Nitrobenzyl-based fluorescent photocages for spatial and temporal control of signalling lipids in cells.

Here we present a set of fluorescent cages prepared by tethering fluorescent dyes to a photolabile group. The developed molecules enable caging of signalling lipids, their delivery to specific cellular membranes, with further imaging, quantification, and controlled photorelease of active lipids in living cells.

Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research.

Rational design of fluorescent membrane probes for apoptosis based on 3-hydroxyflavone.

Environment-sensitive probes constitute powerful tools for monitoring changes in the physico-chemical properties of cell plasma membranes. Among these probes, 3-hydroxyflavone probes are of great interest due to their dual emission and ratiometric response. Here, three probes derived from the parent F2N12S were designed, characterized and applied to monitor the membrane changes occurring during apoptosis. These three probes were designed to orient the dye vertically in the membrane. They differ by the length of their alkyl chains (from 4 to 8 carbons), which were included to optimize their affinity to the lipid membranes. Among these three probes, the one with medium chain length (hexyl) showed the best affinity to model and cell membranes, while the one with the longest alkyl chains (octyl) did not efficiently stain the membranes, probably due to aggregation. The new probes were found to be more sensitive than F2N12S to both the lipid phase and surface charge in lipid vesicles and to loss of lipid order in cell plasma membranes after cholesterol extraction. The one with the shortest (butyl) chains was found to be the most sensitive to apoptosis, while the one with medium-length (hexyl) chains was the brightest. Interestingly, apoptosis induced by different agents led to similar spectroscopic effects to those produced by the loss of lipid order and change in the surface charge, confirming that apoptosis decreases the lipid order and increases the negative surface charge in the outer leaflet of cell membranes. In conclusion, these studies report the relationship between the probe structures and their sensitivity to lipid order, surface charge and apoptosis and propose new probes for membrane research.

Detection of apoptosis through the lipid order of the outer plasma membrane leaflet.

Cell plasma membranes of living cells maintain their asymmetry, so that the outer leaflet presents a large quantity of sphingomyelin, which is critical for formation of ordered lipid domains. Here, a recently developed probe based on Nile Red (NR12S) was applied to monitor changes in the lipid order specifically at the outer leaflet of cell membranes. Important key features of NR12S are its ratiometric response exclusively to lipid order (liquid ordered vs. liquid disordered phase) and not to surface charge, the possibility of using it at very low concentrations (10-20nM) and the very simple staining protocol. Cholesterol extraction, oxidation and sphingomyelin hydrolysis were found to red shift the emission spectrum of NR12S, indicating a decrease in the lipid order at the outer plasma membrane leaflet. Remarkably, apoptosis induced by three different agents (actinomycin D, camptothecin, staurosporine) produced very similar spectroscopic effects, suggesting that apoptosis also significantly decreases the lipid order at this leaflet. The applicability of NR12S to detect apoptosis was further validated by fluorescence microscopy and flow cytometry, using the ratio between the blue and red parts of its emission band. Thus, for the first time, an environment-sensitive probe, sensitive to lipid order, is shown to detect apoptosis, suggesting a new concept in apoptosis sensing.

Dipolar 3-methoxychromones as bright and highly solvatochromic fluorescent dyes.

Herein, three environment-sensitive (solvatochromic) fluorescent dyes presenting a strong electron acceptor 3-methoxychromone unit and varied electron donor 2-aryl were developed. All three dyes showed remarkable polarity-dependent shifts of the emission maximum, which increase with extension of the dye conjugation. For the 3-methoxychromone bearing a 7-(diethylamino)-9,9-dimethylfluoren-2-yl donor group the difference between the excited and the ground state dipole moments, estimated from the Lippert-Mataga expression, reached 20 D, which is among the largest reported for neutral dipolar fluorophores. Moreover, the new dyes are characterized by significant two-photon absorption cross-section (up to 450 GM) and large fluorescence quantum yields. The strong decrease in the fluorescence quantum yields of the dyes in polar protic solvents was observed together with the increase in the non-radiative deactivation rates, which can originate from twisted intramolecular charge transfer and intermolecular proton transfer phenomena. In comparison to the parent 3-hydroxychromone derivatives, the new dyes presented significantly improved photostability, which confirms that photodegradation of 3-hydroxychromones occurs from a product of the excited-state intramolecular proton transfer (phototautomer). Finally, an application of the new dyes for probing local binding site polarity of serum albumin was shown. This new class of fluorescent dyes may serve as attractive building blocks for future molecular sensors utilizing environment-sensitive fluorophores.

Switchable nile red-based probe for cholesterol and lipid order at the outer leaflet of biomembranes.

Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is present mainly at the outer leaflet of cell membranes, monitoring its lipid order requires molecular probes capable to bind specifically at this leaflet and exhibit negligibly slow flip-flop. In the present work, such a probe was developed by modifying the solvatochromic fluorescent dye Nile Red with an amphiphilic anchor group. To evaluate the flip-flop of the obtained probe (NR12S), we developed a methodology of reversible redox switching of its fluorescence at one leaflet using sodium dithionite. This method shows that NR12S, in contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles and living cells with negligible flip-flop in the time scale of hours. Moreover, the emission maximum of NR12S in model vesicles exhibits a significant blue shift in liquid ordered phase (sphingomyelin-cholesterol) as compared to liquid disordered phase (unsaturated phospholipids). As a consequence, these two phases could be clearly distinguished in NR12S-stained giant vesicles by fluorescence microscopy imaging of intensity ratio between the blue and red parts of the probe emission spectrum. Being added to living cells, NR12S binds predominantly, if not exclusively, their plasma membranes and shows an emission spectrum intermediate between those in liquid ordered and disordered phases of model membranes. Importantly, the emission color of NR12S correlates well with the cholesterol content in cell membranes, which allows monitoring the cholesterol depletion process with methyl-beta-cyclodextrin by fluorescence spectroscopy and microscopy. The attractive photophysical and switching properties of NR12S, together with its selective outer leaflet staining and sensitivity to cholesterol and lipid order, make it a new powerful tool for studying model and cell membranes.

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: effects of cholesterol depletion and apoptosis.

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.