A fluorescent sterol probe study of human serum low-density lipoproteins

The fluorescent sterol probe, ergosta-5,7,9,(11),22-tetraen-3 beta-ol (dehydroergosterol), was utilized as a cholesterol analog to label human serum low-density lipoproteins (LDL). Quenching of dehydroergosterol fluorescence by KI indicated that most of the fluorophore was either buried within the outer phospholipid monolayer of LDL or within the neutral lipid core of LDL. The steady-state anisotropy of dehydroergosterol in LDL detected the cholesteric core phase transition near 30 degrees C. Fluorescence lifetime decays for dehydroergosterol contained two components, both below and above the cholesteric phase transition, with the major lifetime component near 1 ns. Neither lifetime component underwent a detectable change in duration at the core phase transition temperature. Time-correlated fluorescence anisotropy decays of dehydroergosterol indicated a single rotational correlation time near 1.7 ns, which was unaffected by the core phase transition. Time-correlated anisotropy decays also suggested hindered rotation of dehydroergosterol in LDL. These results indicate that unesterified cholesterol is primarily located in the outer phospholipid monolayer of LDL, with the majority of cholesterol not in direct contact with the aqueous phase.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

A fluorescent probe study of the solubilisation of cholesterol by bile salt-phospholipid micelles

The fluorescent probe dansyl cadaverine has been shown to bind strongly to mixed bile salt-phospholipid micelles containing unsaturation in the fatty acyl chains. Incorporation of cholesterol into the mixed micelles reduces the number of molecules of bound dansyl cadaverine without altering the binding affinity. These results suggest a tighter packing of the hydrocarbon matrix of the micelles in the presence of cholesterol.     

A novel adaptive fluorescent probe for cell labelling

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.     

Perturbation of egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles by n-alkanols. A fluorescent probe study

The perturbing effects of n-alkanols (pentanol, decanol and tetradecanol) in egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles were studied with five fluorescent probes, 1-(4'-trimethylaminophenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene, and 2-, 7-, and 12-(9-anthroxyloxy)stearic acid (2-, 7-, and 12-AS). These probes localize at various depths in the membrane, enabling study of the membrane-order gradient. Phase-modulation fluorescence spectroscopy was used to measure steady-state anisotropies, excited-state lifetimes and differential polarized lifetimes from which the limiting hindered anisotropies (r infinity) and the logarithm of the rotational rate (log R) were calculated. The probes that localize at about the same depth in the membrane (TMA-DPH and 2-AS, diphenylhexatriene and 12-AS) generally, but not always, showed similar changes in r infinity and log R with added alkanols. However, the absolute values of r infinity and log R were usually different. The inconsistencies are attributed to differences in the probes' sizes, structures, photophysical properties and perturbing abilities. The perturbation of membranes by alkanols is chain-length-dependent. Pentanol disorders the membrane at all depths but is more effective in the membrane center than nearer to the polar headgroups of the phospholipids, tetradecanol can be accommodated into the membrane without effect or with increased order and the effects of decanol are intermediate between pentanol and tetradecanol. Our results with alkanols indicate that: a single perturber can have different effects on membrane order at different depths in the bilayer; the perturbation is observed at and distant from the perturbers' location in the membrane, and the bilayer center is more susceptible to perturbation by alkanols than the region of the bilayer near the phospholipid headgroups.     

A highly sensitive,cell-membrane-permeable fluorescent probe for glutathione

Glutathione (GSH) is a primary intracellular antioxidant. Here, we developed a novel, highly sensitive fluorescent probe for GSH, designated DNs-HMRG, whose fluorescence is regulated by two distinct switching mechanisms, intramolecular spirocyclization and photo-induced electron transfer (PeT). DNs-HMRG showed good cell permeability, and a rapid increase in fluorescence intensity was observed when it was applied to living cells. Further, taking advantage of the fact that the intracellular GSH level in tumor tissue is higher than that in normal tissue, we employed this probe for rapid (within a few tens of seconds) in vivo detection of tiny tumor nodules (less than 1 mm in diameter) in tumor-bearing mice. This probe is expected be a powerful tool in various biological applications, especially studies on redox status.     

