bromide. For both nematic and isotropic drops, we observe a linear decrease of the drop size with time as well as convective flows and self-propelled motions. The solubilization is accompanied by the appearance of small aqueous droplets within the nematic or isotropic drop. At low temperatures, nematic drops expell small nematic droplets into the aqueous environment. Smectic drops show the spontaneous formation of filament-like structures which resemble the myelin figures observed in lyotropic lamellar systems. In all cases, the liquid crystal drops become completely solubilized, provided the weight fraction of the liquid crystal in the system is not larger than a few percent. The solubilization of the liquid crystal drops is compared with earlier studies of the solubilization of alkanes in ionic surfactant solutions.

Unjamming and emergent nonreciprocity in active ploughing through a compressible viscoelastic fluid.

A dilute suspension of active Brownian particles in a dense compressible viscoelastic fluid, forms a natural setting to study the emergence of nonreciprocity during a dynamical phase transition. At these densities, the transport of active particles is strongly influenced by the passive medium and shows a dynamical jamming transition as a function of activity and medium density. In the process, the compressible medium is actively churned up - for low activity, the active particle gets self-trapped in a cavity of its own making, while for large activity, the active particle ploughs through the medium, either accompanied by a moving anisotropic wake, or leaving a porous trail. A hydrodynamic approach makes it evident that the active particle generates a long-range density wake which breaks fore-aft symmetry, consistent with the simulations. Accounting for the back-reaction of the compressible medium leads to (i) dynamical jamming of the active particle, and (ii) a dynamical non-reciprocal attraction between two active particles moving along the same direction, with the trailing particle catching up with the leading one in finite time. We emphasize that these nonreciprocal effects appear only when the active particles are moving and so manifest in the vicinity of the jamming-unjamming transition.

Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes.

Localized fluxes, production, and/or degradation coupled to limited diffusion are well known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, together with localized membrane rupture, suffice for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that, in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion.

Self-Reproduction and Darwinian Evolution in Autocatalytic Chemical Reaction Systems.

Understanding the emergence of life from (primitive) abiotic components has arguably been one of the deepest and yet one of the most elusive scientific questions. Notwithstanding the lack of a clear definition for a living system, it is widely argued that heredity (involving self-reproduction) along with compartmentalization and metabolism are key features that contrast living systems from their non-living counterparts. A minimal living system may be viewed as "a self-sustaining chemical system capable of Darwinian evolution". It has been proposed that autocatalytic sets of chemical reactions (ACSs) could serve as a mechanism to establish chemical compositional identity, heritable self-reproduction, and evolution in a minimal chemical system. Following years of theoretical work, autocatalytic chemical systems have been constructed experimentally using a wide variety of substrates, and most studies, thus far, have focused on the demonstration of chemical self-reproduction under specific conditions. While several recent experimental studies have raised the possibility of carrying out some aspects of experimental evolution using autocatalytic reaction networks, there remain many open challenges. In this review, we start by evaluating theoretical studies of ACSs specifically with a view to establish the conditions required for such chemical systems to exhibit self-reproduction and Darwinian evolution. Then, we follow with an extensive overview of experimental ACS systems and use the theoretically established conditions to critically evaluate these empirical systems for their potential to exhibit Darwinian evolution. We identify various technical and conceptual challenges limiting experimental progress and, finally, conclude with some remarks about open questions.