Biology subjects

Zeno, W. F.

Publications and source records attributed to Zeno, W. F..

12 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↗

bioRxiv2026

Cholesterol differentially regulates α-synuclein binding across membrane packing regimes

-Synuclein (Syn) is an intrinsically disordered protein that preferentially binds anionic membranes with lipid packing defects. Cholesterol is an abundant membrane component that regulates packing and organization within membranes, yet its effect on Syn binding remains unclear as prior studies report both cholesterol-mediated enhancement and suppression. Here, we investigated whether these conflicting effects reflect differences in the intrinsic packing state of the phospholipid bilayer. Using a quantitative fluorescence microscopy-based binding assay, we measured Syn binding preferences among reconstituted phosphatidylcholine/phosphatidylserine membranes with varied cholesterol content, lipid tail chemistry, and vesicle curvature. We found that cholesterols effect depended on the underlying packing regime of the membrane. In defect-rich membranes, cholesterol reduced Syn binding, consistent with cholesterol tightening lipid packing and reducing Syn-accessible defects. In membranes with intermediate defect content, cholesterol enhanced binding, whereas tightly packed membranes remained largely insensitive to cholesterol except when high cholesterol content was combined with high membrane curvature. Curvature further shaped these responses, with high curvature compressing cholesterol-dependent differences between membrane compositions. These results show that cholesterol does not universally promote or inhibit Syn binding. Instead, cholesterol regulates Syn-membrane interactions through a packing-regime-dependent mechanism shaped by both lipid tail chemistry and membrane curvature. This framework helps reconcile opposing reports in the literature and highlights membrane physical state as a key determinant of how cholesterol modulates Syn binding. SignificanceSyn is a membrane-binding protein associated with Parkinsons disease, but the role of cholesterol in regulating its membrane interactions has remained unclear. Some studies report that cholesterol enhances Syn association with membranes, whereas others show that cholesterol suppresses it. This work helps explain why both outcomes can occur. We show that cholesterols effect depends on the membranes underlying packing state: cholesterol can reduce, enhance, or have little effect on Syn binding depending on the membrane environment. These findings shift the question from whether cholesterol is generally pro- or anti-binding to how cholesterol reshapes the membrane physical states that control Syn association.

biophysics↗

bioRxiv2026

Membrane Phase, Charge, and Curvature Regulate α-Synuclein Binding Dynamics

-Synuclein (Syn) is an intrinsically disordered protein whose interactions with lipid membranes are central to both its physiological function and its role in synucleopathies. While membrane charge, phase, and curvature are each known to influence Syn binding, these properties are typically examined independently, leaving their combined effects on both equilibrium and dynamic membrane association unresolved. Here, we systematically investigate how membrane phase and charge jointly regulate Syn binding, curvature sensitivity, and exchange dynamics using fluorescence microscopy, circular dichroism spectroscopy, and fluorescence recovery after photobleaching (FRAP), complemented by coarse-grained molecular dynamics simulations. Under zwitterionic conditions, Syn preferentially binds highly curved gel-phase membranes, driven by curvature-dependent enrichment of packing defects arising from faceted vesicle morphologies. Incorporation of anionic lipids selectively enhances binding in liquid-phase membranes while attenuating curvature-dependent partitioning in gel-phase membranes. Dynamic measurements reveal that membrane phase and charge also govern the stability of membrane-associated Syn, with gel-phase membranes and anionic lipids promoting kinetically stabilized states. Simulations show that curvature-induced defect formation is strongly amplified in gel-phase membranes but largely insensitive to charge. These findings establish that Syn-membrane interactions are governed by a cooperative interplay between membrane phase, curvature, and charge and highlight the importance of resolving both thermodynamic and kinetic contributions to protein-membrane binding.

