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

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.

HydroFlippers respond to membrane compression and hydration in the same fluorescence lifetime imaging microscopy histogram: the responses do not correlate.

The detection and study of membrane mechanics in living cells is a topic of current concern.^1–14 To enable this research, appropriate chemistry tools, that is small-molecule fluorescent probes that allow imaging of membrane tension, are needed.¹⁵ With the direct imaging of physical forces being intrinsically impossible, design strategies toward such probes have to focus on the suprastructural changes caused by changes in membrane tension.¹⁵ These suprastructural changes are divers, often interconnected, and vary with the composition of the membrane.^15–25 Beyond the fundamental lipid compression and decompression, they include changes in membrane curvature, from rippling, buckling and budding to tubules extending from the membrane and excess lipid being ejected. Of similar importance are changes in membrane organization, particularly tension-induced phase separation and mixing, i.e. assembly and disassembly of microdomains. Consequences of these suprastructural changes include microdomain strengthening and softening and changes in membrane hydration and viscosity.^16–25The currently most developed fluorescent flipper probes have been introduced^26,27 to image membrane tension by responding to a combination of mechanical compression and microdomain assembly in equilibrium in the ground state.¹⁵ Extensive studies, including computational simulations,²⁸ have shown that flipper probes align non-invasively along the lipid tails of one leaflet and report changes in membrane order and tension as changes in fluorescent lifetimes and shifts of excitation maxima.¹⁵ Among other candidates, solvatochromic probes respond off-equilibrium in the excited state to changes in membrane hydration and have very recently been considered for the imaging of membrane tension in living cells.^29–36 So far not considered to image tension, ESIPT probes also report off equilibrium in the excited state on membrane hydration, but for different reasons.^37,38 Mechanosensitive molecular rotors respond off equilibrium in the excited state to changes in microviscosity.^{17,30,32,39–53} The same principle holds for the planarization of bent, papillon or flapping fluorophores.^54–57 The response of all possible probes to tension can further include less desired changes in positioning and partitioning between different domains, not to speak of more catastrophic probe aggregation, precipitation, disturbance of the surrounding membrane structure, and so on. Although the imaging of membrane tension is conceivable in principle with most of above approaches, the complex combination of parameters that has to be in place can thus far only be identified empirically, followed by much optimization.¹⁵The force-induced suprastructural changes are accompanied by the alteration in several unrelated physical properties of membranes. It is, for instance, well documented that membrane hydration increases with membrane disorder, from solid-ordered (S_o) to liquid-disordered (L_d) phases.^29,58 Increasing cholesterol content decreases membrane hydration in solid- and liquid-ordered membranes.⁵⁹ However, studies in model membranes also indicate that membrane hydration and membrane fluidity do not necessarily correlate.⁵⁹ The dissection of the individual parameters contributing to the response of fluorescent membrane tension probes would be important for probe design and understanding of their responses, but it remains a daunting challenge. In this study, we introduce fluorescent flipper probes that simultaneously report on mechanical membrane compression and membrane hydration at equilibrium in the ground state. Changes of both in response to changes in membrane tension and membrane composition are determined in various organelles in living cells.The dual hydration and membrane tension probes are referred to as HydroFlippers to highlight the newly added responsiveness to membrane hydration. The mechanosensing of lipid compression in bilayer membranes by flipper probes has been explored extensively.¹⁵ Fluorescent flippers²⁷ like 1 are designed as bioinspired⁶⁰ planarizable push–pull probes²⁶ (Fig. 1). They are constructed from two dithienothiophene fluorophores that are twisted out of co-planarity by repulsion of methyls and σ holes on sulfurs^61,62 next to the twistable bond. The push–pull system is constructed first from formal sulfide and sulfone redox bridges in the two twisted dithienothiophenes. These endocyclic donors and acceptors are supported by exocyclic ones, here a trifluoroketone acceptor and a triazole donor.⁶³ To assure stability, these endo- and exocyclic donors are turned off in the twisted ground state because of chalcogen bonding and repulsion, respectively.⁶²Open in a separate windowFig. 