Periodic Reporting for period 1 - ReMembrane (How do tetraspanin proteins organize, shape, and remodel biological membranes?)
Periodo di rendicontazione: 2023-04-01 al 2025-09-30
Our bodies rely on the precise shaping and remodeling of cell membranes to perform fundamental biological processes such as fertilization and cell-cell communication. These shape changes are actively regulated by membrane-associated proteins, yet the physical principles underlying their function remain poorly understood. Uncovering these mechanisms is not only essential for advancing our basic understanding of biology, but also for enabling targeted therapeutic interventions in processes ranging from reproduction to viral infection.
Among the most intriguing membrane-associated proteins are tetraspanins (TSPANs)—a family of four-pass transmembrane proteins found in nearly all cell types. TSPANs have been implicated in a wide range of physiological and pathological processes, including cell-cell fusion, viral entry, immune signaling, and the formation of newly discovered organelles called migrasomes, which mediate intercellular communication. Despite their biological importance, the ways in which TSPANs exert their function—particularly how their localization and activity are influenced by physical membrane properties like curvature and tension—are still largely unknown.
This project aims to fill this critical gap by applying cutting-edge biophysical tools to directly test how TSPANs respond to and modulate membrane mechanics. Specifically, we will combine optical tweezers, micropipette aspiration, confocal microscopy, and atomic force microscopy (AFM) to develop quantitative, high-resolution assays that reconstitute key TSPAN-mediated events from the bottom up. This approach will allow us to isolate and characterize the individual roles of membrane tension and curvature in TSPAN function.
Our objectives are threefold:
1. To reveal the effect of membrane tension on the formation of TSPN domains, using a novel AFM-based assay.
2. To explain the mechanism by which TSPNs drive migrasome formation, a new mode of cell-cell communication.
3. To characterize how TSPNs mediate membrane fusion, with applications to both fertilization and viral entry.
By tackling these aims, the project will generate transformative insights into how membrane mechanics shape biological function, revealing new paradigms for membrane remodeling in health and disease. The findings have the potential to impact several fields simultaneously: from reproductive biology (e.g. through the development of novel contraceptives and infertility treatments), to virology (e.g. new targets for antiviral therapies), to membrane biophysics. Furthermore, the fundamental principles uncovered here may apply to a broad range of processes in which tetraspanins are implicated, including cancer metastasis and immune cell activation.
Among the most intriguing membrane-associated proteins are tetraspanins (TSPANs)—a family of four-pass transmembrane proteins found in nearly all cell types. TSPANs have been implicated in a wide range of physiological and pathological processes, including cell-cell fusion, viral entry, immune signaling, and the formation of newly discovered organelles called migrasomes, which mediate intercellular communication. Despite their biological importance, the ways in which TSPANs exert their function—particularly how their localization and activity are influenced by physical membrane properties like curvature and tension—are still largely unknown.
This project aims to fill this critical gap by applying cutting-edge biophysical tools to directly test how TSPANs respond to and modulate membrane mechanics. Specifically, we will combine optical tweezers, micropipette aspiration, confocal microscopy, and atomic force microscopy (AFM) to develop quantitative, high-resolution assays that reconstitute key TSPAN-mediated events from the bottom up. This approach will allow us to isolate and characterize the individual roles of membrane tension and curvature in TSPAN function.
Our objectives are threefold:
1. To reveal the effect of membrane tension on the formation of TSPN domains, using a novel AFM-based assay.
2. To explain the mechanism by which TSPNs drive migrasome formation, a new mode of cell-cell communication.
3. To characterize how TSPNs mediate membrane fusion, with applications to both fertilization and viral entry.
By tackling these aims, the project will generate transformative insights into how membrane mechanics shape biological function, revealing new paradigms for membrane remodeling in health and disease. The findings have the potential to impact several fields simultaneously: from reproductive biology (e.g. through the development of novel contraceptives and infertility treatments), to virology (e.g. new targets for antiviral therapies), to membrane biophysics. Furthermore, the fundamental principles uncovered here may apply to a broad range of processes in which tetraspanins are implicated, including cancer metastasis and immune cell activation.
This project investigates the mechanisms by which tetraspanin proteins organize and remodel biological membranes, processes essential to many cellular functions. A central focus is on how membrane tension and curvature influence tetraspanin behavior and domain formation. To address this, we have engineered a novel motorized device capable of applying controlled, equibiaxial stretch to supported lipid membranes. This system allows us to quantify how membrane tension alters the spatial organization and mixing of membrane components, with preliminary data revealing tension-induced modulation of phase-separated membrane domains. Complementing this, we have developed innovative assays to probe the role of membrane curvature in directing tetraspanin localization, particularly during the formation of migrasomes—newly discovered organelles involved in intercellular communication. Our experimental design, supported by theoretical modeling, has elucidated how increased membrane tension can initiate migrasome formation at membrane junctions, explaining preferential formation of migrasomes at retraction fiber branching sites.