A membrane-bound fluorescent probe to detect phospholipid vesicle-cell fusion

Summary Pyrenesulfonylphosphatidylethanolamine has been incorporated into sonicated phospholipid vesicles to provide a fluorescent signal from a membrane-bound probe whose spectrum is sensitive to the local concentration of dye molecules. When vesicle material was taken up by viable mouse splenocytes, the disappearance of the pyrene excimer fluorescence emission peak that accompanied dilution of the vesicle membrane lipid could be quantitated. One can thus measure, by a simple and rapid procedure, a new parameter which is related to the extent of vesicle-cell fusion and which is independent of the transfer of aqueous vesicle contents to the cell cytoplasm.Abbreviations used 6-CF 6-carboxyfluorescein - HBSS Hanks Balanced Salt Solution - DMPC dimyristoylphosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - EYL egg yolk lecithin - PE phosphatidylethanolamine - PEG polyethylene glycol - PLV phospholipid vesicle - PSPE pyrenesulfonylphosphatidylethanolamine     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

A study of carbachol-atropine interaction on intestinal smooth muscle vesicles, using a fluorescent probe.

Plasma membrane vesicles were prepared from guinea pig ileum longitudinal muscle. The vesicles were characterized by electron microscopy and analysis of lipid and protein content. They were shown to be free of gross contamination from actomyosin, sarcoplasmic reticulum, and mitochondria. 8-Anilino-1-naphthalene sulphonic acid (ANS) binding characteristics were similar to those found in other membranes. Both carbachol and atropine increased the fluorescence of ANS bound to this membrane, the maximum increase for atropine being greater than that for carbachol. Since neither drug effected the apparent affinity constant for the ANS-membrane interaction. It may be assumed that the increased fluorescence was due to an increase in the number of ANS binding sites. The carbachol-dependent increase in ANS fluorescence was blocked noncompetitively by atropine but not by tubocurarine or diphenhydramine. These latter two antagonists also increased ANS fluorescence but at much higher concentrations than either carbachol or atropine. Neither atropine nor carbachol increased ANS fluorescence on either erythrocyte ghosts or liposomes (prepared from a lipid extract of the muscle membrane).     

Conformational study of low molecular weight RNAs in solution by fluorescent probe

Pseudouridine psi 73 in yeast 5.8S RNA was modified with 4-bromomethyl-7-methoxycoumarin(BMC). Temperature dependence of fluorescence intensity was measured and it was compared with UV-thermal melting curve. The region around psi 73 melts partially. Fluorescence intensity changes by titration with Mg2+ and it quenches largely by added iodide ion. The results are compared with those of BMC-modified psi 55 of tRNAPhe and psi 50 of 5S RNA reported in previous paper1-4.     

Deep-red fluorescent imaging probe for bacteria

A versatile deep-red fluorescent imaging probe is described that is comprised of a bis(zinc(II)-dipicolylamine) targeting unit covalently attached to a pentamethine carbocyanine fluorophore with Cy5-like spectroscopic properties. A titration assay based on fluorescence resonance energy transfer is used to prove that the probe selectively associates with anionic vesicle membranes whose composition mimics bacterial cell membranes. Whole-body optical imaging experiments show that the probe associates with the surfaces of both Gram-positive and Gram-negative bacteria cells, and it can target the site of bacterial infection in a living mouse. In vivo accumulation at the infection site and subsequent clearance occurs more quickly than a structurally related near-infrared bis(zinc(II)-dipicolylamine) probe. The fact that the same deep-red probe molecule can be used for spectroscopic assays, cell microscopy, and in vivo imaging studies, is an important and attractive technical feature.     

Membrane permeable esterase-activated fluorescent imaging probe

An esterase-triggered probe 2 derived from a cyanine-based pH sensitive dye was developed for cell labeling. Permeation of probe 2 into cells and subsequent hydrolytic activation by cellular esterases result in a bright fluorescent intracellular signal.