Biology subjects

Fletcher, D. A.

Publications and source records attributed to Fletcher, D. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Deep Learning-based Modeling Enhances Efficacy of Natural Ligand CAR Binders Targeting CD70

CD70 is well-recognized as a promising "pan-cancer: chimeric antigen receptor (CAR) T-cell target. Prior work has shown that a "natural ligand" (NL)-based CAR targeting CD70, employing its physiological interaction partner CD27, may have therapeutic advantages over antibody-based CARs. Yet while antibody-based CARs are routinely optimized by affinity maturation of their scFv, whether the binding sequence of an NL CAR can be engineered to improve its function remains unexplored. Here, we combined deep learning with physics-based modeling to redesign residues at the CD27:CD70 interface, identifying a CD27 variant, "N88A", which enhances the efficacy of CD70-targeting CAR T-cells across models of acute myeloid leukemia, multiple myeloma, and renal cell carcinoma. Biophysical approaches, including molecular dynamics simulations, support a mechanism of increased binder conformational freedom underlying potency enhancement. Our work presents CD27N88A CAR T-cells as a promising new therapeutic option and proposes that computational modeling could be applied to enhance efficacy of other NL-based immunotherapies.

cancer biology↗

bioRxiv2026

Ups and downs of liquid-liquid transitions in GUV membranes from osmolarity and aspiration tensions

Lipid membranes undergo liquid-liquid phase separation to form micron-scale domains. Several physical parameters affect this phase transition. For example, a decrease in temperature causes membranes to demix, as does an increase in hydrostatic pressure. However, measurements to determine how tension affects membrane phase separation have yielded conflicting results. Experiments that have applied osmotic pressure differences to a population of vesicles have reported an increase in the membrane's miscibility transition temperature. Conversely, experiments that have applied micropipette aspiration or substrate stretching to single membranes have reported a decrease. Here, we find that both osmotic pressure and micropipette aspiration can increase transition temperatures. We discuss how membrane pores and hidden areas in membranes present challenges to researchers seeking to quantitatively convert experimentally measured osmolarity differences into membrane tensions. We show that challenges of comparing data from different osmotic pressure experiments can be mitigated by renormalizing osmolarity differences, specifically by dividing by the exterior osmolarity. We discuss our results in the context of existing theoretical predictions and in light of four known effects of increasing tension on vesicle membranes: 1) a reduction in hidden area of tubes and aggregates, 2) a reduction in out-of-plane thermal fluctuations, 3) an increase in the area per lipid, and 4) the formation of pores. First, hidden area can explain why vesicles can sustain high osmolarity differences. Next, suppression of thermal fluctuations and increases in the area per lipid may account for shifts in miscibility transition temperatures. Finally, pores can explain time dependences. Overall, our results highlight the need for new theory and simulation that unify predictions of how transition temperatures vary over all four regimes of membrane tension.

biophysics↗

bioRxiv2026

Environment-driven active transport of influenza A virus

Biological media such as airway mucus and extracellular matrix are usually viewed as transport barriers that particles cross by passive diffusion or with internal engines. We show instead that a particle can move actively by modifying the landscape it traverses, creating environmental memory and directional cues in two and three dimensions. Influenza A virus (IAV) realizes this principle through its envelope proteins hemagglutinin (HA) and neuraminidase (NA), which bind and cleave sialylated glycan receptors, respectively. Combining theory, simulations and single-virus tracking, we connect bind--cleave kinetics and HA--NA organization to macroscopic transport. Cleavage dissipates chemical free energy, biases rebinding to the edited landscape and leaves a trail that shapes future encounters. In heterogeneous receptor landscapes, multivalent binding biases motion toward higher receptor density, while receptor destruction by NA can amplify this bias by sharpening the contrast sampled by HA. Experiments on reconstituted glycan membranes show that IAV steps are biased up local receptor gradients, as predicted. The theory suggests that virion-to-virion variability can distribute transport functions across a population, providing a physical hedge against complex receptor environments. Together, these results establish environment-driven active matter as a mechanism for motorless transport powered and guided by chemical modification of the environment.

