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HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

ABSTRACT: HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push-pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push-pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.

SUBMITTER: Garcia-Calvo J

PROVIDER: S-EPMC8849034 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

García-Calvo José J López-Andarias Javier J Maillard Jimmy J Mercier Vincent V Roffay Chloé C Roux Aurélien A Fürstenberg Alexandre A Sakai Naomi N Matile Stefan S

Chemical science 20220203 7

HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push-pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydrati ...[more]

PMID: 35308858

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Project description:Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration, and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T- or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction. Upon ligation, these receptor-ligand bonds transmit piconewton (pN) mechanical forces that are generated, in part, by the cytoskeleton. Importantly, these forces expose cryptic sites within mechanosensitive proteins and modulate the binding kinetics (on/off rate) of receptor-ligand complexes to further fine-tune mechanotransduction and the corresponding cell behavior. Over the past three decades, two categories of methods have been developed to measure cell receptor forces. The first class is traction force microscopy (TFM) and micropost array detectors (mPADs). In these methods, cells are cultured on elastic polymers or microstructures that deform under mechanical forces. The second category of techniques is single molecule force spectroscopy (SMFS) including atomic force microscopy (AFM), optical or magnetic tweezers, and biomembrane force probe (BFP). In SMFS, the experimenter applies external forces to probe the mechanics of individual cells or single receptor-ligand complexes, serially, one bond at a time. Although these techniques are powerful, the limited throughput of SMFS and the nN force sensitivity of TFM have hindered further elucidation of the molecular mechanisms of mechanotransduction. In this Account, we introduce the recent advent of molecular tension fluorescence microscopy (MTFM) as an emerging tool for molecular imaging of receptor mechanics in living cells. MTFM probes are composed of an extendable linker, such as polymer, oligonucleotide, or protein, and flanked by a fluorophore and quencher. By measuring the fluorescence emission of immobilized MTFM probes, one can infer the extension of the linker and the externally applied force. Thus, MTFM combines aspects of TFM and SMFS to optically report receptor forces across the entire cell surface with pN sensitivity. Specifically, we provide an in-depth review of MTFM probe design, which includes the extendable "spring", spectroscopic ruler, surface immobilization chemistry, and ligand design strategies. We also demonstrate the strengths and weaknesses of different versions of MTFM probes by discussing case studies involving the pN forces involved in epidermal growth factor receptor, integrin, and T-cell receptor signaling pathways. Lastly, we present a brief future outlook, primarily from a chemists' perspective, on the challenges and opportunities for the design of next generation MTFM probes.

Dataset Information

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

Publications

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

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