Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

Mg~(2+)加强嵌有H~+-ATP酶脂酶体脂质分子的堆积(packing)

用亲脂性的灵敏的荧光MC 540标记在有Mg~(2+)(1mM)与无Mg~(2+)条件下重建的线粒体H~+-ATP酶脂酶体,后者的荧光强度较前者增加30%左右.这提示,含Mg~(2+)的脂酶体的脂质分子间的堆积紧密度增加.在N-AF系列(n=27和16)探剂与MC 540之间的能量转移效率,又以反应靠近脂双层表面变化的2-AP与MC 54O之间最高.这进一步表明,含Mg~(2+)的脂酶体具有较适合流动性是与Mg~(2+)通过调节靠近脂双层表面的脂质分子具有适度的堆积相关的.这对阐明我们已提出的Mg~(2+)促进线粒体H~+-ATP酶重建作用模型进一步提供了较直接的证据.     

Effect of vanadium compounds on the lipid organization of liposomes and cell membranes

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia–reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.     

Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin

We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.     

pH-sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules

Negatively charged liposomes are endocytosed by the coated vesicle system and accumulate in acidic intracellular vesicles. Liposomes that become unstable at acidic pH improve cytoplasmic delivery of membrane-impermeant macromolecules such as calcein (CAL) and FITC dextran (18 or 40 kDa). Oleic acid (OA): phosphatidylethanolamine (PE) (3:7 mole ratio) liposomes become permeable to CAL at pH less than 7.0. Control liposomes of phosphatidylserine:PE or OA:phosphatidylcholine are stable at pH 4-8. OA:PE liposomes promote cytoplasmic delivery of encapsulated CAL to CV-1 cells, as evidenced by the emergence of diffuse, cytoplasmic CAL fluorescence. Delivery requires metabolic energy and is partially inhibited by chloroquine or monensin, which raise the pH of intracellular vesicles.     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Merocyanine 540 as a fluorescent probe of membranes: staining of electrically excitable cells.

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.     

Staining of Viable and Nonviable Myotubes and of Myofibrils by the Fluorescent Dye Merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5–10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.