Biology subjects

Stachowiak, J. C.

Publications and source records attributed to Stachowiak, J. C..

15 recordsLinked to original sources

bioRxiv2026

Curvature sensing and generation by diverse membrane-tethered polymers can be explained by a unifying physical model

Cell membranes are decorated by dense, polymeric macromolecules such as proteins with bulky intrinsically disordered regions, glycosylated proteins, and glycopolymers. Experiments have illustrated that the curvature of cellular membranes, which plays a role in diverse biological processes, can be regulated by the local density of these membrane-tethered polymers and their response to environmental changes. However, the mechanisms underlying the feedback between solution conditions, membrane curvature, and the local density and molecular weight of membrane-tethered polymers remain largely unexplored. In this work, using a combination of theory and experiment, we show that the ionic strength of the surrounding solution and the degree of ionization both play important roles in regulating the conformation of the membrane-tethered polymers, which in turn influences their membrane curvature generation capabilities. Specifically, we show that for negatively charged, disordered proteins and glycans, the thickness of the polymer brush decreases with ionic strength but increases with grafting density and degree of ionization, resulting in enhanced sensitivity to membrane curvature and an amplified ability to drive membrane curvature at lower ionic concentrations. Our findings reveal a mechanism by which membrane-tethered polymers generate membrane curvature to minimize free energy, with the extent of curvature generation modulated by the polymer grafting density and layer thickness, as well as by the ionic strength of the surrounding solution. The implications of these findings are broad, suggesting that perturbations to biopolymer charge, such as sialylation of glycans and phosphorylation of disordered proteins, could actively modulate membrane curvature induction in diverse cellular processes from endocytosis to cell motility.

biophysics↗

bioRxiv2025

Anillin-dependent actin assembly at PML NBs protects genome stability

Nuclear filamentous actin (F-actin) is emerging as a key regulator of genome stability1-6. During replication stress, transient bursts of nuclear actin assembly in S-phase promote fork remodelling and repair3,4, but how these bursts are initiated, regulated, and whether nuclear F-actin also act on DNA lesions inherited across cell cycles, remains unknown. Here, we show that replication stress induces nuclear actin assembly at liquid-liquid phase-separated (LLPS) promyelocytic leukaemia nuclear bodies (PML NBs), driven by the actin-binding protein Anillin. Although best known as a cytokinesis factor, Anillin localises to PML NBs in interphase, where it concentrates monomeric actin (G-actin) and establishes a favourable environment for spontaneous actin assembly. Filament growth within these condensates remodels PML NB morphology and composition, releasing sequestered AKT into the nucleoplasm. AKT is subsequently phosphorylated and activated, and this nuclear AKT activity is required to regulate 53BP1 nuclear bodies in G1. These protective LLPS compartments shield under-replicated DNA inherited from the stressed S-phase until the following S-phase. This prevents premature engagement by aberrant repair pathways that would otherwise generate toxic intermediates and culminate in mitotic failure. Together, these findings define a condensate-to-filament pathway in which nuclear F-actin dynamically reorganises phase-separated compartments to safeguard genome stability across cell cycles.

cell biology↗

bioRxiv2025

A balance between nucleating and elongating actin filaments controls deformation of protein condensates

Protein condensates use multivalent binding and surface tension to assemble actin filaments into diverse architectures, reminiscent of filopodia and stress fibers. During this process, nucleation of new filaments and elongation of existing filaments inherently compete for a shared pool of actin monomers. Here we show that a balance between these competing processes is required to deform condensates of VASP, an actin binding protein, into structures with high aspect ratios. Addition of magnesium, which promotes filament nucleation, helped actin to deform condensates into high aspect ratio structures. In contrast, addition of profilin, which inhibits filament nucleation, allowing existing filaments to elongate, caused actin to assemble into ring-like bundles that failed to substantially increase condensate aspect ratio. Computational modeling helped to explain these results by predicting that a group of short linear filaments, which can apply asymmetric pressure to the condensate boundary, is needed to increase condensate aspect ratio. In contrast, a small number of long filaments with the same total actin content should fail to overcome the droplet surface tension, forming a ring-like bundle. To test these predictions, we introduced gelsolin, which severed log filaments within rings, creating new barbed ends. The resulting set of shorter filaments regained the ability to deform condensates into high aspect ratio structures. Collectively, these results suggest that a balance of actin filament nucleation and elongation is required to deform protein condensates. More broadly, these findings illustrate how protein condensates can balance multiple kinetic processes to direct the assembly of diverse cytoskeletal architectures.

