膜張力およびドメイン構造報告のための蛍光ペプチド

Cell mechanics play significant roles in all aspects of cell function. While many types of fluorescent linear force sensors inserted in cellular fibrillar elements have been developed, few tools are available to track two-dimensional tension in cell membranes. Here, we present a novel principle for tension detection using a fluorescent probe based on the scaffold of the GsMTx4 peptide from Grammostola venom. Previously, we have shown that amphipathic GsMTx4 binds to lipids and inhibits mechanosensitive channels by inserting more deeply into the membrane at tensions near activation thresholds, thereby acting as a buffer clamping lateral pressure in the bilayer. We leverage this property of GsMTx4 to redistribute between the 'shallow' and 'deep' immersion states, thereby designing probes with a fluorescent moiety that increases quantum yield in nonpolar environments. GsMTx4 analogs carrying fluorescent groups at the two positions increase fluorescence intensity in osmotically shocked liposomes and aspirated giant vesicles in a near-linear fashion in response to physiological bilayer tensions. The responses show dependence on membrane composition, particularly lipid charge and the presence of lipid-ordering components, such as sphingomyelin and cholesterol. Langmuir compression isotherms recorded in the presence of NBD analogs indicated initial incorporation into the monolayer, followed by sharp expulsion at the monolayer-bilayer equivalence pressure, with correlated changes in monolayer compressibility and fluorescence, illustrating the basic principle of probe action. The probes show promise for monitoring tension in biological membranes at low, non-inhibitory concentrations. Experiments with native cell-derived membrane vesicles reveal heterogeneous baseline staining and tension responses, underscoring the probes' selectivity for distinct membrane domains.

Cell mechanics are crucial for all cell functions, including division, survival, migration, and differentiation. Although many versions of fluorescent linear force sensors have been developed for cytoskeletal and ECM elements, few tools exist to monitor two-dimensional tension in cell membranes. Many cells are motile, actively deforming their membrane, supported and driven by the underlying cytoskeleton. There is a two-order-of-magnitude discrepancy between membrane tension estimates from the tether formation technique and the tensions that activate common mechanosensitive channels in most cells. This discrepancy highlights the need for non-invasive membrane probes that can independently measure membrane tension, especially since it can be highly localized and dynamic. Here, we introduce such probes and a new principle for tension measurement.