1The dual sensing cycle of HydroFlippers 1–5, made to target the indicated MOIs in living cells and responding to membrane compression by planarization and to membrane hydration by dynamic covalent ketone hydration. With indication of excitation maxima (ref. 63) and fluorescence lifetimes (this study).Mechanical planarization of the flipper probe establishes conjugation along the push–pull systems, electrons flow from endocyclic donors to acceptors, which turns on the exocyclic donors and acceptors to finalize the push–pull system.⁶² This elaborate, chalcogen-bonding cascade switch has been described elsewhere in detail, including high-level computational simulations.⁶² The planar high-energy conformer 1dp excels with red shifted excitation and increased quantum yield and lifetime compared to the twisted conformer 1dt because the less twisted Franck-Condon state favors emission through planar intramolecular charge transfer (PICT) over non-radiative decay through twisted ICT, or conical intersections.¹⁵Flipper probe 1 was considered for dual responsiveness to membrane tension and hydration because of the trifluoroketone acceptor.⁶³ Dynamic covalent hydration of 1dt yields hydrate 1ht.^64–76 Blue-shifted excitation and short lifetime of 1ht are not expected to improve much upon planarization because the hydrate is a poor acceptor and thus, the push–pull system in 1hp is weak. The dynamic covalent chemistry of the trifluoroketone acceptor has been characterized in detail in solution and in lipid bilayer membranes.⁶³To explore dual responsiveness to membrane tension in any membrane of interest (MOI) in living cells, HydroFlippers 2–5 were synthesized. While HydroFlipper 1 targets the plasma membrane (PM), HydroFlippers 2–4 were equipped with empirical targeting motifs.⁷⁷ HydroFlipper 5 terminates with a chloroalkane to react with the self-labeling HaloTag protein, which can be expressed in essentially any MOI.⁷⁸ Their substantial multistep synthesis was realized by adapting reported procedures (Schemes S1–S4^†).The MOIs labeling selectivity of HydroFlippers was determined in HeLa Kyoto (HK) cells by confocal laser scanning microscopy. Co-localization experiments of flippers 1–4 with the corresponding trackers gave Pearson correlation coefficients (PCCs) >0.80 for the targeting of mitochondria, lysosomes and the endoplasmic reticulum (ER, Fig. S4–S6^†). HydroFlipper 5 was first tested with stable HGM cells, which express both HaloTag and GFP on mitochondria (referred to as 5_M).^78,79 The well-established chloroalkane penetration assay demonstrated the efficient labeling of HaloTag protein by 5 as previously reported HaloFlippers (Fig. S3^†).⁷⁸ By transient transfection, HydroFlippers 5 were also directed to lysosomes (5_L), Golgi apparatus (GA, 5_G)⁸⁰ and peroxisomes (5_P) with HaloTag and GFP expressed on their surface.⁷⁸ PCCs >0.80 for co-localization of flipper and GFP emission confirmed that MOI labeling with genetically engineered cells was as efficient as with empirical trackers (Fig. S7–S11^†).Dual imaging of membrane compression and hydration was envisioned by analysis of fluorescence lifetime imaging microscopy (FLIM) images using a triexponential model (Fig. 2).⁸¹ FLIM images of ER HydroFlipper 4 in iso-osmotic HK cells were selected to illustrate the concept (Fig. 3a). Contrary to classical flipper probes, the fluorescence decay curve of the total FLIM image (Fig. 2a, grey) showed a poor fit to a biexponential model (Fig. 2a, cyan, b). Consistent with their expected dual sensing mode, a triexponential fit was excellent (Fig. 2a, dark blue, c). Lifetimes τ_1i = 4.3 ns (†) were obtained besides background. This three-component model was then applied to every pixel of FLIM images (Fig. 3c). The resulting reconvoluted FLIM histogram revealed three clearly separated populations for τ₁ (red), τ₂ (green), and background (τ₃, blue, Fig. 2d). Maxima of these three clear peaks were at the lifetimes estimated by triexponential fit of the global decay curve, thus demonstrating the validity of the methodology at necessarily small photon counts. Irreproducible fitting would give randomly scattered data without separated peaks.Open in a separate windowFig. 2(a) Fluorescence decay curve (grey, corresponding to the total image, not to a single pixel) with biexponential (cyan) and triexponential fit (dark blue). (b, c) Residual plots for bi- (b) and triexponential fit (c). (d) Histogram with the intensities associated with the τ₁ (red), τ₂ (green), and τ₃ (blue, background) components obtained by triexponential fit of the fluorescence decay curve of each pixel of the FLIM image, fit to Gaussian function (black solid curves).Open in a separate windowFig. 3FLIM images of HK cells labelled with ER flipper 4 before (a, c) and after (b, d) hyper-osmotic shock, showing average lifetimes τ_av (a, b) and τ₁ (c, d) from triexponential reconvolution; scale bars = 10 μm. (e) Distribution of the photon counts associated with the τ₁ component of 4 in HK cells after triexponential reconvolution of FLIM images before (c, τ_1i) and after (d, τ_1h) hyper-osmotic shock, showing decreasing lifetimes for τ₁ (4d). (f) The dehydration factor dh_i defined as total integrated photon counts for τ₁ (Στ₁) divided by Στ₂ (i.e., dh_i = area Στ_1i/area Στ_2i) for 4 in strongly hydrated ER (dh_i < 2, turquoise) and 1 in weakly hydrated plasma membrane (dh_i > 6, purple) of HK Kyoto cells under iso-osmotic conditions.Dual response of HydroFlippers to changes in membrane tension^a