biophysics↗

bioRxiv2025

Light-Controlled Synthetic Communication Networks via Paired Connexon Nanopores

Living cells employ dynamic networks for intercellular communication and cooperation, leading to tissue-wide activity. One emerging challenge in the field of bottom-up synthetic biology is emulating such sophisticated behaviors in liposome-based synthetic cells (SCs). Fabricating communication networks in lipid bilayer-based SCs remains a challenge as signaling molecules must transit through two consecutive membranes to transfer information between different SCs. Here, we address this obstacle by engineering connexin channels that directly connect the lumens of adhering SC membranes. We focus on orthogonal channel-forming connexins, namely connexin 43 and connexin 32, and re-design their channel activity to be UV- and near IR-responsive, respectively. By combining engineered connexins into a single SC assembly, we demonstrate orthogonal transfer of reactive signaling molecules between SCs, giving rise to unique reaction products and network states in a wavelength-dependent manner - an important step toward synthetic communication networks.

synthetic biology↗

bioRxiv2024

Transmembrane coupling accelerates the growth of liquid-like protein condensates

Timely and precise assembly of protein complexes on membrane surfaces is essential to the physiology of living cells. Recently, protein phase separation has been observed at cellular membranes, suggesting it may play a role in the assembly of protein complexes. Inspired by these findings, we observed that protein condensates on one side of a planar suspended membrane spontaneously colocalized with those on the opposite side. How might this phenomenon contribute to the assembly of stable transmembrane complexes? To address this question, we examined the diffusion and growth of protein condensates on both sides of membranes. Our results reveal that transmembrane coupling of protein condensates on opposite sides of the membrane slows down condensate diffusion while accelerating condensate growth. How can the rate of condensate growth increase simultaneously with a decrease in the rate of condensate diffusion? We provide insights into these seemingly contradictory observations by distinguishing between diffusion-limited and coupling-driven growth processes. While transmembrane coupling slows down diffusion, it also locally concentrates condensates within a confined area. This confinement increases the probability of condensate coalescence and thereby enhances the overall rate of growth for coupled condensates, substantially surpassing the growth rate for uncoupled condensates. These findings suggest that transmembrane coupling could play a role in the assembly of diverse membrane-bound structures by promoting the localization and growth of protein complexes on both membrane surfaces. This phenomenon could help to explain the efficient assembly of transmembrane structures in diverse cellular contexts. SignificanceProtein assemblies that span biological membranes are critical to cellular physiology. In the past decade, liquid-like protein condensates, which are flexible, multivalent protein assemblies, have been discovered on diverse membrane surfaces. Recently, we observed that protein condensates on opposite sides of a membrane spontaneously colocalize to form coupled, transmembrane complexes. Interestingly, while transmembrane coupling slows down the diffusion of membrane-bound condensates, it substantially accelerates their growth by strongly localizing interactions between them. These findings suggest that transmembrane coupling of protein condensates may play a role in promoting the robust assembly of membrane-bound protein complexes in crowded, complex cellular environments.

biophysics↗

bioRxiv2023

Liquid-like condensates mediate competition between actin branching and bundling

Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch versus becoming bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells. SIGNIFICANCE STATEMENTReorganization of actin filaments allows cells to migrate, which is required for embryonic development, wound healing, and cancer metastasis. During migration, the leading-edge of the cell consists of needle-like protrusions of bundled actin, which emanate from a sheet of branched actin. Given that the proteins responsible for both architectures are present simultaneously, what determines whether actin filaments will be branched or bundled? Here we show that liquid-like condensates, composed of both branching and bundling proteins, can mediate the inherent competition between these fundamentally different ways of organizing actin networks. This work demonstrates that by tuning the composition of condensates, we can recapitulate the transition from branched to bundled networks, a key step in cell migration.