biophysics↗

bioRxiv2026

Amphiphilic Peptide Fusion Promotes Endocytic Uptake of Nanodiscs

A major limitation across nanoparticle delivery platforms is sequestration within endosomal compartments, which restricts access to intracellular targets despite efficient cellular uptake. Here, we show that peptide architecture can be used to control intracellular trafficking and reduce endosomal accumulation in lipid-protein nanocarriers. Specifically, we fuse R6W3 (RRWWRRWRR), an amphipathic cell penetrating peptide, to the N- or C- terminus of the nanodisc scaffold proteins and systematically evaluate its impact on membrane interactions and cellular behavior. Structural and biophysical characterization confirms that R6W3 incorporation preserves nanodisc assembly and protein-lipid interactions, enabling direct attribution of functional differences to peptide-driven interfacial effects. R6W3-functionalized nanodiscs exhibit enhanced binding and cellular uptake, with N-terminal fusion producing the strongest interfacial interactions. In live cells, R6W3-functionalization increases endocytic activity, evidenced by increased formation of clathrin-coated pits and intracellular colocalization with clathrin-coated vesicles. Notably, R6W3-funtionalized nanodiscs display reduced accumulation in early endosomes relative to unmodified nanodiscs, indicating decreased endosomal sequestration following endosomal uptake. These trafficking differences translate to functional outcomes, as doxorubicin-loaded, R6W3-functionalized nanodiscs achieve greater cytotoxicity than unmodified controls at equivalent concentrations. Together, these results establish peptide architecture as a design parameter for controlling intracellular trafficking and overcoming endosomal bottlenecks, providing a broadly applicable strategy for improving nanocarrier- based delivery systems.

bioengineering↗

bioRxiv2026

Graphene Quantum Dots Mitigate Oxidative Stress in Bacteria

Manufacturing and storage processes can expose microbes to oxidative stress, reducing viability and limiting their use in biotechnological applications. Here, we evaluate graphene quantum dots (GQDs) containing hydroxyl and carboxyl groups as protective additives that mitigate peroxide-induced oxidative stress in Escherichia coli. GQDs did not adversely affect bacterial growth under basal conditions and restored growth in the presence of hydrogen peroxide. Using the membrane-partitioning fluorescent probe C11-BODIPY, we found that GQDs reduced peroxide-induced oxidation in bacterial membranes. We further used redox-sensitive roGFP2 probes to monitor intracellular oxidative stress and found that GQDs suppressed intracellular hydrogen peroxide accumulation and attenuated disruption of glutathione redox homeostasis. Together, these results show that GQDs protect bacteria by limiting peroxide-driven oxidative damage at both membrane and intracellular levels. This work supports the potential use of GQDs as protective additives for microbial formulations that are susceptible to oxidative stress.

microbiology↗

bioRxiv2026

The disordered and structured regions of α-Synuclein contribute to membrane remodeling synergistically

-Synuclein (Syn) remodels cellular membranes through interactions that involve both its structured, membrane-binding N-terminal domain (NTD) and intrinsically disordered C-terminal domain (CTD). While the amphipathic NTD helix is known to insert into lipid bilayers and generate curvature, the contribution of the acidic CTD remains unclear. Here, we dissect the individual and cooperative roles of these domains using Supported Bilayers with Excess Membrane Reservoir (SUPER) templates to quantify membrane remodeling via membrane fission and membrane morphological deformations (i.e., membrane budding and tubulation). We show that both the NTD and CTD independently remodel membranes, while full-length Syn exhibits greater remodeling ability than either the NTD or CTD in isolation. This result demonstrates a synergistic amplification between helix insertion of the NTD and the tethered, disordered CTD. To further probe the mechanism of membrane remodeling by the CTD, we modulated the chain length of the protein, the bulk ionic strength of the solution (i.e., charge screening), and applied relevant polymer scaling laws for disordered proteins. Our results suggest that the membrane remodeling mechanism for the disordered CTD is electrostatic in nature, stemming from protein-protein repulsion at elevated binding densities. Together, our findings reveal a cooperative energetic mechanism in which N-terminal helix insertion biases membrane curvature and the disordered, C-terminal domain adds an additional electrostatic component that helps to overcome the free energy barrier for membrane bending.

