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。     

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。     

不同pH值缓冲液中蚕豆花药小孢子的有丝分裂状况

近年来随着细胞内pH值测定技术的改进,对胞内pH值生理影响的研究报告显著增多。在动物细胞方面,有报告指出细胞在分裂时胞内pH值有明显升高的变化。并有报告指出外源pH值可以通过影响胞内pH值而影响细胞的分裂。在植物细胞,也     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时, 其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质, 并从多个方面对其应用进行了概括, 包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等, 且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时,其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质,并从多个方面对其应用进行了概括,包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等,且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

质膜Na^＋／H^＋逆向转运蛋白与植物耐盐性

土壤盐碱化是造成农作物减产的主要原因之一。质膜Na^＋／H^＋逆向转运蛋白能够介导植物根部Na^＋的外排和体内Na^＋的长距离运输, 并能够调控细胞K＋的稳态平衡及细胞内pH值和Ca^2＋的转运, 因此其在植物耐盐性方面具有重要作用。该文概述了植物质膜Na^＋／H^＋逆向转运蛋白的分子结构、功能、表达调控及其与植物耐盐性关系等方面的研究进展, 并对今后有关该蛋白的主要研究方向作了分析和展望。     

绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

植物细胞Ca2+荧光蛋白指示剂研究进展

Ca2+作为第二信使参与了植物生长和发育过程的调控,不同生物和非生物胁迫信号均可诱导胞内Ca2+变化.对Ca2+在信号转导作用中的认识主要来自于细胞内Ca2+浓度测定.水母发光蛋白和基于荧光蛋白的Ca2+荧光指示剂作为检测细胞Ca2+信号的手段是近年发展起来的新方法.本文综述了水母发光蛋白和基于荧光蛋白的Ca2+荧光指示剂的发展、测量原理、优点与不足及其在细胞Ca2+信号转导中的应用研究进展.     

植物生长素通过ABP1促进细胞膜泡的外排运输

为了研究植物生长素结合蛋白ABP1(auxin binding protein 1)对膜泡运输的调控,将烟草生长素结合蛋白基因ABP1 cDNA分别构建成可诱导型表达的过表达和干扰表达载体,并将绿色荧光蛋白GFP与烟草分泌载体膜蛋白SCAMP2(secretory carrier membrane protein 2)融合进行细胞的膜泡标记,转化植物模式细胞BY-2后分别获得了转ABP1和antiABP1的两类膜泡标记转基因细胞系。以雌二醇诱导ABP1、antiABP1表达后,结合生长素处理,通过扫描激光共聚焦观察了细胞的膜泡运输变化。当诱导ABP1在细胞内过量表达后,以吲哚-3-乙酸(indole-3-acetic acid,IAA)处理细胞,在细胞核膜及周围内质网膜、细胞质膜以及其他细胞内膜系统都观察到强烈的荧光信号,说明细胞内膜泡运输更为活跃;当诱导antiABP1在细胞内干扰表达时,在细胞核附近维持有较强烈的荧光信号,而细胞质膜及两细胞间隔的荧光信号明显减弱,表明抑制ABP1表达显著抑制了细胞膜泡的外排运输。在ABP1经诱导过表达后,加入IAA处理细胞,在0~6 min时间段内间隔性观察了细胞膜泡对生长素的时间响应,在这段时间内细胞核周围及内膜系统的荧光信号明显增强,细胞质膜的荧光强度没有明显的变化,表明细胞核与内膜系统间存在活跃的膜泡运输,内膜系统向细胞质膜间的外排膜泡运输也逐渐加强。因此,可以证明ABP1参与生长素信号响应,增强细胞膜泡的外排运输。     

大豆液泡膜H+-ATPase功能与构象关系的初步研究

大豆液泡膜V型H⁺-ATPase是ATPases中的一种,它在植物细胞的生长发育中有重要的作用.利用竹红菌乙素(HB）和KI这两种分别猝灭蛋白质疏水区域内源荧光和亲水区域内源荧光的荧光猝灭剂,在不同pH值、温度条件下对纯化的大豆液泡膜V型ATPase进行荧光猝灭实验,初步探讨了V型H⁺-ATPase的水解活性同其蛋白质折叠状态间的关系.研究表明,通过比较不同pH值、温度条件下蛋白质疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）,发现当环境pH值、温度偏离酶的最适pH值和温度时,蛋白质的内源荧光强度降低且疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）降低,说明伴随着酶的水解活性降低,蛋白质的折叠状态发生了变化.我们认为蛋白质在膜内的折叠状态变化是酶失活机制的一个重要方面,为植物的抗冻和抗盐研究提供了一定的参考.     

Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.     

Analysis of Golgi pH in Chinese hamster ovary cells using ratiometric pH-sensitive fluorescent proteins

Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4–6.5; trans-Golgi: 5.9–6.0; and cytoplasm: 7.1–7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control.     

Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

The pH‐sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside‐out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation‐resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady‐state levels and is compatible with kinetic analysis of endocytosis in live cells.     

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.     

The use of pHluorins for optical measurements of presynaptic activity

Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.     

High-resolution imaging of Ca2+ , redox status, ROS and pH using GFP biosensors

Many plant response systems are linked to complex dynamics in signaling molecules such as Ca(2+) and reactive oxygen species (ROS) and to pH. Regulatory changes in these molecules can occur in the timeframe of seconds and are often limited to specific subcellular locales. Thus, to understand how Ca(2+) , ROS and pH form part of plants' regulatory networks, it is essential to capture their rapid dynamics with resolutions that span the whole plant to subcellular dimensions. Defining the spatio-temporal signaling 'signatures' of these regulators at high resolution has now been greatly facilitated by the generation of plants expressing a range of GFP-based bioprobes. For Ca(2+) and pH, probes such as the yellow cameleon Ca(2+) sensors (principally YC2.1 and 3.6) or the pHluorin and H148D pH sensors provide a robust suite of tools to image changes in these ions. For ROS, the tools are much more limited, with the GFP-based H(2) O(2) sensor Hyper representing a significant advance for the field. However, with this probe, its marked pH sensitivity provides a key challenge to interpretation without using appropriate controls to test for potentially coupled pH-dependent changes. Most of these Ca(2+) -, ROS- and pH-imaging biosensors are compatible with the standard configurations of confocal microscopes available to many researchers. These probes therefore represent a readily accessible toolkit to monitor cellular signaling. Their use does require appreciation of a minimal set of controls but these are largely related to ensuring that neither the probe itself nor the imaging conditions used perturb the biology of the plant under study.     

M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins

Background

Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.

Methodology/Principal Findings

Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.

Conclusions/Significance

Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.     

Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress

For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca(2+)]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca(2+)] in the apoplast and in the cytoplasm were studied under salt and "drought" (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells.     

Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight''s response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.     

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by ¹H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo ¹H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15–60 min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5 h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.