In parallel, we have advanced our understanding of membrane fusion, a critical event in processes such as fertilization and viral entry, by creating a tension-controlled fusion assay. This method integrates micropipette aspiration with fluorescence microscopy to monitor fusion intermediates under varying tension conditions. We were able to demonstrate that membrane tension increases the energy barrier for hemifusion, thereby inhibiting lipid mixing. Our technical developments, including a method for equibiaxial stretching of supported membranes and novel assays to probe curvature-enhanced protein interactions, constitute essential tools to study how tetraspanin proteins respond to membrane tension and curvature. Collectively, these achievements provide a solid experimental foundation to reveal the mechanisms by which tetraspanins mediate membrane remodeling and function in key physiological processes.
In parallel, we have advanced our understanding of membrane fusion, a critical event in processes such as fertilization and viral entry, by creating a tension-controlled fusion assay. This method integrates micropipette aspiration with fluorescence microscopy to monitor fusion intermediates under varying tension conditions. We were able to demonstrate that membrane tension increases the energy barrier for hemifusion, thereby inhibiting lipid mixing. Our technical developments, including a method for equibiaxial stretching of supported membranes and novel assays to probe curvature-enhanced protein interactions, constitute essential tools to study how tetraspanin proteins respond to membrane tension and curvature. Collectively, these achievements provide a solid experimental foundation to reveal the mechanisms by which tetraspanins mediate membrane remodeling and function in key physiological processes.
Our work significantly advances the state of the art in understanding how tetraspanin proteins sense and respond to membrane curvature and tension. Curvature describes the shape of a membrane, where thin tubes are characterized by high curvature, while tension reflects membrane stretching - both are key properties that influence how proteins organize and function on the membrane surface. We developed several novel experimental platforms to probe these effects with high precision. Our curvature-sensitive protein sorting assay allowed us to dissect the specific domains within TSPAN4 responsible for membrane curvature sensing and lateral protein interactions. These findings provide critical insights into how TSPANs form functional assemblies on membranes—an essential step toward understanding their role in processes such as migrasome formation. Our biomimetic assay for tension-induced bulging at tubular junctions sheds light on a previously uncharacterized early step of migrasome biogenesis, demonstrating how a sudden increase in membrane tension can trigger bulge formation preferentially at junctions of retraction fibers. This model, validated by theoretical predictions and live-cell imaging, provides a new framework for identifying molecular players and physical cues involved in the initiation of migrasome formation.
In parallel, we introduced a method for studying the effect of membrane tension on domain formation in supported bilayers, using a motorized device that enables equibiaxial stretching of elastic substrates. This system is currently being applied to TSPAN-containing membranes. We also advanced the field of membrane fusion by building a tension-controlled fusion assay using micropipette-aspirated vesicles and membrane-coated beads. Our results demonstrated that increased tension raises the energy barrier for lipid mixing. Moreover, by applying this approach to asymmetric membranes, we revealed that membrane asymmetry can introduce a preferred direction for fusion—an unanticipated finding with implications for directional fusion processes in biological membranes. Finally, our study of tension propagation in cellular membranes uncovered that intracellular pressure and membrane “crumpling” control the rate at which tension changes spread across the membrane. These results advance our understanding of the physical principles governing membrane organization and remodeling and provide the foundation for essential mechanistic insight into how membrane tension and curvature regulate tetraspanin-mediated processes such as migrasome biogenesis and membrane fusion.
In parallel, we introduced a method for studying the effect of membrane tension on domain formation in supported bilayers, using a motorized device that enables equibiaxial stretching of elastic substrates. This system is currently being applied to TSPAN-containing membranes. We also advanced the field of membrane fusion by building a tension-controlled fusion assay using micropipette-aspirated vesicles and membrane-coated beads. Our results demonstrated that increased tension raises the energy barrier for lipid mixing. Moreover, by applying this approach to asymmetric membranes, we revealed that membrane asymmetry can introduce a preferred direction for fusion—an unanticipated finding with implications for directional fusion processes in biological membranes. Finally, our study of tension propagation in cellular membranes uncovered that intracellular pressure and membrane “crumpling” control the rate at which tension changes spread across the membrane. These results advance our understanding of the physical principles governing membrane organization and remodeling and provide the foundation for essential mechanistic insight into how membrane tension and curvature regulate tetraspanin-mediated processes such as migrasome biogenesis and membrane fusion.