Ganglioside-Selective Mechanosensitive Fluorescent Membrane Probes

The development of fluorescent probes to image forces in cells is an important challenge in chemistry and biology. Planarizable push-pull probes have been introduced recently for this purpose. To provide most valuable information on forces in complex systems, these mechanosensitive flipper' probes will have to be localized by molecular recognition of targets of interest. Here we report fluorescent flippers that selectively recognize gangliosides on the surface of lipid bilayer membranes by formation of dynamic covalent boronate esters. The original flipper probes were equipped with 2-fluorophenyl boronic acids and benzoboroxoles using consecutive triazole and oxime ligation. Evaluation was done in large unilamellar vesicles composed of EYPC/SM/CL/GM 40:40-x:20:x to obtain mixed membranes with separate liquid-disordered (L-d) and ganglioside (GM) containing liquid-ordered (L-o) domains. With increasing GM concentration, fluorescence intensities increased and excitation maximum shifted to the red. Deconvolution of the spectra confirmed that these changes originate from a migration of the flipper probes from L-d to L-o domains upon binding to the gangliosides and thus the planarization in the more ordered environment. Control mechanophores without boronic acids failed to show the same response, and fructose partially inhibited the ganglioside sensitivity. These results demonstrate that it is possible to selectively accumulate mechanosensitive flipper probes in L-o domains and, more generally, that probe localization in complex membranes is possible and matters.