Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential

Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.     

Energy transfer to analyse membrane-integrated mitoxantrone in BCRP-overexpressed cells

The binding and the diffusion of mitoxantrone (MTX) through the plasma membrane was performed by F?rster resonance energy transfer (FRET) from the membrane fluorescent donor (4Di-10ASP) to the co-localized acceptor MTX. The MTX addition to living 4Di-10ASP-tagged cells resulted in the rapid quenching of the probe emission (1s), revealing the MTX binding to the outer leaflet. Then, a slower quenching (about 90s) occurred which corresponded to the MTX flip-flop into the inner leaflet. Changes of MTX integration into the plasma membrane were described in BCRP-overexpressed cells (HCT-116R) treated with (i) the BCRP inhibitor fumitremorgin C (FTC), (ii) cyclosporin A (CSA) and (iii) benzyl alcohol (BA). Treatments with FTC or CSA showed 80% and 40% higher flip-flop of MTX from the outer to the inner leaflet of HCT-116R cells. The addition of BA clearly increased the MTX integration into both outer and inner leaflets. Confocal fluorescence microscopy displayed that FTC, CSA and BA enhanced MTX accumulation in HCT-116R. In conclusion, Fumitremorgin C and agents modulating MTX accumulation resulted in higher MTX integration in the resistant cell membrane and could disrupt the membrane cohesion. This energy transfer method appears well-adapted to describe the drug diffusion through the plasma membrane of living cells.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.     

Structural and dynamic characterization of the interaction of the putative fusion peptide of the S2 SARS-CoV virus protein with lipid membranes

The SARS coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In the present work, we report a study of the binding and interaction with model membranes of a peptide pertaining to the putative fusion domain of SARS-CoV, SARS FP, as well as the structural changes that take place in both the phospholipid and the peptide molecules upon this interaction. From fluorescence and infrared spectroscopies, the peptide ability to induce membrane leakage, aggregation and fusion, as well as its affinity toward specific phospholipids, was assessed. We demonstrate that SARS FP strongly partitions into phospholipid membranes, more specifically with those containing negatively charged phospholipids, increasing the water penetration depth and displaying membrane-activity modulated by the lipid composition of the membrane. Interestingly, peptide organization is different depending if SARS FP is in water or bound to the membrane. These data suggest that SARS FP could be involved in the merging of the viral and target cell membranes by perturbing the membrane outer leaflet phospholipids and specifically interacting with negatively charged phospholipids located in the inner leaflet.     

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.     

Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super‐resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane‐targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long‐chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short‐chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.     

Anticancer β-hairpin peptides: membrane-induced folding triggers activity

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.     

Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells

A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4–10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells.     

Colorimetric and ratiometric fluorescent chemosensor based on diketopyrrolopyrrole for selective detection of thiols: an experimental and theoretical study

A colorimetric and ratiometric fluorescent thiol probe was devised with diketopyrrolopyrrole (DPP) fluorophore. The probe gives absorption and emission at 523 and 666 nm, respectively. In the presence of thiols, such as cysteine, the absorption and emission band shifted to 479 and 540 nm, respectively. Correspondingly, the color of the probe solution changed from purple to yellow, and the fluorescence changed from red to yellow. The emission intensity at 540 nm was enhanced by 140-fold. The Stokes shift of probe 1 (107 nm) is much larger than the unsubstituted DPP fluorophore (56 nm). Mass spectral analysis demonstrated that besides the expected Michael addition of thiols to the C═C bonds, the CN groups of the malonitrile moieties also react with thiols to form 4,5-dihydrothiazole structure. Probe 1 was used for fluorescence imaging of intracellular thiols. In the presence of thiols, both the green and red channel of the microscopy are active. With removal of the intracellular thiols, signal can only be detected through the red channel; thus, ratiometric bioimaging of intracellular thiols was achieved. The ratiometric response of probe 1 was rationalized by DFT calculations. Our complementary experimental and theoretical studies will be useful for design of ratiometric/colorimetric molecular probes.     

The effect of Alzheimer's Aβ aggregation state on the permeation of biomimetic lipid vesicles

Alzheimer's disease is characterized by the aggregation and deposition of the Aβ peptide. This 40 or 42 residue peptide is the product of the proteolysis of the amyloid precursor protein membrane protein and is able to assemble to form ordered, stable amyloid fibrils as well as small, soluble, and potentially cytotoxic oligomers. The toxicity of the oligomers may be associated with the ability to bind to and affect the integrity of lipid membranes. In this novel work, we have monitored and compared the ability of the potent Aβ42 peptide, the less amyloidogenic Aβ40 peptide, and a variant with reduced amyloidogenicity to bind to and affect the integrity of membranes using dye-filled synthetic vesicles. We reveal that the potency of the assemblies reduces with incubation time of the peptide and that maximal effect occurs when a particular species is apparent by electron microscopy. We have investigated the effect of lipid vesicle composition, and our results suggest that charge on the vesicles is important and that binding may partly be mediated by the GM1 ganglioside receptors expressed in the outer leaflet of vertebrate membranes.     