biophysics↗

bioRxiv2023

Sizes, Conformational fluctuations, and SAXS profiles for Intrinsically Disordered Proteins

The preponderance of Intrinsically Disordered Proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, folded proteins, RNA, and DNA for functional purposes, have made it important to quantitatively characterize their biophysical properties. Toward this end, we developed the transferable Self-Organized Polymer (SOP-IDP) model to calculate the properties of several IDPs. The values of the radius of gyration (Rg) obtained from SOP-IDP simulations are in excellent agreement (correlation coefficient of 0.96) with those estimated from SAXS experiments. For AP180 and Epsin, the predicted values of the hydrodynamic radii (Rhs) are in quantitative agreement with those from Fluorescence Correlation Spectroscopy (FCS) experiments. Strikingly, the calculated SAXS spectra for thirty-six IDPs are also nearly superimposable on the experimental profiles. The dependence of Rg and the mean end-to-end distance (Ree) on chain length, N, follows Florys scaling law, R {approx} aN 0.588 ( = g, and e), suggesting that globally IDPs behave as synthetic polymers in a good solvent. The values of ag, and ae are 0.20 nm and 0.48 nm respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, are negligible for Rg and Ree. In contrast, only by accounting for the finite sizes of the IDPs, the dependence of experimentally measurable Rh on N can be quantitatively explained using{nu} = 0.588. Although Flory scaling law captures the estimates for Rg, Ree, and Rh accurately, the spread of the simulated data around the theoretical curve is suggestive of of sequence-specific features that emerge through a fine-grained analysis of the conformational ensembles using hierarchical clustering. Typically, the ensemble of conformations partitiones into three distinct clusters, having different equilibrium populations and structural properties. Without any further readjustments to the parameters of the SOP-IDP model, we also obtained excellent agreement with paramagnetic relaxation enhancement (PRE) measurements for -synuclein. The transferable SOP-IDP model sets the stage for several applications, including the study of phase separation in IDPs and interactions with nucleic acids.

biophysics↗

bioRxiv2022

Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

Liquid-liquid phase separation of proteins has recently been observed on the surfaces of biological membranes, where it plays a role in diverse cellular processes, from assembly of focal adhesions and the immunological synapse, to biogenesis of trafficking vesicles. Interestingly in each of these cases, proteins on both surfaces of the membrane are thought to participate, suggesting that protein phase separation could be coupled across the membrane. To explore this possibility, we used an array of freestanding planar lipid membranes to observe protein phase separation simultaneously on both surfaces of lipid bilayers. When proteins known to engage in phase separation bound to the surfaces of these membranes, two-dimensional, protein-rich phases rapidly emerged. These phases displayed the hallmarks of a liquid, coarsening over time by fusing and re-rounding. Interestingly, we observed that protein-rich domains on one side of the membrane colocalized with those on the other side, resulting in transbilayer coupling. How do liquid-like protein phases communicate across the lipid bilayer? Our results, based on lipid probe partitioning and the differential mobility of proteins and lipids, collectively suggest an entropic coupling mechanism, which relies on the ability of protein phase separation to locally reduce the entropy of the underlying lipid membrane, most likely by increasing lipid packing. Regions of reduced entropy then colocalize across the bilayer to minimize the overall free energy of the membrane. These findings suggest a previously unknown mechanism by which cellular signals originating from one side of the membrane, triggered by protein phase separation, can be transferred to the opposite side.

biophysics↗

bioRxiv2022

Light-Activated Assembly of Connexon Nanopores in Synthetic Cells

During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Non-living systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One non-living system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexin nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.