biophysics↗

bioRxiv2026

Automated detection of Loa loa: a field trial in Cameroon

Onchocerciasis (river blindness) is targeted for elimination through mass administration (MDA) of ivermectin (IVM) to endemic populations. In areas where loiasis, caused by the blood-borne filarial parasite Loa loa, is co-endemic, IVM MDA faced significant challenges because individuals harboring more than 30,000 L. loa microfilaria (mf)/mL of blood are at high risk of developing serious adverse events (SAEs) that can sometimes be fatal. An alternative strategy was developed for safe IVM distribution: the so-called 'Test and Not Treat' (TaNT), which identifies and excludes individuals with high mf density from IVM MDA and treats only those with minimal risk. TaNT requires a point-of-care diagnostic device to rapidly quantify L. loa parasites in field conditions. The NTDscope is a handheld device which captures bright-field videos of whole blood in capillaries. An onboard algorithm detects and counts the live microfilaria of L. loa via movement of red blood cells. This paper describes (i) a new detection algorithm; and (ii) a May 2025 field trial in Cameroon using the NTDscope with new algorithm (550 patients). Results for the TaNT use case (i.e. flagging cases with L. loa mf densities > 30,000 mf/mL): 94% to 97% sensitivity, and 94% to 97% specificity. The results indicate that the NTDscope and new algorithm provide a greater margin of safety than the predecessor device, and can potentially offer rapid, effective field detection of high mf infections to enable scalability of the TaNT strategy.

pathology↗

bioRxiv2026

Bead-based RNA Detection with Cas13a: Multi-scale Design and Validation

CRISPR-based assays offer sensitive and specific nucleic acid detection for a broad range of diagnostic and surveillance applications. While standard solution-based assays are simple, they typically lack the ability to detect multiple targets individually without the need for more complex instrumentation and multi-step workflows. Surface-based strategies offer greater potential for multiplexing since the identity of a target can be associated with a location on a slide or a specific bead, as in the case of DNA arrays or Luminex bead assays. However, the design principles for optimizing surface-based CRISPR assays have yet to be developed. Here we present a multi-scale analytical framework integrating nanoscale enzyme kinetics, microscale diffusion constraints, and macroscale assay parameters that we use to develop a bead-based Cas13a assay where the bead becomes fluorescent when the target RNA for that bead is detected. The assay, which we call SurfCas, uses surface-bound guide RNAs and reporter RNAs to detect one or more soluble target RNAs in a one-pot assay. We show that SurfCas has a sensitivity approaching bulk reactions, and we demonstrate multiplexed detection of target RNAs in a complex biological matrix. The design principles we establish for surface-based CRISPR assays offer guidance on how to further improve SurfCas sensitivity and scale multiplexed detection of target RNAs in a single reaction.

bioengineering↗

bioRxiv2026

LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs

CRISPR diagnostics have emerged as powerful tools for detecting infectious diseases, with the RNA endonuclease Cas13a enabling sensitive and specific, amplification-free RNA detection through collateral trans-cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun", would address these challenges and expand assay design space. Here, we introduce Light-Uncaged Cas13a (LUCas), a light controllable system that directly gates Cas13a using a photocleavable interfering guide RNA (pc-igRNA) that suppresses trans-cleavage activity even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an approximately 100-fold suppression of trans-cleavage activity prior to photo-activation. Importantly, LUCas also suppresses target-independent background activity, enabling a predictive, background-limited determination of assay sensitivity. Using measured kinetic parameters, we predict and experimentally validate the limit-of-detection of the LUCas system. Finally, we demonstrate a multiplexed detection strategy termed "temporal barcoding," which enables quantitative detection of viral co-infections in a single bulk reaction. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.

bioengineering↗

bioRxiv2026

From biting to engulfment: curvature-actin coupling controls phagocytosis of soft, deformable targets