Synthetic peptides with selective affinity for apoptotic cells

The appearance of anionic phosphatidylserine (PS) in the outer monolayer of the plasma membrane is a universal indicator of the early/intermediate stages of cell apoptosis. The most common method of detecting PS on a cell surface is to use the protein annexin V; however, in certain applications there is a need for alternative reagents. Recent research indicates that rationally designed zinc 2,2'-dipicolylamine (Zn2+-DPA) coordination complexes can mimic the apoptosis sensing function of annexin V. Here, a series of fluorescently-labelled, tri- and pentapeptides with side chains containing Zn2+-DPA are prepared and shown to selectively bind to anionic vesicle membranes. Fluorescein-labelled versions of the peptides are used to detect apoptotic cells by fluorescence microscopy and flow cytometry.     

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.     

Nanoscale imaging of domains in supported lipid membranes

The formation of domains in supported lipid membranes has been studied extensively as a model for the 2D organization of cell membranes. The compartmentalization of biological membranes to give domains such as cholesterol-rich rafts plays an important role in many biological processes. This article summarizes experiments from the author's laboratory in which a combination of atomic force microscopy and near-field scanning optical microscopy is used to probe phase separation in supported monolayers and bilayers as models for membrane rafts. These techniques are used to study binary and ternary lipid mixtures that have gel-phase or liquid-ordered domains that vary in size from tens of nanometers to tens of micrometers, surrounded by a fluid-disordered membrane. Examples are presented in which these models are used to investigate the distribution of glycolipid membrane raft markers and the preference for peptide and protein localization in ordered versus fluid membrane phases. Finally, the enzyme-mediated restructuring of membranes containing liquid-ordered domains provides an in vitro model for the coalescence of membrane rafts to give signaling platforms. Overall, the results demonstrate the importance of using techniques that can probe the nanoscale organization of membranes and of combining techniques that yield complementary information. Furthermore, the ability of supported lipid bilayers to model some aspects of membrane compartmentalization provides an important approach to understanding natural membranes.     

Emission ratiometric imaging of intracellular zinc: design of a benzoxazole fluorescent sensor and its application in two-photon microscopy

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.     

Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.     

Ratiometric Fluorescent Probe Based on Novel Red-emission BODIPY for Determination of Bovine Serum Albumin

A novel red-emission boron-dipyrromethene（BODIPY） dye with a pyrrole ring was synthesized simply via one-pot reaction. The spectral properties of it were investigated under the conditions of different solvents. The results show that the as-prepared BODIPY dye is extremely sensitive to solvent polarity, and the fluorescent emission enhances with the decrease of solvent polarity. In aqueous buffer, the addition of bovine serum albumin leads to a ratiometric change in absorption spectra with an association constant of 1.16×10^6 L/mol. Meanwhile, the fluorescence emission increases greatly at 622 nm but changes slightly at 575 nm. The response time is very short（less than 3 min）, and the changes of color can be noticed by naked eyes. Bovine serum albumin can be detected by this ratiometric fluorescence probe, but other proteins or enzymes cannot be detected by this method, which indicates that this novel dye has high selectivity towards bovine serum albumin. The reason is that bovine serum albumin has suitable hydro- phobic cavities for binding with the dye. In addition, the dye molecule can penetrate cell membrane easily and make a fast fluorescent stain, which makes it a potential probe for living-cell fluorescence imaging.     

Ratiometric Fluorescence Imaging of Cellular Polarity: Decrease in Mitochondrial Polarity in Cancer Cells

Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λ_abs=426 and 561 nm) and two emission maxima—a strong green emission (λ_em=467 nm) and a weak red emission (642 nm in methanol)—when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (R_r=0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.     

Zwitterionic Character and Lipid Composition Determine the Behaviour of Glycosylphosphatidylinositol Fragments in Monolayers

Glycosylphosphatidylinositols (GPIs) are complex glycolipids found in free form or anchoring proteins to the outer leaflet of the cell membrane in eukaryotes. GPIs have been associated with the formation of lipid rafts and protein sorting on membranes. The presence of a conserved glycan core with cell-specific modifications together with lipid remodelling during biosynthesis suggest that the properties of the glycolipids are being fine-tuned. We synthesized a series of GPI fragments and evaluated the interactions and arrangement of these glycolipids in monolayers as a 2-D membrane model. GIXD and IRRAS analyses showed the need of N-acetylglucosamine deacetylation for the formation of hydrogen bonds to obtain highly structured domains in the monolayers and an effect of the unsaturated lipids in formation and localization of the glycolipids within or between membrane microdomains. These results contribute to understand the role of these glycolipids and their modifications in the organization of membranes.