Quantitative assessment of peptide-lipid interactions.: Ubiquitous fluorescence methodologies

Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug.     

Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis

Studying mechanisms of drug antitumor action is complicated by the lack of noninvasive methods enabling direct monitoring of the state and interactions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming this problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, 8-bis-[([2-(2-hydroxyethyl)-amino]ethyl)amino]-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spectra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target complexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affecting drug fluorescence. MITOX exposed to aqueous intracellular environment, MITOX bound to hydrophobic cellular structures, complexes of MITOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These states and complexes are known to be immediately related to the antitumor action of the drug. The results obtained present a basis for the subsequent quantitative analysis of concentration and time-dependent accumulation of free and bound MITOX within different compartments of living cancer cells.     

三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较

目的：对三套荧光显微成像系统在国产新型光敏剂HMME亚细胞定位研究中的应用特点及适用范围进行了比较与评价。方法：分别应用LSCM、CCD、ICCD荧光显微成像系统,选择特异性细胞器荧光探针Rhodamine-123、DIOC6(3)标记细胞内线粒体和内质网。采用细胞器-细胞荧光强度比值法,对HMME进行单细胞内分布的定性与定量研究。结果：LSCM和CCD成像系统能采集到浓度达到160μg／ml时的HMME的荧光图像,获得荧光探针图像信息显示所标记的细胞内线粒体和内质网平均荧光强度比值(J1/J2值)都明显高于细胞内J1/J2值。而ICCD成像系统只需HMME浓度为5μg/ml,荧光图像特点都呈胞浆中荧光强度较高且分布不均,细胞核区荧光较弱的中空现象。ICCD系统对细胞器探针荧光图像在空间分辨上不理想。结论：LSCM与CCD成像系统限于其探测灵敏度,对于弱荧光性光敏剂,适用于其高孵育浓度条件下的亚细胞定位研究。二者获得的结果相一致：孵育24h,HMME在鼠肺内皮细胞线粒体和内质网有分布而几乎不进入细胞核。ICCD成像系统可不受孵育浓度条件的限制,实现光敏剂极微弱荧光的有效探测,但空间分辨率较低。     

Rapid microspectrofluorimetry for biochemical and metabolic studies in single living cells

A rapid multichannel microspectrofluorometer (e.g., to NAD(P)H, fluorescent probes) can be operated on a topographic mode for the evaluation of intracellular metabolic topography or on a spectral mode for the individual or simultaneous intracellular spectral analysis of various fluorochromes. The fluorescence emission spectra of the living cells, as well as difference spectra (spectra after intracellular microelectrophoretic addition of substrate minus before)_are analyzed under various conditions, and provide a direct proof that the fluorescence observed is that of NAD(P)H. The spectral changes which accompany treatment with substrate (e.g., glucose-6-P) can be further followed in cells incubated with other probes (e.g., acridine orange). Repeated and quite reversible transients of NAD(P) reduction—reoxidation may be observed in cells having absorbed acridine orange following repetitive additions of substrate. The spectral response to substrate is also comparatively studied in cells grown in presence of agents affecting the cell cycle (e.g., dibutyryl cyclic AMP, bleomycin).     

Plasmodium falciparum: Organelle-specific acquisition of lipoic acid

Lipoic acid is an essential cofactor of multienzyme complexes that are integral to energy metabolism, amino acid degradation and folate metabolism. In recent years it has been shown that the malaria parasite Plasmodium falciparum possesses organelle-specific pathways that guarantee the lipoylation of their multienzyme complexes which occur in the mitochondrion (LA salvage) and in a plastid-like organelle, the apicoplast (LA biosynthesis). The unique distribution of the lipoylation machineries and the unique metabolic requirements of the parasites present a situation that is potentially exploitable for new ways to improve malaria control.     