Phagocytosis is a core innate immune process that clears targets spanning a wide range of mechanical properties, yet the role of target mechanics in recognition and engulfment remains unclear. Here, we combine theoretical modeling and experiments to reveal how target stiffness governs distinct modes of phagocyte-target interaction. We develop a membrane-based simulation framework in which both the engulfing cell and its target are deformable and undergo large shape changes, while actin-driven protrusions are regulated by curvature-sensitive membrane complexes. The model predicts three mechanical regimes with increasing target stiffness: (i) biting (trogocytosis), where part of the target is extracted; (ii) pushing, where the target is displaced rather than engulfed; and (iii) complete engulfment. We validate these predictions in epithelial clearance of apoptotic targets in vivo and macrophage engulfment of Giant Unilamellar Vesicles (GUVs) and lymphoma cells. Together, our results identify target mechanics as a key regulator of clearance and cell-cell interactions. Significance statementPhagocytosis is essential for immune defence, yet the physical principles governing engulfment of deformable targets remain poorly understood. Most theoretical models assume rigid particles, appropriate for phagocytosis of bacteria or fungi. When phagocytes engage dying cells or antibody-opsonised cancer cells, these targets undergo substantial shape changes during phagocytosis. We develop a theoretical model to simulate cell-cell interactions, enabling a mechanistic exploration of phagocytosis of soft targets. We utilize a model in which cytoskeletal protrusive activity is guided by curvature-sensitive membrane complexes, and show that target membrane rigidity dictates whether targets are fully engulfed, pushed away, or partially bitten. These mechanically driven dynamic regimes are validated experimentally using artificial elastic beads, GUVs, and lymphoma cells, both in vivo and in vitro.

biophysics↗

bioRxiv2026

Overcoming steric inhibition of antibody-dependent phagocytosiswith tall adhesions

Macrophages recognize and phagocytose opsonized target cells, including those coated with IgG antibodies. This process relies on binding of IgG to Fc{gamma} receptors (Fc{gamma}R) expressed on the macrophage surface, resulting in formation of a phagocytic synapse. Since the surface of both macrophages and target cells are densely packed with macromolecules of diverse sizes, most of which are not directly involved in phagocytic signaling, it is possible for tall bystander proteins to sterically interfere with Fc{gamma}R engagement. Here, we use cell-like target particles to show that bystander proteins can inhibit phagocytosis by blocking synapse formation. We then demonstrate that adding a tall binding protein to the target particle can overcome inhibition by the crowded environment and substantially recover phagocytosis, a process we call kinetic enhancement. Using a cell-free system of giant unilamellar vesicles and synthetic binders, we demonstrate that kinetic enhancement is a tunable feature of interface formation that can determine whether short binders engage, and we present theory and computer simulations to explain the nonmonotonic dependence of phagocytosis on tall binding protein surface density. These findings point to a strategy for overcoming surface crowding on phagocytic targets by re-engineering transition states with tall adhesion proteins, one that could be used to promote short receptor binding at other cell-cell junctions. Significance StatementMacrophages contribute to our immune defenses by phagocytosing pathogens and diseased cells. To accomplish this, they must first establish close contacts between receptors on their membranes and antibodies or other ligands decorating target cells. However, macrophage binding to the target can be disrupted by the presence of tall neighboring proteins and glycans-- bystander molecules--that sterically prevent the two surfaces from coming into close contact. Counterintuitively, this inhibition can be overcome by the addition of even taller binding proteins between the macrophage and target cell, albeit at low concentrations. Using live cell and in vitro experiments, theory, and computer simulations, we show that tall binders can promote close contact that enables phagocytosis, even in the presence of bystander proteins that would normally block close contact.

biophysics↗

bioRxiv2025

Actin conformation dynamics precede force generation in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) are cells of the adaptive immune system that are able to recognise and kill cancerous or virally infected target cells. The biochemical basis of CTL-mediated killing has been examined in great detail. Recent work has underscored the importance of physical forces in T cell function too, but how CTLs sense and exert force during migration and killing is not fully understood. Here, by expressing actin conformation probes based on the CH domain of utrophin, we directly visualize regions of altered F-actin conformation in primary CTLs during migration and killing. By combining these probes with traction force microscopy, we correlate external force with the internal cytoskeleton. We show that actin conformation is regulated upstream of force production, and that force exertion at the cytotoxic immune synapse temporally follows actin conformation dynamics. Our work offers novel insight into the relationship between cellular force production and F-actin conformation in important primary immune cells.

