pH敏感的荧光探针及在活细胞中的应用

细胞内的pH是细胞内多种酶活性和生理活动的重要调节因素,准确、动态的监测细胞内pH变化对研究细胞内的活动至关重要。一些荧光小分子可以感应pH的变化,同时具有较高的灵敏度和特异性,对细胞损伤较小且标记操作简单,已逐渐发展成为一种监测细胞内pH变化的有效方法。本文主要介绍目前常用pH敏感的荧光探针及其在活细胞研究中的进展。     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Macromolecular spin pH-probes on the basis of human serum albumin

Human serum albumin has been chemically modified by two different spin pH-sensitive labels of the imidazoline series containing in their structure alkylating and carboxyl groups, respectively. The ESR spectra of spin-labeled proteins are sensitive to pH of the medium. The pK values of spin-labeled proteins measured by the ESR method are: pKI = 3.2 +/- 0.1; pKII = 4.75 +/- 0.1. The resulting macromolecular spin pH probes may be used for measuring the local values of pH by the ESR technique within the pH range of 1.8-6.2.     

Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry.

BACKGROUND: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. AIM: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. METHODS: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn ADP Flow Cytometer. RESULTS: The pHi clamping conditions were optimized as 140 mM potassium and 10 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. CONCLUSION: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

超分辨显微成像技术在活细胞成像中的应用与发展

超分辨显微成像技术(super-resolution microscopy,SRM)可以绕过光学衍射极限对成像分辨率的限制,让以前观察不到的纳米级结构实现可视化,这一重大研究进展推动了现代生命科学和生物医学研究的进步与发展.细胞是生物体的基本组成单位,对活细胞内部的细微结构和动力学过程进行研究是掌握生命本质必不可少的途径.但由于成像原理或条件的限制,早期的SRM技术在活细胞成像应用方面受到了不同程度的限制.近几年来,随着SRM和相关技术的发展,SRM在活细胞成像研究中的应用也越来越多.本文简要介绍目前常见的几种SRM技术的基本原理和特点,并在此基础上着重阐述它们在活细胞成像应用中所取得的最新研究进展和发展方向.     

细胞内pH值测定方法的研究进展

很多细胞的活动都对pH的变化十分敏感。pH值的有效控制对维持正常的细胞生理活动十分重要。如何有效监测细胞内pH值是很多细胞生物学的重要研究内容之一,如在研究细胞内转运蛋白、Ca2+离子等的变化活动时均需要测定细胞内的pH值,其相关的研究已有100多年的历史。本文将介绍目前几种细胞内pH值的主要测定方法,包括:弱酸弱碱分布法、核磁共振法、微电极法、荧光探针法等;每一种方法将从相关方法、技术的原理、特点、应用、局限性和注意事项等着手,将各个方法的优缺点进行横向的比较。本文还将重点探讨免疫探针法的最新进展,将报道一种最新的基于碳纳米点技术和荧光染料结合的pH定量测定计,还将介绍SNARF的两种最新衍生染料SNARF-F和SNARF-Cl的性能及其应用。     

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.     

Measuring magnetosomal pH of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using pH-sensitive fluorescent proteins

Magnetotactic bacteria synthesize uniform-sized and regularly shaped magnetic nanoparticles in their organelles termed magnetosomes. Homeostasis of the magnetosome lumen must be maintained for its role accomplishment. Here, we developed a method to estimate the pH of a single living cell of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using a pH-sensitive fluorescent protein E²GFP. Using the pH measurement, we estimated that the cytoplasmic pH was approximately 7.6 and periplasmic pH was approximately 7.2. Moreover, we estimated pH in the magnetosome lumen and cytoplasmic surface using fusion proteins of E²GFP and magnetosome-associated proteins. The pH in the magnetosome lumen increased during the exponential growth phase when magnetotactic bacteria actively synthesize magnetite crystals, whereas pH at the magnetosome surface was not affected by the growth stage. This live-cell pH measurement method will help for understanding magnetosome pH homeostasis to reveal molecular mechanisms of magnetite biomineralization in the bacterial organelle.     

