Allen P. Liu

A central challenge in quantum physics is to understand the structural properties of many-body systems, both in equilibrium and out of equilibrium. For classical systems, we have a unified perspective which connects structural properties of systems at thermal equilibrium to the Markov chain dynamics that mix to them. We lack such a perspective for quantum systems: there is no framework to translate the quantitative convergence of the Markovian evolution into strong structural consequences.

The coordination of microtubules (MTs) and actin filaments is essential for cytoskeletal organization, but the factors that affect their integration remain unclear. Here, we reconstitute cytoskeletal networks in giant unilamellar vesicles to characterize MT-actin crosstalk mediated by tau, a microtubule-associated protein. We show that tau promotes the organization of MTs into diverse architectures, including bundles, clusters, and networks, depending on its concentration and vesicle size. In vitro assays confirm that, while tau binds and bundles MTs, it does not directly bundle actin. However, tau facilitates MT-actin colocalization in the presence of actin crosslinkers with distinct properties. Fascin, which forms rigid actin bundles, significantly enhances MT-actin colocalization with tau, whereas α-actinin, which forms flexible actin bundles, induces colocalization in vesicles but not in bulk conditions. By combining cellular reconstitution and coarse-grained simulations of composite network assembly in both vesicles and bulk conditions, our findings reveal how tau-mediated cytoskeletal integration is governed by bundle mechanics and spatial confinement, providing insights into cytoskeletal organization within reconstituted synthetic cell-like systems.

pH is a critical parameter in biological systems, with acidic environments often serving as hallmarks of pathological conditions such as cancer, infection, and metabolic disorders. Here, we developed a pH-responsive synthetic cell capable of protein synthesis and release under acidic conditions. The system was constructed by integrating three molecular modules: a proton channel for pH sensing, a pH-responsive single-stranded DNA (ssDNA) that releases a trigger ssDNA upon acidification, and a toehold switch RNA that activates translation in response to the trigger ssDNA. During integration, we discovered that adjusting the annealing length between the pH-responsive and trigger strands was critical for enabling the acid-triggered protein synthesis. Using this strategy, we successfully demonstrated acid-responsive protein expression within synthetic cells. To further explore applications, we embedded the synthetic cells in a hydrogel to endow pH-responsive behavior to materials and coupled pH-responsive protein translation with a cell-penetrating peptide technology for selective release of proteins.

A virtual screen combining molecular simulations, alchemical calculations, Gaussian process regression, and Bayesian optimization discovers elastin-like polypeptides predicted to form stable bilayer vesicles for synthetic cells.

SUMMARY The coordination of microtubules (MTs) and actin filaments is essential for cytoskeletal organization, yet the factors that affect their integration remain unclear. Here, we reconstitute cytoskeletal networks in giant unilamellar vesicles to characterize MT-actin crosstalk mediated by tau, a microtubule-associated protein. We show that tau promotes the organization of MTs into diverse architectures, including bundles, clusters, and networks, depending on its concentration and vesicle size. In vitro assays confirm that while tau binds and bundles MTs, it does not directly bundle actin. However, tau facilitates MT-actin colocalization in the presence of actin crosslinkers with distinct properties. Fascin, which forms rigid actin bundles, significantly enhances MT-actin colocalization with tau, whereas α-actinin, which forms flexible actin bundles, induces colocalization in vesicles but not in bulk conditions. By combining cellular reconstitution and coarse-grained simulations of composite network assembly in both vesicles and bulk conditions, our findings reveal how tau-mediated cytoskeletal integration is governed by bundle mechanics and spatial confinement, providing insights into cytoskeletal organization within reconstituted synthetic cell-like systems.

