Biology subjects
Publications and source records attributed to Liu, A. P..
We present a membrane-associated, cell-free biosensing reporter based on rapalog-induced split sfCherry reconstitution. Rapalog increased fluorescence in solution, lipid vesicles, and supported lipid bilayers. Antibodies binding to epitope tags near the dimerization interface reduced the fluorescence signal, consistent with steric interference with reporter assembly.
The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.
Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.
Lipid membrane-bound synthetic cells provide programmable, cell-like compartments for on-demand biomolecule production. However, most cell-free gene expression systems in synthetic cells are regulated by user-imposed cues rather than signals associated with environmental or physiological states. Here, we present an acidity-transducing synthetic cell that converts external pH changes into nucleic acid information to drive in situ protein synthesis. The system integrates gramicidin A proton channels for pH sensing, triplex-forming single-stranded DNA (ssDNA) that releases a trigger ssDNA upon acidification, and a toehold switch RNA that activates translation in response to the released trigger ssDNA. This work further reveals that tuning the annealing length between the pH-responsive and trigger ssDNAs controls the trigger-release pH, which is critical for enabling acid-triggered protein synthesis. The synthetic cells retain their pH-responsive activity when embedded in alginate hydrogels, creating acidity-responsive materials that synthesize proteins in situ rather than release preloaded cargo. Cell-penetrating peptide tagging further enables selective protein release and target-specific binding. This work establishes a molecular transduction strategy for programming synthetic cell materials to sense environmentally relevant acidity and generate functional biomolecules on demand.
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 -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, 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.
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 TRAPs 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.
The identification and control of high-risk host cell proteins (HCPs) in biotherapeutics development is crucial for ensuring product quality and shelf life. Specifically, HCPs with hydrolase activity can cause degradation of excipient polysorbates (PS), leading to a decrease in the shelf life of the drug product. In this study, we systematically optimized every step of an activity-based protein profiling (ABPP) workflow to identify trace amounts of active polysorbate-degradative enzymes (PSDEs) in biotherapeutics process intermediates. Evaluation of various parameters during sample preparation pinpointed optimal pH level and fluorophosphonate (FP)-biotin concentration. Moreover, the combined use of a short liquid chromatography gradient and the fast-scanning Parallel Accumulation Serial Fragmentation (PASEF) methodology increased sample throughput without compromising identification coverage. Tuning Trapped Ion Mobility Spectrometry (TIMS) parameters further enhanced sensitivity. In addition, we evaluated various data acquisition modes including PASEF combined with data-dependent acquisition (DDA PASEF), data-independent acquisition (diaPASEF), or parallel reaction monitoring (prm-PASEF). By employing the newly optimized ABPP workflow, we successfully identified PSDEs at a concentration as low as 10 parts per billion (ppb) in a drug substance sample. Finally, the new workflow enabled us to detect a PSDE that could not be detected with the original workflow during a PS degradation root-cause investigation.
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.
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.
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.
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.
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.
Plasma membrane tension functions as a global physical organizer of cellular activities. Technical limitations of current membrane tension measurement techniques have hampered in-depth investigation of cellular membrane biophysics and the role of plasma membrane tension in regulating cellular processes. Here, we develop an optical membrane tension biosensor by repurposing an E. coli mechanosensitive channel via insertion of circularly permuted GFP (cpGFP), which undergoes a large conformational rearrangement associated with channel activation and thus fluorescence intensity changes under increased membrane tension.
Cellular unjamming is the collective fluidization of cell motion and has been linked to many biological processes, including development, wound repair, and tumor growth. In tumor growth, the uncontrolled proliferation of cancer cells in a confined space generates mechanical compressive stress. However, because multiple cellular and molecular mechanisms may be operating simultaneously, the role of compressive stress in unjamming transitions during cancer progression remains unknown. Here we investigate which mechanism dominates in a dense, mechanically stressed monolayer. We find that long-term mechanical compression triggers cell arrest in benign epithelial cells and enhances cancer cell migration in transitions correlated with cell shape, leading us to examine the contributions of cell-cell adhesion and substrate traction in shape-dependent unjamming transitions. We show that cadherin-mediated cell-cell adhesion regulates differential cellular responses to compressive stress and predominantly controls unjamming transitions in dense monolayers. Importantly, compressive stress does not induce the epithelial--mesenchymal transition in unjammed cells. Using traction force microscopy, traction forces are attenuated in compressed cells in the bulk of the monolayer regardless of cell type and motility. Intercellular adhesion thus is the dominant regulator of compression-induced unjamming transitions and may impact collective cell motion in tumor development and breast cancer progression.
Actin networks polymerize and depolymerize to construct highly organized structures, thereby, endowing the mechanical phenotypes found in a cell. It is generally believed that the amount of filamentous actin and actin network architecture determine cytoplasmic viscosity and elasticity of the whole cell. However, the intrinsic complexity of a cell and numerous other endogenous cellular components make it difficult to study the differential role of distinct actin networks in regulating cell mechanics. Here, we model a cell by using giant unilamellar vesicles (GUVs) encapsulating actin filaments and networks assembled by various actin crosslinker proteins. Perturbation of these cytoskeletal vesicles using AC electric fields revealed that deformability depends on lumenal viscosity and actin network architecture. While actin-free vesicles exhibited large electromechanical deformations, deformations of GUVs encapsulating actin filaments were significantly dampened. The suppression of electrodeformation of actin-GUVs can be similarly recapitulated by using aqueous PEG 8000 solutions at different concentrations to modulate viscosity. Furthermore, alpha actinin-crosslinked actin networks resulted in decreased GUV deformability in comparison to actin filament-encapsulating GUVs, and membrane-associated actin networks through the formation of dendritic actin cortex greatly dampened electrodeformation of GUVs. These results highlight the organization of actin networks regulates the mechanics of GUVs and shed insights into the origin of differential deformability of cells.
Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through SpyTag/SpyCatcher interaction. Inclusion of split fluorescent protein fragments in the designed InterSpy toolkit allows tracking the formation of membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. We first demonstrate InterSpy by testing split protein designs using a mammalian cell-free expression system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with a desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, since InterSpy is fully genetically encoded, the engineered system is adapted to cells and showcased. InterSpy demonstrates the power of cell-free expression systems in functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful for synthetic biology where cell-cell contacts and communication are recreated in a controlled manner using minimal components.
Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as -actinin and fascin as well actin nucleator Arp2/3 complex largely determine the architecture and connectivity of actin network patterns; consequently, they regulate network remodeling and membrane shape changes. Membrane constriction in animal cell cytokinesis proceeds by assembly and contraction of a contractile ring pattern rich in -actinin and myosin at the equator of the cell cortex, with which the ring is contiguous. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that, depending on vesicle size and concentrations of -actinin and fascin, actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane enhances the interaction of the contractile networks with lipid membrane but does not change the architecture of the patterns. A membrane-bound actomyosin ring exerts force and constricts the membrane. An Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex and contribute to myosin-driven clustering of the cortex and consequently membrane deformation. An active gel theory unifies a model for the observed membrane constriction and protrusion induced by the membrane-bound actomyosin networks.