cell biology↗

bioRxiv2024

SurFlex Microscopy: Measuring Flexibility of Surface-Tethered Biomolecules

The flexibility of tethered molecules, such as those bound to biological membranes, is an important property that can influence molecular height, mobility, and accessibility. However, quantifying the flexibility of surface-tethered biomolecules in aqueous environments has been difficult due to a lack of experimental tools. Here we introduce SurFlex microscopy, a method based on fluorescence anisotropy that exploits the relationship between the conformational dynamics of a tethered molecule and the rotational diffusion of an attached fluorophore to extract information about molecular flexibility. By analyzing the polarization state of photons emitted after polarized excitation, we quantify apparent molecular flexibilities that include effects of tethering, self-interactions and buffer conditions. We first demonstrate the capabilities of SurFlex microscopy by measuring the flexibility of bilayer-tethered single-stranded DNA (ssDNA) of different lengths and nucleotide sequences. We find that sequence significantly impacts ssDNA flexibility, consistent with theoretical estimates, with weak intramolecular interactions in random sequences leading to higher apparent stiffness. Interestingly, we show that a pathological DNA sequence linked to Huntingtons disease exhibits unusual flexibility despite intramolecular interactions. We next extend SurFlex microscopy to live cells by measuring surface glycoprotein flexibility on red blood cells using fluorescent lectins. We show that trypsinization decreases glycan fluctuations, demonstrating that modifications to the cell surface can alter the flexibility of remaining surface molecules. SurFlex microscopy provides a new tool for quantifying molecular flexibility that can be used to study the role of tethered surface molecules in fundamental biological processes. Significance statementBiomolecules immobilized on one end play crucial roles in diverse cellular processes, from cell-cell signaling through surface receptors to the formation of DNA secondary structures. However, measuring biomolecular flexibility on surfaces has remained challenging. Here we present SurFlex microscopy, a technique that uses fluorescence anisotropy to quantify the flexibility of surface-anchored molecules. By analyzing the rotational dynamics of fluorophores attached to the ends of fluctuating biomolecules, SurFlex microscopy can be used to quantify persistence length. We demonstrate its capabilities by measuring sequence-dependent flexibility of DNA and crowding-dependent changes in glycan flexibility on native cell surfaces. This method opens new avenues for understanding how biomolecular flexibility influences key biological processes, such as those at cell surfaces during cell-cell contact formation and subsequent signaling.

biophysics↗

bioRxiv2024

Cell surface crowding is a tunable biophysical barrier to cell-cell fusion

Cell-cell fusion is fundamental to developmental processes such as muscle formation, as well as to viral infections that cause pathological syncytia. An essential step in fusion is close membrane apposition, but cell membranes are crowded with proteins, glycoproteins, and glycolipids, all of which must be cleared before a fusion pore can be nucleated. Here, we find that cell surface crowding drastically reduces fusogenicity in multiple systems, independent of the method for driving fusion. We estimate that cell surface crowding presents an energetic barrier to membrane apposition on the scale of [~]100kBT, greater than that of bare membrane fusion. We show that increasing cell surface crowding reduces fusion efficiency of PEG-mediated and fusogen-mediated cell-cell fusion, as well as synthetic membranes under force. Interestingly, we find that differentiating myoblasts naturally decrease cell surface crowding prior to fusion. Cell surface crowding presents an underappreciated biophysical barrier that may be tuned developmentally and could be targeted externally to control tissue-specific cell-cell fusion.

biophysics↗

bioRxiv2024

Target cell tension regulates macrophage trogocytosis

Macrophages are known to engulf small membrane fragments, or trogocytose, target cells and pathogens, rather than fully phagocytose them. However, little is known about what causes macrophages to choose trogocytosis versus phagocytosis. Here, we report that cortical tension of target cells is a key regulator of macrophage trogocytosis. At low tension, macrophages will preferentially trogocytose antibody-opsonized cells, while at high tension they tend towards phagocytosis. Using model vesicles, we demonstrate that macrophages will rapidly switch from trogocytosis to phagocytosis when membrane tension is increased. Stiffening the cortex of target cells also biases macrophages to phagocytose them, a trend that can be countered by increasing antibody surface density and is captured in a mechanical model of trogocytosis. This work suggests that a distinct molecular pathway for trogocytosis is not required to explain differences in trogocytosis among target cell types and points to a mechanism for target cells to modulate trogocytosis.

biophysics↗

bioRxiv2023

Application of an NMR/crystallography fragment screening platform for the assessment and rapid discovery of new HIV-CA binding fragments

Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pK values of 5.3 and 5.4 respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/569544v2_ufig1.gif" ALT="Figure 1"> View larger version (6K): org.highwire.dtl.DTLVardef@1c7043corg.highwire.dtl.DTLVardef@e73044org.highwire.dtl.DTLVardef@11c1d9org.highwire.dtl.DTLVardef@1cd99e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

The Quest to Identify USP8 Inhibitors for Parkinsons Disease, a PAINful Experience.