Cytoplasmic abundant heat-soluble (CAHS) proteins, a stress-responsive intrinsically disordered protein from tardigrades, have been discovered to form gel-like networks providing structural support during dehydration, thus enabling anhydrobiosis. However, the mechanism by which CAHS proteins protect the dehydrating cellular membrane remains enigmatic. Using giant unilamellar vesicles (GUVs) as a model membrane system, here we show that encapsulated CAHS12 undergoes a reversible structural transformation that reinforces membrane integrity and preserves encapsulated components, mimicking natural anhydrobiosis. CAHS12-containing GUVs demonstrated stability for weeks and mechanical robustness under dehydration, elevated temperature, and osmotic stresses. Molecular simulations suggest that CAHS12 forms a filamentous network within the vesicle lumen that mitigates membrane collapse and preserves compartmental architecture. Synthetic cells with cell-free transcription-translation capabilities withstand desiccation and recover biochemical activities, akin to the tun state of the tardigrade. This discovery opens up synthetic cell applications in bioengineering, cold-chain-independent biomanufacturing, and adaptive biointerfaces.

Nature has already solved many challenges that synthetic biology seeks to address. At the same time, high-throughput platforms and artificial intelligence-driven design tools are redefining the landscape of synthetic biology. By integrating evolutionary insights with new enabling technologies, we are poised to move beyond modifying existing traits to engineering entirely new cellular functions. This series of vignettes explores how phenotypic engineering, inspired by nature's most extreme adaptations, represents the next frontier in synthetic biology. We first make a case for the importance of continuing to explore nature by examining how foundational discoveries in molecular biology, motivated by curiosity to understand how life works rather than immediate application, laid the groundwork for modern applications in biology. Next, we discuss how evolution produced novel cell types with extraordinary properties and how leveraging these innovations can enable the creation of radical phenotypes beyond natural evolutionary constraints. Then, we explore the potential of artificial cells, constructed from the bottom up, as experimental platforms for studying genotype-phenotype relationships and testing evolutionary principles in real time. Finally, we argue that engineering radical phenotypes and emergent traits will require better investment in basic science, infrastructure, and graduate training to avoid bottlenecking innovation. History has shown that committing to basic science results in transformative solutions with far-reaching societal and economic benefits. By drawing inspiration from evolution and expanding synthetic biology beyond traditional model systems, we can push current boundaries to open new avenues for fundamental discovery and technological advances to stimulate the bioeconomy.

Synthetic cells are prevalent models for understanding and recapitulating complicated functions of natural cells such as DNA replication and protein expression. Lipid-based vesicles are widely employed but limited in real-world applications due to its fragility under mechanic forces or osmotic pressures. Elastin-like polypeptides (ELPs), composed of repetitive (VPGXG) sequences present alternative building block for synthetic cells with structural stability and tolerance of harsh environmental stress. In this work, we present a high-throughput virtual screening pipeline combining coarse-grained simulations, alchemical free energy calculations, Gaussian process regression, and Bayesian optimization to traverse a library of amphiphilic diblock ELPs for mutant sequences predicted to form thermodynamically stable bilayer vesicles. From our screening campaign, we have identified a range of novel ELP candidates with enhanced predicted stability. Analysis of our screening data exposes new rational design principles that suggest incorporating particular guest residues in hydrophilic blocks -- including histidine, tyrosine, and threonine -- and in hydrophobic blocks -- including alanine, phenylalanine, cysteine, and isoleucine -- to enhance the thermodynamic stability of ELP bilayer vesicles. The computational pipeline greatly accelerates the discovery of ELP building blocks for synthetic cells, exposes new design principles for these molecules, and furnishes a transferable framework for designing peptides with desirable structural or functional properties.

Creating artificial cells with a dynamic cytoskeleton, akin to those in living cells, is a major goal in bottom-up synthetic biology. In this study, we demonstrate the in situ polymerization of microtubules encapsulated in giant polymer-lipid hybrid vesicles (GHVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and an amphiphilic block copolymer. The block copolymer is comprised of poly(cholesteryl methacrylate-co-butyl methacrylate) as the hydrophobic block and either poly(6-O-methacryloyl-D-galactopyranose) or poly(carboxyethyl acrylate) as the hydrophilic extension. Depending on the concentrations of guanosine triphosphate (GTP) or its slowly hydrolyzable analog, guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP), different microtubule morphologies are observed, including encapsulated microtubule networks, spike protrusions, as well as membrane-associated or aggregated microtubules. Overall, this work represents a step forward in mimicking the cellular cytoskeletons and uncovering the influence of membrane composition on microtubule morphologies.

Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, a protein-based platform termed TEV Protease-mediated Releasable Actin-binding Protein (TRAP) is designed and constructed for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins is demonstrated. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.

Tumor metastasis is a complex phenomenon that poses significant challenges to current cancer therapeutics. While the biochemical signaling involved in promoting motile phenotypes is well understood, the role of biomechanical interactions has recently begun to be incorporated into models of tumor cell migration. Specifically, we propose the unjamming transition, adapted from physical paradigms describing the behavior of granular materials, to better discern the transition toward an invasive phenotype. In this review, we introduce the jamming transition broadly and narrow our discussion to the different modes of 3D tumor cell migration that arise. Then we discuss the mechanical interactions between tumor cells and their neighbors, along with the interactions between tumor cells and the surrounding extracellular matrix. We center our discussion on the interactions that induce a motile state or unjamming transition in these contexts. By considering the interplay between biochemical and biomechanical signaling in tumor cell migration, we can advance our understanding of biomechanical control in cancer metastasis.

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with spatial responses. Herein, a light-activated contact-dependent communication scheme for synthetic cells is designed and demonstrated. A split luminescent protein is utilized to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. The modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

We show that thermal states of local Hamiltonians are separable above a constant temperature. Specifically, for a local Hamiltonian <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$H$</tex> on a graph with degree <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\mathfrak{g}$</tex>, its Gibbs state at inverse temperature <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\beta$</tex>, denoted by <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\rho=e^{-\beta H}/\text{tr}(e^{-\beta H})$</tex>, is a classical distribution over product states for all <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\beta &lt; 1/ (c \mathfrak{{g}})$</tex>, where <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$c$</tex> is a constant. This sudden death of thermal entanglement upends conventional wisdom about the presence of short-range quantum correlations in Gibbs states. Moreover, we show that we can efficiently sample from the distribution over product states. In particular, for any <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\beta &lt; 1/(c\mathfrak{g}^{3})$</tex>, we can prepare a state <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\varepsilon$</tex> -close to <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\rho$</tex> in trace distance with a depth-one quantum circuit and <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\text{poly}(n)\log(1/\varepsilon)$</tex> classical overhead. <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sup><sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sup> In independent and concurrent work, Rouzé, França, and Alhambra [37] obtain an efficient quantum algorithm for preparing high-temperature Gibbs states via a dissipative evolution.

Constructing molecular classifiers that enable cells to recognize linear and nonlinear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effects of biological constraints such as resource limitation and competitive binding on the function of those classifiers have been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working based on the principles of molecular sequestration between the sigma factor and its antisigma molecule. We then investigate how the output of the biomolecular perceptron, i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multilayer perceptron neural networks and outline design principles that enable the construction of nonlinear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects.

Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, we designed and constructed a protein-based platform termed TEV Protease-mediated Releasable Actin-binding protein (TRAP) for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. We demonstrated TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.

