Fluorescent Biomembrane Probe for Ratiometric Detection of Apoptosis

Abstract

Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4'-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

“…It exhibits only a limited flip-flop in the membranes of living cells during the time course of our experiments (0.5-1 h), as the absence of time-dependent changes in its emission spectrum was shown at this time scale. (Shynkar et al 2007).…”

“…In all these studies it was shown that the ratio of intensities of the two emission bands changed dramatically in response to apoptosis. The ability of producing bright fluorescence image of cellular membrane is seen by both confocal (Shynkar et al 2007) and two-photon (Oncul et al 2010) microscopy. Figure 9 demonstrates the results obtained by flow cytometry.…”

“…Excitation wavelength was 400 nm. Final probe concentration was 100 nM (Shynkar et al 2007) Cytotechnology (2013) 65:157-172 167 Thus, the advantages of this new technology can be summarized as follows (Demchenko 2012):…”

A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress

“…Consequently, a wide range of fluorescent probes suitable for probing multiple properties of lipid membranes was developed,13 including probes for sensing membrane potential and fluidity,14 for detecting lipid order in the outer lipid leaflet of the lipid bilayer15 and for sensing changes in the membrane during apoptosis 16. In this work, we utilized BODIPY‐C 10 17, 18 (Figure 1), a fluorophore that belongs to a group of dyes termed ‘molecular rotors’ that have viscosity‐dependent fluorescence quantum yields, lifetimes,19, 20 and depolarization 21, 22.…”

Specificity and Kinetics of α-Synuclein Binding to Model Membranes Determined with Fluorescent Excited State Intramolecular Proton Transfer (ESIPT) Probe

“…The intensity ratio of the normal (N*) and tautomeric bands is highly sensitive to the environment properties (50,51). Diethylamino-3-hydroxyflavone (FE) discriminates between protic and aprotic environments as well as different membrane types (48,(51)(52)(53) and thus constitutes a promising tool for monitoring protein-membrane association (54). We have previously reported that FE reveals differences in the supramolecular organization of amyloid fibrils (55) and the utility of a related ESIPT probe (FC) as a reporter of AS aggregation (56).…”