biophysics↗

bioRxiv2026

Clathrin is an Intrinsic Driver of Membrane Fission

Clathrin-mediated endocytosis is a primary internalization pathway where adaptor proteins recruit clathrin and bend the plasma membrane to form clathrin coated vesicles. Since clathrin does not bind membranes directly, its contributions to membrane remodeling remain unclear. Using an in vitro system that recruits clathrin directly to lipid membranes, we show that clathrin alone can drive end-to-end membrane budding and vesicle fission. Surprisingly, stronger clathrin assembly (i.e., higher membrane association) yields weaker membrane fission, indicating that membrane remodeling depends not simply on how much clathrin is recruited, but on how the resulting lattice is assembled. Brownian dynamics simulations, together with experimentally measured shifts in clathrin cage diameter, suggest that this counterintuitive behavior arises at least in part from assembly-dependent changes in intrinsic lattice curvature. This framework extends to adaptor-mediated recruitment, with clathrin either assisting or opposing membrane fission in an adaptor-dependent manner. Together, these findings support a mechanism by which coat protein mechanics regulate membrane remodeling.

biophysics↗

bioRxiv2025

Membrane curvature enhances lipid peroxidation in a composition-dependent manner

Excessive production of reactive oxygen species (ROS) in cells results in oxidative stress, which can promote lipid oxidation in cellular membranes. This oxidation of membrane lipids accompanies various diseases and can even result in cell death through processes such as ferroptosis. The complex compositions and diverse morphologies of cellular membranes make understanding the mechanisms of lipid oxidation challenging, especially when attempting to investigate membrane composition and curvature simultaneously. Here, we utilize reconstituted lipid membranes and the fluorescent oxidation probe C11-BODIPY to quantify oxidation in lipid bilayers as functions of both lipid composition and membrane curvature. By tethering synthetic lipid vesicles to glass substrates, we were able to monitor oxidation of the fluorescent probe on a per vesicle basis using fluorescence microscopy. Our results demonstrate that highly curved membranes markedly increase both the rate and extent of oxidation across diverse membrane compositions. This effect arises from greater exposure of lipid tails to the aqueous environment, which allows more efficient transport of ROS into the hydrophobic core of the bilayer. Compositional effects on oxidation are most pronounced in membranes with low curvature (i.e., greater than 100 nm diameter) and become progressively weaker as curvature increases. We found that low to moderate cholesterol levels (i.e., 10-25 mol%) suppress curvature-dependent oxidation by tightening lipid packing, whereas high cholesterol content (i.e., 50 mol%) restores curvature sensitivity by influencing lateral lipid mobility. Together, these findings establish membrane curvature and lipid composition as interdependent determinants of oxidative susceptibility, offering new insight into how cells regulate or resist oxidative stress. Statement of significanceOxidative stress drives lipid oxidation in cellular membranes, but the combined influence of membrane curvature and composition remains poorly defined. Using reconstituted lipid vesicles and a fluorescent oxidation probe, we show that oxidation in lipid bilayers is enhanced in smaller vesicles (i.e., highly curved membranes). The oxidation rate increases with unsaturated lipids, while cholesterol suppresses this effect. Measurements of membrane packing and diffusivity support these findings, demonstrating how curvature and composition together govern membrane susceptibility to oxidative damage. These results provide new insight into the physicochemical basis of membrane stability under oxidative stress and have broad implications for understanding the vulnerability of curved cellular membranes.

biophysics↗

bioRxiv2025

Recombinant protein platform for high-throughput investigation of peptide-liposome interactions via fluorescence anisotropy depolarization

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. Altogether, this recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for detailed investigation of the specific membrane biochemical parameters facilitating binding.

molecular biology↗

bioRxiv2024

Lipid Packing Defects are Necessary and Sufficient for Membrane Binding of alpha-Synuclein

