Harnessing Dual-Fluorescence Lifetime Probes to Validate Regulatory Mechanisms of Organelle Interactions

Abstract

Organelles are dynamic yet highly organized to preserve cellular homeostasis. However, the absence of time-resolved molecular tools for simultaneous dual-signal imaging of two organelles has prevented scientists from elucidating organelle interaction regulatory mechanisms on a nanosecond timescale. To date, the regulatory mechanisms governing the interaction between endoplasmic reticulum (ER) and autophagosomes are unknown. In this study, we propose a strategy for developing dualfluorescence lifetime probes localized to the endoplasmic reticulum and autophagosomes to investigate their interaction regulatory mechanisms. Using the robust probe CF2, we investigated the regulatory mechanisms between ER and autophagosomes and discovered the following: (i) motile autophagosome in ER tips drives the ER tubule to grow and slide; (ii) the ER reticulate tubule forms a three-way junction centered on the autophagosome; (iii) ER autophagy is a type of cell damage index during drug-induced apoptosis. Thus, this study advances our knowledge of organelle interaction regulatory mechanisms, shedding light on the identification of therapeutic targets for neurodegenerative diseases.

Fluorescent Flippers: Small‐Molecule Probes to Image Membrane Tension in Living Systems

“…[120] Molecular rotors show increasing intensity and lifetimes with increasing membrane order (Figure 13Bc) because twisting in the excited state for non-radiative decay through formal TICT excited states or other mechanisms decelerates with increasing viscosity (Figure 13Bd). [121][122][123][124][125][126][127][128][129][130][131][132] Although not studied so far in membranes, many probes for nucleic acid structures, [133] or "aggregationinduced-emission" [134] could operate in a similar manner. The origin of the mechanosensitivity of molecular rotors, responding to the kinetics of excited-state deplanarization (Figure 13Bd), is different from flipper probes, which mainly operate by ground-state planarization (Figure 3Bc).…”

“…[149] Very recently, 40 was introduced as planar push-pull rotor that might image membrane tension in model GUV membranes with unusually large changes of lifetimes, and target the ER. [132] Referred to as flappers [135] or papillons, [139,140] mechanophores that deplanarize by bending rather than twisting are attractive candidates for imaging membrane tension. Constant excitation maxima in model membranes reveal that high membrane order fails to planarize dihydrodibenzo-[a,c]phenazines papillons 41 in the ground state, presumably because of the too high energy costs (Figure 13Cf).…”

“…The arguably best explored solvatochromic probes show red‐shifted emission with decreasing membrane order (Figure 13Ad) because water, that is membrane hydration, stabilizes the ICT excited state (Figure 13Ac) [120] . Molecular rotors show increasing intensity and lifetimes with increasing membrane order (Figure 13Bc) because twisting in the excited state for non‐radiative decay through formal TICT excited states or other mechanisms decelerates with increasing viscosity (Figure 13Bd) [121–132] . Although not studied so far in membranes, many probes for nucleic acid structures, [133] or “aggregation‐induced‐emission” [134] could operate in a similar manner.…”

Recent Advances in Synthetic Routes to Azacycles

“…N -heterocycles, which contain nitrogen atoms, have attracted much attention from scientists because of their unique properties and diverse utilization. N -heterocycles have been employed in many industries, including as dyes, agrochemicals, and materials [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. In pharmaceuticals, small-molecule drugs contain nitrogen-containing heterocycles and exhibit diverse bioactivities including anti-Alzheimer’s, antivirus, and anticancer behavior [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ].…”

Naphthalimide Fluorescent Skeleton for Facile and Accurate Quantification of Glutathione

“…The traditional method for intracellular GSH measurement is the enzymatic recycling assay. Glutathione reductase (GR) and NADPH are required during the operation, so this method is costly with tedious and time-consuming sample pretreatment. , Although LC-MS is also widely used for the detection of thiols, cost-effective and portable measurement is not allowed. , Fluorescent probes have recently emerged as indispensable and ideal tools to monitor molecular events for real-time monitoring in multiple research fields due to their simple operation, high selectivity, and good biocompatibility. − To date, plenty of fluorescent probes for detecting GSH have been reported based on different mechanisms, e.g., cleavage of sulfonamides/sulfonate esters/Se–N bond, disulfide bond exchange, aryl substitution reactions, Michael addition followed by cyclization, and metal ion replacement followed by coordination. − In addition, several probes for the real-time quantification of GSH have been developed. − The fly in the ointment is that thiol-containing proteins are ubiquitous in cells (up to 70% of the total cellular thiols), the interference of which is not considered by most GSH probes in cell imaging. In addition, there are relatively few studies using probes to quantify the total GSH (tGSH), GSH, and GSSG in cells and tissues.…”