Fluorescent Flipper Probes: Comprehensive Twist Coverage

Abstract

To image the membrane tension in living cells, planarizable push–pull probes have been introduced. The first operational probe is built around two dithieno[3,2‐b:2′,3′‐d]thiophenes (DTTs) that are twisted out of co‐planarity and polarized with donors and acceptors at either end. In this report, the chemical space available for the twisting of “flipper probes” is assessed comprehensively. The result is, not surprisingly, that every atom matters: Removal of one methyl group in the twist region yields probes that planarize already in solution and are thus less sensitive to membrane tension. Addition of one or more carbons in the same region hinders non‐interfering probe alignment along lipid tails and thus partitioning into lipid bilayer membranes as well as mechanosensitivity. However, substitution of one methyl by an isosteric trifluoromethyl group in the twist region, achieved by quite substantial multistep organic synthesis, yields excitation maxima that shift over +100 nm to the red in response to increasing order of the surrounding membrane. This record redshift comes with record changes in fluorescence intensity and lifetime, high push–pull transition dipoles and higher rotational barriers. Supported by distinct dependence on viscosity and twist of the push–pull probes, kinetic competition between dark, fully twisted and bright, fully planarized relaxed excited states emerges as unifying origin of fluorescence quantum yields.

Photocleavable Fluorescent Membrane Tension Probes: Fast Release with Spatiotemporal Control in Inner Leaflets of Plasma Membrane, Nuclear Envelope, and Secretory Pathway

“…It thus indicated that, as soon as photoreleased from the ER surface within a few minutes (Figures 2 B,C, S2), flipper 1 re‐distributes among all membrane leaflets accessible by free diffusion (Figures 2 D, S4). Only the most ordered membrane, here the PM, is labeled because planarization of flipper 1 in such membranes suppresses competing non‐radiative decay and thus increases emission intensity decisively, [5] as previously illustrated in FLIM images and computational simulations of phase‐separated membranes [12, 27, 28] (Figure 2 B,C).…”

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells

“…The emission maximum around 600 nm is nearly mechano-insensitive because the emission always occurs from a fully planarized excited state. 6 This combination of planarization and polarization in equilibrium in the ground state often occurs in nature, from lobster pigmentation 15 − 17 to the chemistry of vision, 18 − 20 but is unexplored in the design of fluorescent probes. Most other fluorescent membrane probes operate in the excited state by different mechanisms, such as intramolecular charge transfer (ICT), twisted intramolecular charge transfer (TICT), excited-state intramolecular proton transfer (ESIPT), photoinduced electron transfer (PET), Förster resonance energy transfer (FRET), and the like.…”

Twisting and tilting of a mechanosensitive molecular probe detects order in membranes

“…1b ), and the appearance of some vibrational finestructure. 16 Flipper-TR is composed of (i) a triazole-carboxylate hydrophilic head for positioning into the membrane and stability, and (ii) a DTT-based chromophore responsible for the spectroscopic behavior. It has been hypothesized that the two DTT flippers may respond to lateral forces by switching between planar and nonplanar conformations as a result of twisting around the “mechanosensitive” dihedral Θ ( Fig.…”