Fluorescent Probes for HOCl Detection in Living Cells

Russian Journal of Bioorganic Chemistry - Hypochlorous acid (HOCl) plays an important role in the immune system not only protecting the organism from pathogens, but also, due to its high...     

Colorful Protein-Based Fluorescent Probes for Collagen Imaging

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni²⁺-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.     

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.     

Uptake and Compartmentation of Fluorescent Probes by Plant Cells

Several fluorescent compounds are now being used as probes forstudying plant transport processes. This review considers thepotential mechanisms of uptake of such probes with particularemphasis on their subsequent compartmentation within the cell.Physico-chemical parameters, such as the dissociation constant(pK_a) and polarity (log k^ow) of the dye molecule provide importantguides as to the likely permeability of the plasmalemma to differentfluorochromes and an ion-trap mechanism may explain the accumulationof many fluorescent probes by plant cells. However, physico-chemicalparameters alone do not always explain the subsequent compartmentationof fluorescent probes within the cell. Evidence is accumulatingthat many anionic fluorescent probes may cross the plasmalemmain the undissociated state, followed by carrier-mediated transportof the anion across the tonoplast. In the specialized case ofthe highly dissociated dye, Lucifer Yellow CH (LYCH), the physico-chemicalproperties of the molecule would predict that it should be unableto cross membranes. Despite this, there have been several reportsof the movement of LYCH from the apoplast to the vacuole ofplant cells. Fluid-phase endocytosis has been implicated inthe vacuolar accumulation of LYCH and also a range of high-molecularweight, purified fluorescent conjugates. This evidence is discussedin the light of some reports that membrane-impermeant dyes,including LYCH, may cross the tonoplast following their microinjectioninto the cytoplasm.     

钙荧光探剂的研究及其在生命科学中的应用

钙荧光探剂测量活细胞胞浆游离Ｃａ２＋浓度的方法在钙研究中已成为一种越来越重要的技术。特别是由于新的一代荧光探剂的合成和激光共聚焦显微镜的发展,使其应用更加广泛。由于国内使用这种技术的实验室逐渐增多,本文将系统介绍钙荧光探剂的发展、测量原理和方法、新的常用钙荧光探剂的比较及其在生命科学中的应用。     

Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes

We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGlu_h, iGlu_m, and iGlu_l cover a broad range of K_d-s (5.8 μM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a K_d of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGlu_u and iGlu_f had monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlu_m and iGlu_l, the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGlu_h and Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion-limited rate constant to iGlu_h and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding.     

Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes

We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.     

Reaction Fields in the Environment of Fluorescent Probes: Polarity Profiles in Membranes

Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains.     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Strategies for Positioning Fluorescent Probes and Crosslinkers on Formyl Peptide Ligands

Abstract

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1–3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.     

Fluorescent Labeling of Oligonucleotide Probes for Double Indicator Microarray Hybridization Analysis

Russian Journal of Bioorganic Chemistry - Herein, we present a synthesis of a molecular construct based on the polysulfonated indocarbocyanine dye and the lysine. The chemical structure of the...     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.     

Characterization of a New Series of Fluorescent Probes for Imaging Membrane Order

Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.     

Synthesis,Photophysical Properties,and Cytotoxicity of Rhodamine Based Fluorescent Probes

Russian Journal of Bioorganic Chemistry - Rhodamine derivatives are heterocyclic compound, related to the fluorescent probe having a profound application in the field of biotechnology. Herein, we...     

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ∼390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room- and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.     

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

Fluorescent Probes DPH,TMA-DPH and C17-HC Induce Erythrocyte Exovesiculation

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.