Biology subjects

Cornell, C. E.

Publications and source records attributed to Cornell, C. E..

8 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

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↗

bioRxiv2025

Micron-scale, liquid-liquid phase separation in ternary lipid membranes containing DPPE

Micron-scale, liquid-liquid phase separation occurs in membranes of living cells, with physiological consequences. To discover which lipids might support phase separation in cell membranes and how lipids might partition between phases, miscibility phase diagrams have been mapped for model membranes. Typically, model membranes are composed of ternary mixtures of a lipid with a high melting temperature, a lipid with a low melting temperature, and cholesterol. Phospholipids in ternary mixtures are chosen primarily to favor stable membranes (phosphatidylcholines and sphingomyelins) or add charge (phosphatidylglycerols and phosphatidylserines). A major class of phospholipids missing from experimental ternary diagrams has been the phosphatidylethanolamines (PEs). PE-lipids constitute up to 20 mol% of common biological membranes, where they influence protein function and facilitate membrane fusion. These biological effects are often attributed to PEs smaller headgroup, which leads to higher monolayer spontaneous curvatures and higher melting temperatures. Taken alone, the higher melting points of saturated PE-lipids imply that liquid-liquid phase separation should persist to higher temperatures in membranes containing PE-lipids. Here, we tested that hypothesis by substituting a saturated PE-lipid (DPPE) for its corresponding PC-lipid (DPPC) in two well-studied ternary membranes (DOPC/DPPC/cholesterol and DiphyPC/DPPC/cholesterol). We used fluorescence microscopy to map full ternary phase diagrams for giant vesicles over a range of temperatures. Surprisingly, we found no micron-scale, liquid-liquid phase separation in vesicles of the first mixture (DOPC/DPPE/cholesterol), and only a small region of liquid-liquid phase separation in the second mixture (DiphyPC/DPPE/cholesterol). Instead, coexisting solid and liquid phases were widespread, with the solid phase enriched in DPPE. An unusual feature of these ternary membranes is that solid and liquid-ordered phases can be distinguished by fluorescence microscopy, so tie-line directions can be estimated throughout the phase diagram, and transition temperatures to the 3-phase region (containing a liquid-disordered phase, a liquid-ordered phase, and a solid phase) can be accurately measured. SIGNIFICANCE STATEMENTUnder physiological conditions, yeast vacuole membranes phase separate into liquid phases. The resulting domains are microns in size, and they are important for the cells function. Yeast membranes contain a significant fraction of lipids with phosphatidylethanolamine headgroups. It was unknown whether these lipids enhanced liquid-liquid phase separation or hindered it. Here, we produced model membranes containing a saturated phosphatidylethanolamine lipid, we mapped miscibility phase diagrams over broad temperature ranges, and we compared our results to existing diagrams for membranes of other lipid types. We were surprised to find that the new lipid suppressed liquid-liquid phase separation compared to a lipid with a larger, phosphatidylcholine headgroup. Previous simulations incorporated phosphatidylethanolamine lipids, and our results provide an experimental basis for future simulations.

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

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development

Cells tend to divide along the direction in which they are longest, as famously stated by Oscar Hertwig in 1884 in his long axis rule1,2. The orientation of the mitotic spindle determines the cell division axis3, and Hertwigs long axis rule is usually ensured by forces stemming from microtubules4. Pulling on the spindle from the cell cortex can give rise to unstable behaviors5,6, and we here set out to understand how Hertwigs long axis rule is realized in early embryonic divisions where cortical pulling forces are prevalent. We focus on early C. elegans development, where we compressed embryos to reveal that cortical pulling forces favor an alignment of the spindle with the cells short axis. Strikingly, we find that this misalignment is corrected by an actomyosin-based mechanism that rotates the entire cell, including the mitotic spindle. We uncover that myosin-driven contractility in the cytokinetic ring generates inward forces that align it with the short axis, and thereby the spindle with the long axis. A theoretical model together with experiments using slightly compressed mouse zygotes suggest that a constricting cytokinetic ring can ensure Hertwigs long axis rule in cells that are free to rotate inside a confining structure, thereby generalizing the underlying principle.

developmental biology↗

bioRxiv2021

Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cells normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Recent studies suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a key role in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether yeast stringently regulate the phase transition and how they respond to new physical conditions. Here, we measure transition temperatures - an increase of roughly 15{degrees}C returns vacuole membranes to a state that appears uniform across a range of growth temperatures. We find that broad populations of yeast grown at a single temperature regulate the transition to occur over a surprisingly narrow temperature range. Moreover, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. Specifically, we test how levels of ergosterol, the main sterol in yeast, induce or eliminate membrane domains. We isolate vacuoles from yeast during their rapid stage of growth, when their membranes do not natively exhibit domains. We find that membrane domains materialize when ergosterol is depleted, contradicting the assumption that increases in ergosterol cause membrane phase separation in vivo, and in agreement with prior studies that use artificial and cell-derived membranes. SIGNIFICANCE STATEMENTPhase separation in membranes creates domains enriched in specific components. To date, the best example of micron-scale phase separation in the membrane of an unperturbed, living cell occurs in a yeast organelle called the vacuole. Recent studies indicate that the phases are functionally important, enabling yeast survival during periods of cellular stress. We have discovered that yeast regulate this phase transition; the temperature at which membrane components mix into a single phase is ~15{degrees}C above the growth temperature. To maintain this offset, yeast may tune the level of ergosterol (a molecule that is structurally similar to cholesterol) in their membranes. We find that reducing sterol levels in vacuole membranes causes them to phase separate, in contrast to previous assumptions.

biophysics↗

bioRxiv2020

Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography

Images of micron-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been lacking, leading to an inability to answer key questions. For example, researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two new methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under native solution conditions. The first method is optimized for probe-free applications: domains are directly distinguished from the surrounding membrane by their thickness. This technique measures area fractions of domains with quantitative accuracy, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to the first direct and quantitative imaging of submicron membrane domains under native conditions. SIGNIFICANCE STATEMENTFluorescence micrographs that capture the sizes, shapes, and distributions of liquid domains in model membranes have provided high standards of evidence to prove (and disprove) theories of how micron-scale domains form and grow. Corresponding theories about smaller domains have remained untested, partly because experimental methods of identifying submicron domains in vesicles under native solvent conditions have not been available. Here we introduce two such methods. Both leverage cryo-electron tomography to observe membrane features far smaller than the diffraction limit of light. The first method is probe-free and identifies differences in thicknesses between liquid domains and their surrounding membranes. The second method identifies membrane regions labeled by an electron-dense, fluorescent protein, which enables direct comparison of fluorescence micrographs with cryo-electron tomograms.

biophysics↗