A dual organelle-targeting mechanosensitive probe

Abstract

Cells are responsive to the mechanical environment, but the methods to detect simultaneously how different organelles react in mechanobiological processes remain largely unexplored. We herein report a dual organelle-targeting fluorescent probe, ( E )-1-[3-(diethoxyphosphoryl)propyl]-4-[4-(diethylamino)styryl]pyridin-1-ium bromide (ASP-PE), for mechanical mapping in live cells. ASP-PE is aggregation-induced emission active and is sensitive to the local mechanical environment. It targets the plasma membrane (PM) and intracellular mitochondria in cells by its phosphonate moiety and pyridinium. In this work, through ASP-PE staining, changes of membrane tension in the PM and mitochondria in response to varied osmotic pressure and substrate stiffness are visualized using fluorescence lifetime imaging microscopy. The mechanobiological importance of actin filaments and microtubules in the PM and mitochondria is also investigated using this probe. Computational simulations are applied to study the sensing mechanism of the probe. This study introduces a unique tool for mapping the membrane tension in the PM and mitochondria together, providing us great opportunities to study organelle’s interactions in mechanobiology.

Fluorogenic In Situ Thioacetalization: Expanding the Chemical Space of Fluorescent Probes, Including Unorthodox, Bifurcated, and Mechanosensitive Chalcogen Bonds

“…The resulting push–pull system causes a large red-shift of the absorption maximum and increases fluorescence lifetime, intensity, and quantum yield. This mode of action is contrary to other membrane probes that report off equilibrium in the excited state. − In lipid bilayer membranes, these changes report on an increasing order from liquid-disordered (L d ) to liquid-ordered (L o ) and solid-ordered (S o ) membranes (Figure b). Tension applied to biomembranes increases fluorescence lifetimes because the probe response is dominated by membrane reorganization, particularly tension-induced phase separation (Figure c).…”

“…Fluorescent flippers have been introduced as small-molecule fluorescent probes to image membrane tension in living systems (Figure a–c) . For the imaging of biomembrane function, − membrane tension − is particularly interesting but also particularly demanding because physical forces are detectable only through the suprastructural changes they cause . Inspired by nature, we have considered the concept of planarizable push–pull probes to tackle this challenge .…”

Recent Development of Advanced Fluorescent Molecular Probes for Organelle-Targeted Cell Imaging

“…The same group employed a semi-cyanine unit as the SO 2 -responsive site, a naphthalimide unit and semi-cyanine unit as the fluorophores, and semi-cyanine as well as morpholine groups to target mitochondria and lysosomes, constructing the first dual-targeting organelle-targeted fluorescent probe, DML-P ( 58 , Scheme 7 ), capable of tracing the mitochondria and lysosome SO 2 simultaneously [ 173 ]. Most recently, Ho et al utilized an electron push–pull π-conjugation skeleton and a rarely reported diethyl phosphonate moiety on the pyridinium ring to develop an AIE-based bioprobe, ASP-PE ( 59 , Scheme 7 ), for dual plasma membrane–mitochondria targeting through its phosphonate moiety and pyridinium, visualizing the changes in the membrane tension of the plasma membrane as well as mitochondria in response to varied osmotic pressure and substrate stiffness [ 174 ]. Therefore, this probe revealed the significance of actin and microtubules for plasma membranes and mitochondria in response to mechanobiological stimulations.…”

Hydrophobic Interfacing of Fluorescent Membrane Probes

“…[5] For their design, the bioinspired [51,52] concept of planarizable push-pull probes has been formulated, [5,51] which remains different from other smallmolecule approaches toward tension probes explored later on. [6][7][8]22,25,28,34,53] In brief, flipper probes are composed of two dithienothiophenes that are forced out of co-planarity by electrostatic repulsion from chalcogen-bond donors next to the twistable bond (Figure 1a, r). Mechanical planarization then brings the two dithienothiophenes into conjugation, which induces bathochromic shifts of absorption and excitation maxima and increases in fluorescence lifetime and quantum yield.…”

“…Small‐molecule fluorescent membrane probes are of central importance for bioimaging in living cells [1–5] . Depending on their design, the most common membrane probes respond to viscosity, [6–27] hydration, [1,3,26–40] thickness, [41–44] potential, [4,13–18,26,45–47] membrane order, [1–3,6,8,24,27–33] surface charge [39,40] and surface pH [12,36,48–50] by different mechanisms that mostly operate off equilibrium in the excited state. Fluorescent flippers have been introduced specifically as small‐molecule probes to image membrane tension and order in living systems by mechanical compression in equilibrium in the ground state (Figure 1a–d).…”