Pan Assay INterference compoundS (PAINS) are known to be a source of false positives in High Throughput Screening (HTS) campaigns. This has become a major problem in medicinal chemistry, often resulting in undesirable project outcomes and increased overall cost. Our recent campaign to identify inhibitors of USP8 that could be used in the treatment of Parkinsons disease identified several PAINS that worked via a variety of mechanisms. Herein, we discuss the process developed to identify not only the PAINS but also confirming the interference mechanism causing their activity. We found in this project that our USP8 assay was susceptible to multiple modes of interference, making it difficult to identify genuine hit molecules. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/556294v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@b917a6org.highwire.dtl.DTLVardef@1bfac47org.highwire.dtl.DTLVardef@c78e1dorg.highwire.dtl.DTLVardef@1434fdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Engineered molecular sensors of cell surface crowding

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2-20 fold in live cells compared to a bare membrane surface, resulting in a cell surface osmotic pressure opposing binding of 1 - 4 kPa. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface osmotic pressure may be combined with functional assays to enable further biophysical dissection of the cell surfaceome. Significance StatementCells interact with each other and the extracellular environment through a crowded assembly of polymers on their plasma membranes. The high density of these surface polymers can generate physical crowding that impacts cell function. However, tools to quantify the extent and effect of surface crowding on live cell membranes are lacking. In this work, we design macromolecular sensors that act as direct reporters of cell surface crowding. We combine experiments on reconstituted and live cell surfaces with molecular dynamics simulations to provide a mechanistic understanding of how cell surface crowding reduces binding of soluble molecules, and we show that crowding varies significantly with cell type and is affected by oncogene expression.

biophysics↗

bioRxiv2022

CellScape: Protein structure visualization with vector graphics cartoons

MotivationIllustrative renderings of proteins are useful aids for scientific communication and education. Nevertheless, few software packages exist to automate the generation of these visualizations. ResultsWe introduce CellScape, a tool designed to generate 2D molecular cartoons from atomic coordinates and combine them into larger cellular scenes. These illustrations can outline protein regions in different levels of detail. Unlike most molecular visualization tools which use raster image formats, these illustrations are represented as vector graphics, making them easily editable and composable with other graphics. Availability and ImplementationCellScape is implemented in Python 3 and freely available at https://github.com/jordisr/cellscape. It can be run as a command-line tool or interactively in a Jupyter notebook. Contactjordisr@berkeley.edu

bioinformatics↗

bioRxiv2021

The molecular mechanism of load adaptation by branched actin networks

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Unexpectedly, force slows the rate of filament nucleation by promoting the interaction of nucleation promoting factors with actin filament ends, limiting branch formation. This inhibition is countered by an even larger force-induced drop in the rate of filament capping, resulting in a shift in the balance between nucleation and capping that increases network density. Remarkably, the force dependence of capping is identical to that of filament elongation because they require the same size gap to appear between the filament and load for insertion. These results provide direct evidence that Brownian Ratchets generate force and govern the load adaptation of branched actin networks.

biophysics↗

bioRxiv2020

Variable optical properties of light-harvesting complex II revisited

Understanding photosynthetic light harvesting requires knowledge of the molecular mechanisms that dissipate excess energy in thylakoids. However, it remains unclear how the physical environment of light-harvesting complex II (LHCII) influences the process of chlorophyll de-excitation. Here, we demonstrate that protein-protein interactions between LHCIIs affect the optical properties of LHCII and thus influence the total energy budget. Aggregation of LHCII in the dark altered its absorption properties, independent of the amount of prior light exposure. We also revisited the triplet excited state involved in light-induced fluorescence quenching and found another relaxation pathway involving emission in the green region, which might be related to triplet excited energy transfer to neighboring carotenoids and annihilation processes that result in photoluminescence. LHCII- containing liposomes with different protein densities exhibited altered fluorescence and scattering properties. Our results suggest that macromolecular reorganization affects overall optical properties, which need to be addressed to compare the level of energy dissipation.

plant biology↗