We are delighted to introduce the “Physics of the Cytoskeleton” special issue. This collection of 10 contributed papers (seven research articles, two short reports, and a perspective) highlights physical properties central to understanding cytoskeletal function. These papers showcase a variety of methodologies, including theoretical and computational techniques and experimental approaches using in vitro or cellular systems. Some of the themes in the papers of this issue include multiscale mechanics, self-organization, phase separation, scaling, and molecular modeling. Mostafazadeh and Peng (2024) used mathematical and computational modeling to connect lamin molecular properties to nuclear mechanics. Starting from molecular modeling of force-extension properties of individual lamin dimers, they proceed to construct a 2D network continuum model and then finite element simulations of the entire nucleus. Wubshet et al. (2024) looked at the mechanical properties of confined bundled and branched actin networks encapsulated within giant unilamellar vesicles experimentally through micropipette aspiration. They find a differential response: while linear actin bundles align along the axis of the micropipette following vesicle aspiration, branched bundles remain intact in the vesicle. Mostafazadeh et al. (2024) developed a mechanical model of primary cilia that considers the nine microtubule doublets and the cilium membrane. They find that the deforming cilia result in significant membrane bending stress. This result indicated that transmembrane sensing proteins in cilia may be activated more by membrane curvature than membrane stretching, challenging traditional models of cilium mechanosensing. Matsuda et al. (2024) studied contracting actomyosin networks with in vitro experiments and discrete, filament-level computational modeling. Their findings highlight how actin filament fragmentation due to tensile forces regulates network rupture and contraction. Banerjee et al. (2024) studied the evolution of polymerizing filaments in a two-dimensional layer with steric interactions. They find that kinetic trapping into long-lived collective bending patterns, organized around nematic defects, arises from competition between filament polymerization and bending elasticity. Chauhan et al. (2024) studied the self-organization properties of microtubules and antiparallel crosslinker MAP65 as a function of ionic conditions. They find that monovalent salt changes the morphologies from spindle-like tactoids to long bundles, by modifying MAP65 phase-separation properties. Zimyanin and Redemann (2024) asked how the length of individual microtubules relates to the size of the meiotic spindle. By combining electron tomography and live imaging of Caenorhabditis elegans, they discover a positive correlation between microtubule and spindle lengths. These findings highlight how large-scale spindle properties depend on molecular-level processes regulating microtubule length. Aydin et al. (2024) studied how actin regulatory protein VASP binds to actin filaments through its FAB domain. Using homology modeling and docking simulations, they predict atomistic level binding as well as mutation sites that disrupt the VASP–actin complex. These results are supported by their total internal reflection fluorescence microscopy experiments. Joshi et al. (2024) used molecular modeling to understand how αIIbβ3 integrin undergoes bent to extended conformation transitions to regulate platelet aggregation during hemostasis and thrombosis. Using molecular dynamics simulations to calibrate an elastic network model, they find that integrin extension involves changes in interfaces between functional domains as well as allosteric coupling, highlighting the importance of the lower leg domains. Finally, in their perspective, Mukadum et al. (2024) discuss challenges and opportunities in probing the mechanical regulation of the actin cytoskeleton through atomistic and coarse-grained molecular simulations. This special issue reflects the tradition of quantitative studies in the field of cytoskeletal research, which the journal of Cytoskeleton has repeatedly recognized. Certainly, such studies will continue to play a pivotal role in unraveling the physical principles underlying the complex behavior and properties of cytoskeletal components. We would like to thank all contributing authors of this issue for responding to our call, the anonymous peer reviewers for their valuable insights and suggestions, and Editor-in-Chief Julian Guttman for his enthusiasm and support.

Autonomous generation of energy, specifically adenosine triphosphate (ATP), is critical for sustaining the engineered functionalities of synthetic cells constructed from the bottom-up. In this mini-review, we categorize studies on ATP-producing synthetic cells into three different approaches: photosynthetic mechanisms, mitochondrial respiration mimicry, and utilization of non-conventional approaches such as exploiting synthetic metabolic pathways. Within this framework, we evaluate the strengths and limitations of each approach and provide directions for future research endeavors. We also introduce a concept of building ATP-generating synthetic organelle that will enable us to mimic cellular respiration in a simpler way than current strategies. This review aims to highlight the importance of energy self-production in synthetic cells, providing suggestions and ideas that may help overcome some longstanding challenges in this field.

Abstract Constructing molecular classifiers that enable cells to recognize linear and non-linear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effect of biological constraints such as resource limitation and competitive binding on the function of those classifiers has been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working on the principles of molecular sequestration between the sigma factor and its anti-sigma molecule. We then investigate how the output of the biomolecular perceptron, i.e ., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multi-layer perceptron neural networks and outline design principles that enable the construction of non-linear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects.

construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

We show that thermal states of local Hamiltonians are separable above a constant temperature. Specifically, for a local Hamiltonian $H$ on a graph with degree $\mathfrak{d}$, its Gibbs state at inverse temperature $β$, denoted by $ρ= e^{-βH}/ \operatorname{tr}(e^{-βH})$, is a classical distribution over product states for all $β&lt; 1/(c\mathfrak{d})$, where $c$ is a constant. This proof of sudden death of thermal entanglement resolves the fundamental question of whether many-body systems can exhibit entanglement at high temperature. Moreover, we show that we can efficiently sample from the distribution over product states. In particular, for any $β&lt; 1/( c \mathfrak{d}^2)$, we can prepare a state $\varepsilon$-close to $ρ$ in trace distance with a depth-one quantum circuit and $\operatorname{poly}(n, 1/\varepsilon)$ classical overhead.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, microfluidic chips capable of measuring single-cell mechanics via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. We found slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell signaling in space.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.

Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.

Droplets on nanotextured oil-impregnated surfaces have high mobility due to record-low contact angle hysteresis (∼1-3°), attributed to the absence of solid-liquid contact. Past studies have utilized the ultralow droplet adhesion on these surfaces to improve condensation, reduce hydrodynamic drag, and inhibit biofouling. Despite their promising utility, oil-impregnated surfaces are not fully embraced by industry because of the concern for lubricant depletion, the source of which has not been adequately studied. Here, we use planar laser-induced fluorescence (PLIF) to not only visualize the oil layer encapsulating the droplet (aka wrapping layer) but also measure its thickness since the wrapping layer contributes to lubricant depletion. Our PLIF visualization and experiments show that (a) due to the imbalance of interfacial forces at the three-phase contact line, silicone oil forms a wrapping layer on the outer surface of water droplets, (b) the thickness of the wrapping layer is nonuniform both in space and time, and (c) the time-average thickness of the wrapping layer is ∼50 ± 10 nm, a result that compares favorably with our scaling analysis (∼50 nm), which balances the curvature-induced capillary force with the intermolecular van der Waals forces. Our experiments show that, unlike silicone oil, mineral oil does not form a wrapping layer, an observation that can be exploited to mitigate oil depletion of nanotextured oil-impregnated surfaces. Besides advancing our mechanistic understanding of the wrapping oil layer dynamics, the insights gained from this work can be used to quantify the lubricant depletion rate by pendant droplets in dropwise condensation and water harvesting.

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in Western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with Western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of Western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium-crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano-responsive hydrogels that may be useful in various biomedical applications.

Abstract Stimuli‐responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano‐responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano‐responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium‐crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano‐responsive hydrogels that may be useful in various biomedical applications.

We give a new framework for solving the fundamental problem of low-rank matrix completion, i.e., approximating a rank-$r$ matrix $\mathbf{M} \in \mathbb{R}^{m \times n}$ (where $m \ge n$) from random observations. First, we provide an algorithm which completes $\mathbf{M}$ on $99\%$ of rows and columns under no further assumptions on $\mathbf{M}$ from $\approx mr$ samples and using $\approx mr^2$ time. Then, assuming the row and column spans of $\mathbf{M}$ satisfy additional regularity properties, we show how to boost this partial completion guarantee to a full matrix completion algorithm by aggregating solutions to regression problems involving the observations. In the well-studied setting where $\mathbf{M}$ has incoherent row and column spans, our algorithms complete $\mathbf{M}$ to high precision from $mr^{2+o(1)}$ observations in $mr^{3 + o(1)}$ time (omitting logarithmic factors in problem parameters), improving upon the prior state-of-the-art [JN15] which used $\approx mr^5$ samples and $\approx mr^7$ time. Under an assumption on the row and column spans of $\mathbf{M}$ we introduce (which is satisfied by random subspaces with high probability), our sample complexity improves to an almost information-theoretically optimal $mr^{1 + o(1)}$, and our runtime improves to $mr^{2 + o(1)}$. Our runtimes have the appealing property of matching the best known runtime to verify that a rank-$r$ decomposition $\mathbf{U}\mathbf{V}^\top$ agrees with the sampled observations. We also provide robust variants of our algorithms that, given random observations from $\mathbf{M} + \mathbf{N}$ with $\|\mathbf{N}\|_{F} \le Δ$, complete $\mathbf{M}$ to Frobenius norm distance $\approx r^{1.5}Δ$ in the same runtimes as the noiseless setting. Prior noisy matrix completion algorithms [CP10] only guaranteed a distance of $\approx \sqrt{n}Δ$.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites.

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium-crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano-responsive hydrogels that may be useful in various biomedical applications.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.