synthetic biology↗

bioRxiv2022

Liquid-like assembly of VASP drives actin polymerization and bundling

The organization of actin filaments into bundles is required for cellular processes such as motility, morphogenesis, and cell division. Filament bundling is controlled by a network of actin binding proteins. Recently, several proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might liquid-like condensates contribute to filament bundling? Here, we show that the processive actin polymerase and bundling protein, VASP, forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the droplets surface tension, as predicted by a continuum-scale computational model. However, as actin polymerizes and the ring grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming into a linear bundle. The resulting bundles contain long, parallel actin filaments that grow from their tips. Significantly, the fluid nature of the droplets is critical for bundling, as more solid droplets resist deformation, preventing filaments from rearranging to form bundles. Once the parallel arrangement of filaments is created within a VASP droplet, it propagates through the addition of new actin monomers to achieve a length that is many times greater than the initial droplet. This droplet-based mechanism of bundling may be relevant to the assembly of cellular architectures rich in parallel actin filaments, such as filopodia, stress fibers, and focal adhesions.

biophysics↗

bioRxiv2022

Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Forster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape. SignificanceWith nearly half their surfaces covered by proteins, biological membranes are highly crowded. Rapid diffusion and collision of membrane-bound proteins generates substantial steric pressure that is capable of shaping membrane surfaces. Many proteins involved in membrane remodeling, are intrinsically disordered. Having large hydrodynamic radii, disordered domains could contribute substantially to membrane crowding. However, it is unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. Toward resolving this uncertainty, we have measured steric pressure at membrane surfaces during dynamic membrane remodeling events. Our data indicate that disordered domains generate significant steric pressure through entropic and electrostatic mechanisms, suggesting that they may constitute a critical, yet previously neglected class of membrane remodeling proteins.

biophysics↗

bioRxiv2020

Clathrin senses membrane curvature

The ability of proteins to sense membrane curvature is essential to diverse membrane remodeling processes including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to assemble together at curved membrane sites, leading to local recruitment of the clathrin coat. Because clathrin does not bind to the membrane directly, it has remained unclear whether clathrin plays an active role in sensing curvature or is passively recruited by its adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable to previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly, rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Further, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by two to ten-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface, compared to a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the critical drivers of membrane curvature sensing.

molecular biology↗

bioRxiv2020

Membrane bending by protein phase separation

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder, which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multi-valent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds, but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes. Significance StatementCellular membranes take on an elaborate set of highly curved and bent shapes, which are essential to diverse cellular functions from endocytosis to cell division. The prevailing view has been that membrane bending is driven by proteins with curved shapes, which assemble at the membrane surface to form solid scaffolds. In contrast, here we show that proteins which form liquid-like assemblies on membranes are also potent drivers of bending. These "liquid scaffolds" apply compressive stress to the membrane surface, generating a diverse and dynamic family of membrane shapes. These data, which come at a time when liquid-like protein assemblies are being identified throughout the cell, suggest that liquid-like protein assemblies may play an important role in shaping cellular membranes.

biophysics↗

bioRxiv2020

Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 is mechanically responsive to extracellular mechanical cues and alters its association with cytoskeletal and adhesion-related proteins in response to substrate stiffness and physiological flow.

bioengineering↗

bioRxiv2019

Liquid-like protein interactions catalyze assembly of endocytic vesicles

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As early initiators of endocytosis, Eps15 and Fcho1 are responsible for locally concentrating downstream components on the membrane surface. However, they must also permit dynamic rearrangement of proteins within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1 rely on weak, liquid-like interactions to efficiently catalyze endocytosis. In reconstitution experiments, these weak interactions promote the assembly of protein droplets with liquid-like properties, including rapid coalescence and dynamic exchange of protein components. To probe the physiological role of liquid-like interactions among initiator proteins, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove initiator proteins into liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15 knockout cells. In contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels. Assembly of these structures stalled vesicle budding, likely owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of early initiator proteins provides an optimal catalytic platform for endocytosis.

cell biology↗