-Synuclein (Syn), an intrinsically disordered protein implicated in Parkinsons disease, is thought to initiate aggregation by binding to cellular membranes. Previous studies suggest that anionic lipids are necessary for this binding. However, these studies largely focused on unmodified Syn, while physiological Syn is N-terminally acetylated (NTA). Our work challenges the long-standing paradigm that anionic lipids are necessary for Syn binding by demonstrating that NTA diminishes Syns reliance on anionic membrane charge, revealing that membrane packing defects (i.e., interfacial hydrophobicity) alone can drive membrane binding. Using fluorescence microscopy and circular dichroism spectroscopy, we monitored the binding of NTA-Syn to membrane vesicles with different lipid compositions. Phosphatidylcholine and phosphatidylserine concentrations were varied to control surface charge, while phospholipid tail unsaturation and methylation were varied to modulate lipid packing. We also formulated cholesterol-containing membranes that mimicked the lipid composition of synaptic vesicles. In these membranes, all- atom molecular dynamics simulations were used to visualize and quantify membrane packing defects. Our results demonstrate that membrane packing defects are necessary for NTA-Syn binding and that defect-rich membranes are sufficient for NTA-Syn binding regardless of membrane charge. These findings provide a molecular mechanism by which lipid structural properties, such as poly-unsaturation, can regulate Syn binding to physiological membranes.

biophysics↗

bioRxiv2024

Predicting how lipid composition controls protein curvature sensing with a continuum bilayer membrane model

Cytoplasmic proteins must recruit to membranes to function in processes such as endocytosis and cell division. Many of these proteins recognize not only the chemical structure of the membrane lipids, but the curvature of the surface, binding more strongly to more highly curved surfaces, or curvature sensing. Curvature sensing by amphipathic helices is known to vary with membrane bending rigidity, but changes to lipid composition can simultaneously alter membrane thickness, spontaneous curvature, and leaflet symmetry, thus far preventing a systematic characterization of lipid composition on such curvature sensing through either experiment or simulation. Here we develop and apply a bilayer continuum membrane model that can tractably address this gap, quantifying how controlled changes to each material property can favor or disfavor protein curvature sensing. We evaluate both energetic and structural changes to vesicles upon helix insertion, with strong agreement to new in vitro experiments and all-atom MD simulations, respectively. Our membrane model builds on previous work to include both monolayers of the bilayer via representation by continuous triangular meshes. We introduce a coupling energy that captures the incompressibility of the membrane and the established energetics of lipid tilt. In agreement with experiment, our model predicts stronger curvature sensing in membranes with distinct tail groups (POPC vs DOPC vs DLPC), despite having identical head-group chemistry; the model shows that the primary driving force for weaker curvature sensing in DLPC is that it is thinner, and more wedge shaped. Somewhat surprisingly, asymmetry in lipid shape composition between the two leaflets has a negligible contribution to membrane mechanics following insertion. Our multi-scale approach can be used to quantitatively and efficiently predict how changes to membrane composition in flat to highly curved surfaces alter membrane energetics driven by proteins, a mechanism that helps proteins target membranes at the correct time and place. SignificanceProteins must recruit to membranes for essential biological functions including endocytosis and cell division. In addition to recognizing specific lipid head-groups, many of these proteins also sense the curvature of the membrane, but the strength of sensing is known to vary with distinct membrane compositions. Predicting the dependence of sensing on changes to lipid composition cannot be done a priori due to the multiple material properties, including bilayer thickness, bending rigidity, tilt modulus, spontaneous curvature, and leaflet asymmetry that vary with lipid type. Here we use a multi-scale approach to systematically address this gap, developing a double-leaflet continuum model that is informed by structural deformations from all-atom MD and validated against in vitro experiments. This efficient approach can be applied and extended to quantify how proteins sense and drive membrane curvature across a wide range of membrane bilayers, including distinct leaflet compositions and membrane geometries.

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↗