A broad view of scaffolding suggests that scaffolding proteins can actively control regulation and signaling of multienzyme complexes through allostery

Enzymes often work sequentially in pathways; and consecutive reaction steps are typically carried out by molecules associated in the same multienzyme complex. Localization confines the enzymes; anchors them; increases the effective concentration of substrates and products; and shortens pathway timescales; however, it does not explain enzyme coordination or pathway branching. Here, we distinguish between metabolic and signaling multienzyme complexes. We argue for a central role of scaffolding proteins in regulating multienzyme complexes signaling and suggest that metabolic multienzyme complexes are less dependent on scaffolding because they undergo conformational control through direct subunit–subunit contacts. In particular, we propose that scaffolding proteins have an essential function in controlling branching in signaling pathways. This new broadened definition of scaffolding proteins goes beyond cases such as the classic yeast mitogen-activated protein kinase Ste5 and encompasses proteins such as E3 ligases which lack active sites and work via allostery. With this definition, we classify the mechanisms of multienzyme complexes based on whether the substrates are transferred through the involvement of scaffolding proteins, and outline the functional merits to metabolic or signaling pathways. Overall, while co-localization topography helps multistep pathways non-specifically, allosteric regulation requires precise multienzyme organization and interactions and works via population shift, either through direct enzyme subunit–subunit interactions or through active involvement of scaffolding proteins. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Cytotoxicity of daunorubicin in trisomic (+21) human fibroblasts: relation to drug uptake and cell membrane fluidity

The influence of daunorubicin (DNR) on survival of human normal (S-126) and trisomic, with respect to chromosome 21 (T-164; S-240), skin fibroblasts and some parameters related to it, such as intracellular drug accumulation, distribution and interaction with cell membrane, were studied. The in vitro growth-inhibition assay indicated that DNR was less cytotoxic for trisomic than for normal cells. Comparison of kinetic parameters and intracellular distribution of this compound showed that the uptake and the amount of intracellular free DNR were greater in normal than in trisomic cells. Contrary to this, there were no significant differences between the amount of DNA-bound drug in both types of cells. TMA-DPH and 12-AS fluorescence anisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydrophobic region of plasma membrane in both cell types, but did not influence the fluidity of the outer surface of membrane. We conclude that fibroblasts derived from individuals affected with Down's syndrome are better protected from the damage induced by DNR than normal cells.     

In-Cell Protease Assay Systems Based on Trans-Localizing Molecular Beacon Proteins Using HCV Protease as a Model System

This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s). The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.     

Tumor-stroma interactions: their role in the control of tumor cell invasion

The development and progression of tumors result from the concerted activity not only of tumor cells with neighboring cells e.g., fibroblasts and inflammatory cells. Host-tumor interactions are considered critical in tumor invasion and metastasis. In vitro studies as well as established in vivo models have analysed the reciprocal effects of tumor-host interactions for the tumor invasion process. These studies have shown that modifications in the extracellular matrix composition surrounding the tumors as well as alterations in the expression of tumor cell receptors or in the expression of growth factors/cytokines and proteases, are critical regulators of a developing tumor. We shortly review the most important and well characterized mechanisms involved in the progression of tumor cells through tissues, especially those participating in cellular communication, cell adhesion, and proteolysis.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis

BACKGROUND: Anthracycline resistance is known to be mediated by P-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs. METHODS: The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction. Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution. Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling. RESULTS: An original image analysis procedure was developed to investigate daunorubicin and organelles co-localization. The reliability of the image analysis was controlled through chromatic shift and intensity linearity measurement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated. Although no rational relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells. CONCLUSIONS: In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular vesicles identified as Golgi vesicles and resistance to apoptosis induction.     

Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine- containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.     

The metabolic interaction of cancer cells and fibroblasts – coupling between NAD(P)H and FAD,intracellular pH and hydrogen peroxide

Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment.     

Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.     

A microspectrofluorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism

The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.e. antiproliferative effect of anthralin, has been attributed to its action at the level of mitochondria or at the level of glucose-6-phosphate dehydrogenase which initiates the pentose phosphate shunt (cf. its prominent role in nucleic acid synthesis). Upon addition of 2.3 to 23 microM anthralin to the L cell culture, the characteristic structure of the anthralin anion fluorescence spectrum is recognized almost immediately in the cytoplasm (much weaker in the nucleus) but disappears within minutes. The vital mitochondrial fluorescence probe dimethylaminostyryl-pyridinium-methyl-iodine reveals striking structural alterations of the mitochondria within 15 min after addition of the drug. At the same time, there is a stimulation of the transient NAD(P)+ reduction observed upon microinjection into the L cell of the Krebs' cycle substrate malate, or the pentose cycle substrate 6-phosphogluconate. Specially, the injection of the latter to anthralin-treated cells suggests that upon release of the mitochondrial control, there is a tremendous disruption of metabolic activity which could have profound consequences on the proliferative activity of the cell. These findings, while they open new possibilities for the intracellular evaluation of therapeutic agents, create also a challenge in understanding the complex and dynamic interrelationships between intracellular organelles and bioenergetic or biosynthetic pathways.     

Europium complexes as novel indicators of paracellular diffusion

Measurement of paracellular permeation is an important assay for tight-junction investigations of drug toxicity, especially for metal-based drugs, and routine validation of the integrity of cell monolayers for models of drug absorption. Great efforts have been made in discovery and validation of novel paracellular diffusion indicators. In the present work, we prepared three Eu complexes, i.e., [Eu(dtpa)] (dtpa=diethylenetriaminepentaacetic acid), [Eu(dtpa)(BSA)], and [Eu(dtpa)(PLL)] (PLL=poly(L-lysine)), and tested their permeation properties on Madin?Darby canine kidney (MDCK) cells. The experimental results showed that all three probes were nontoxic to MDCK cells, permeated across MDCK monolayer exclusively via the paracellular pathways, and responded well to the changes on tight junction with high correlation of P(app) values to the decrease of trans-epithelial electric resistance (TEER). In addition, time-resolved fluorescence assays were conducted in a high-sensitivity and background-free mode. All these results confirmed the Eu complexes as novel and practical paracellular indicators.     

Recent developments in ruthenium anticancer drugs

Interest in Ru anticancer drugs has been growing rapidly since NAMI-A ((ImH(+))[Ru(III)Cl(4)(Im)(S-dmso)], where Im = imidazole and S-dmso = S-bound dimethylsulfoxide) or KP1019 ((IndH(+))[Ru(III)Cl(4)(Ind)(2)], where Ind = indazole) have successfully completed phase I clinical trials and an array of other Ru complexes have shown promise for future development. Herein, the recent literature is reviewed critically to ascertain likely mechanisms of action of Ru-based anticancer drugs, with the emphasis on their reactions with biological media. The most likely interactions of Ru complexes are with: (i) albumin and transferrin in blood plasma, the former serving as a Ru depot, and the latter possibly providing active transport of Ru into cells; (ii) collagens of the extracellular matrix and actins on the cell surface, which are likely to be involved in the specific anti-metastatic action of Ru complexes; (iii) regulatory enzymes within the cell membrane and/or in the cytoplasm; and (iv) DNA in the cell nucleus. Some types of Ru complexes can also promote the intracellular formation of free radical species, either through irradiation (photodynamic therapy), or through reactions with cellular reductants. The metabolic pathways involve competition among reduction, aquation, and hydrolysis in the extracellular medium; binding to transport proteins, the extracellular matrix, and cell-surface biomolecules; and diffusion into cells; with the extent to which individual drugs participate in various steps along these pathways being crucial factors in determining whether they are mainly anti-metastatic or cytotoxic. This diversity of modes of action of Ru anticancer drugs is also likely to enhance their anticancer activities and to reduce the potential for them to develop tumour resistance. New approaches to metabolic studies, such as X-ray absorption spectroscopy and X-ray fluorescence microscopy, are required to provide further mechanistic insights, which could lead to the rational design of improved Ru anticancer drugs.     

Interactions of sulindac and its metabolites with phospholipid membranes: An explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO?) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of Sulindac and its metabolites was tested and related with their membrane interactions. Drug–membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

Interactions of sulindac and its metabolites with phospholipid membranes: an explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO*) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of sulindac and its metabolites was tested and related with their membrane interactions. Drug-membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

Quantum dots targeted to the assigned organelle in living cells

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.