Synthesis,structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.     

Receptor-mediated targeting of fluorescent probes in living cells

A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.     

Spin labelled phosphatidyl-MYO-inositols: Synthesis and properties

The paper describes the synthesis of phosphatidyl-myo-inositol mono and bi-radicals, containing spin labelled stearic acid residues, which can be used in studies of membranes and protein lipid interactions. The EPR study of certain physico-chemical properties of the products important for their application as spin probes (aggregation in various media, pH stability range) is reported.     

In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.     

Near-infrared fluorescent nanocapsules with reversible response to thermal/pH modulation for optical imaging

Polymeric near-infrared (NIR) fluorescent nanocapsules were developed, of which the fluorescence exhibited reversible response to local thermal/pH modulation. Our strategy was to use polymeric micelles made of temperature-sensitive Pluronic F-127 to encapsulate an amphiphilic NIR fluorescent dye-indocyanine green (ICG)-within the core and then cross-link the micelle corona by pH-sensitive poly(ethylenimine) (PEI). The size swelling/shrinking property of the micelles induced by temperature decrease/increase was used as a switch to control the fluorescence yield of the nanocapsules. It was found that the fluorescence yield significantly increased with the increase in temperature. The PEI cross-link made the fluorescence yield also sensitive to local pH change and enhanced intracellular delivery of the nanocapsules as well. Preliminary results suggest the NIR fluorescent probes could be potentially used as a contrast agent sensitive to local environment for translational optical imaging/sensing.     

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

Determination of intracellular pH using sensitive, clickable fluorescent probes

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK(a) value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H(+)] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using 'click' chemistry.     

Fluorescent proteins as sensors for cellular functions

The exploitation of green fluorescent protein-based biosensors promises to revolutionize functional imaging of the nervous system. Various approaches have created a multitude of reporters of neuronal activity and of activation of biochemical signaling pathways. Although the number of different probes has increased significantly, the critical step remains to bring these probes from the cuvette through the imaging of single cells to the imaging of whole organisms in vivo. The recent development of new genetically encoded sensors and their functional expression in model organisms are encouraging signs that the field is moving ahead in this direction.     

New pH-sensitive liposomes containing phosphatidylethanolamine and a bacterial dirhamnolipid

Phosphatidylethanolamine-based pH-sensitive liposomes of various compositions have been described as efficient systems for cytoplasmic delivery of molecules into cells. Incorporation of an amphiphile of appropriate structure is needed for the stabilization and performance of these vesicles. Among the wide variety of interesting activities displayed by Pseudomonas aeruginosa dirhamnolipids (diRL), is their capacity to stabilize bilayer structures in phosphatidylethanolamine systems. In this work, X-ray scattering, dynamic light scattering, fluorescence spectroscopy and fluorescence microscopy have been used to study the structure and pH-dependent behaviour of phosphatidylethanolamine/diRL liposomes. We show that diRL, in combination with dioleoylphosphatidylethanolamine (DOPE), forms stable multilamellar and unilamellar liposomes. Acidification of DOPE/diRL vesicles leads to membrane destabilization, fusion, and release of entrapped aqueous vesicle contents. Finally, DOPE/diRL pH-sensitive liposomes act as efficient vehicles for the cytoplasmic delivery of fluorescent probes into cultured cells. It is concluded that DOPE/diRL form stable pH-sensitive liposomes, and that these liposomes are incorporated into cultured cells through the endocytic pathway, delivering its contents into the cytoplasm, which means a potential use of these liposomes for the delivery of foreign substances into living cells. Our results establish a new application of diRL as a bilayer stabilizer in phospholipid vesicles, and the use of diRL-containing pH-sensitive liposomes as delivery vehicles.