Differential operators for harmonic weak Maass forms and the vanishing of Hecke eigenvalues

For integers k ≥ 2, we study two differential operators on harmonic weak Maass forms of weight 2 ? k. The operator ξ_2-k (resp. D ^k-1) defines a map to the space of weight k cusp forms (resp. weakly holomorphic modular forms). We leverage these operators to study coefficients of harmonic weak Maass forms. Although generic harmonic weak Maass forms are expected to have transcendental coefficients, we show that those forms which are “dual” under ξ_2-k to newforms with vanishing Hecke eigenvalues (such as CM forms) have algebraic coefficients. Using regularized inner products, we also characterize the image of D ^k-1.     

Highly Reproducible Formation of a Polymer Single‐Molecule Junction for a Well‐Defined Current Signal

Single‐molecule devices attract much interest in the development of nanoscale electronics. Although a variety of functional single molecules for single‐molecule electronics have been developed, there still remains the need to implement sophisticated functionalization toward practical applications. Given its superior functionality encountered in macroscopic materials, a polymer could be a useful building block in the single‐molecule devices. Therefore, a molecular junction composed of polymer has now been created. Furthermore, an automated algorithm was developed to quantitatively analyze the tunneling current through the junction. Quantitative analysis revealed that the polymer junction exhibits a higher formation probability and longer lifetime than its monomer counterpart. These results suggest that the polymer provides a unique opportunity to design both stable and highly functional molecular devices for nanoelectronics.     

Mixed-potential-type NOx sensor based on YSZ and zinc oxide sensing electrode

Electrochemical sensors using tubular yttria-stabilized zirconia (YSZ) and oxide sensing electrode (SE) were fabricated and examined for NO_x detection at high temperatures. The mixed-potential-type NO_x sensor using ZnO-SE gave the highest sensitivity to NO_x among other single-type oxides tested as SEs in the temperature range of 600–700 °C. The response of the ZnO-attached device was a linear for the logarithm of NO₂ (NO) concentrations from 40 to 450 ppm. The sensing mechanism of the sensor was discussed on the basis of the gas adsorption-desorption behavior, the catalytic activity data, and electrochemical behavior for oxides examined.     

A convenient ring formation of 3-aryl-2,2-dialkyl-2,3-dihydrobenzofurans from phenols and 2-aryl-2,2-dialkylacetaldehydes

A new and simple route for the preparation of 3-aryl-2,2-dialkyl-2,3-dihydrobenzofurans from phenols is described. In the presence of an acid catalyst phenols react with 2-aryl-2,2-dialkylacetaldehydes, prepared in good yield from 2-arylacetonitriles in 2 steps, to give 3-aryl-2,2-dialkyl-2,3-dihydrobenzofurans. Electron-donating substituents were required on the phenols in order to give 3-aryl-2,2-dialkyl-2,3-dihydrobenzofurans in good yield.     

Synthesis of water-soluble porphyrin and the corresponding highly planar benzoporphyrin without meso-substituents

Water-soluble bicycloporphyrin and benzoporphyrin having octacarboxyl groups are synthesized from the corresponding octaester porphyrin by simple hydrolyzing and heating procedures. These compounds are characterized by UV-vis, NMR and mass spectroscopies. The benzoporphyrin octacarboxylic acid exhibits extremely high planarity in spite of its poly-substitution.     

Evidence of new high-pressure magnetic phases in Fe–Pt Invar alloy

To investigate the magnetic properties of disordered Fe₇₀Pt₃₀ Invar alloy under high pressure, measurements of the real part of the AC susceptibility (χ) were made under pressure up to 7.5 GPa in the temperature range 4.2–385 K using a cubic anvil high-pressure apparatus. The Curie temperature (T_C) decreased with increasing pressure, and then, two new high-pressure magnetic phases appeared. These results show that the ferromagnetism of Fe–Pt Invar alloy becomes weaker, and the antiferromagnetic interaction becomes dominant with increasing pressure.