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1.
《Biophysical journal》2022,121(7):1156-1165
Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.  相似文献   

2.
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.  相似文献   

3.
We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.  相似文献   

4.
Summary A number of methods have been developed to measure intracellular pH (pHi) because of its importance in intracellular events. A major advance in accurate pHi measurement was the development of the ratiometric fluorescent indicator dye, 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We have used a fluorescence multi-well plate reader and a ratiometric method for determining pHi in primary cultures of rabbit corneal epithelial (CE) cells with BCECF. Fluorescence was measured at excitation wavelengths of 485±11 nm and 395±12.5 nm, with emission detected at 530±15 nm. Cells grown in multi-well plates were loaded with 4 μM BCECF for 30 min at 37° C. Resting pHi was 7.34±0.03 (2 cultures, N=5 wells). Changes in pHi determined with the fluorescence multi-well plate reader after the addition and removal of NH4Cl or sodium lactate were comparable to changes in cells analyzed with a digitized fluorescence imaging system. A concentration-response relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionophore. Low doses of ionomycin (2.5–5 μM), produced a prolonged acidification; 7.5 μM ionomycin produced a transient acidification; and 10 μM ionomycin resulted in a slight alkalinization. We conclude that accurate pHi measurements can be obtained with a ratiometric method with BCECF in a multi-well plate reader. This technology may simplify screening studies evaluating effects of hormones, growth factors, or toxicants on pHi homeostasis.  相似文献   

5.
P Mariot  P Sartor  J Audin  B Dufy 《Life sciences》1991,48(3):245-252
Intracellular pH (pHi) can now be measured at the single cell level using dual emission wavelength microspectrofluorimetry with the fluorescent pH indicator SNARF 1 and its membrane permeant acetoxymethyl ester (SNARF 1/AM). We measured pHi of individual pituitary cells under both basal and stimulated conditions. The emitted fluorescence of SNARF 1 probe was calibrated following experimental manipulations of pHi in two types of rat pituitary cells. The calibration curves obtained in the two cell types were identical. We observed a Gaussian distribution of individual pHi with a wide dispersion (6.95 to 8) in the two cell populations. TRH (10(-7) M) and ionomycin (5 microM) induced a transient acidification followed by a sustained alkalinization, whereas K+ (50 mM) depolarization only exerted a transient acidification. These results show that the dual emission pH indicator SNARF 1 can be used to reliably investigate changes in pHi in individual endocrine cells.  相似文献   

6.
In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.  相似文献   

7.
Each of the three plasma membrane Ato proteins is involved in ammonium signalling and the development of yeast colonies. This suggests that although these proteins are homologous, they do not functionally substitute for each other, but may form a functional complex. Here, we present a detailed combined FRET, FLIM and photobleaching study, which enabled us to detect interactions between Ato proteins found in distinct compartments of yeast cells. We thus show that the proteins Ato1p and Ato2p interact and can form complexes when present in the plasma membrane. No interaction was detected between Ato1p and Ato3p or Ato2p and Ato3p. In addition, using specially prepared strains, we were able to detect an interaction between molecules of the same Ato protein, namely Ato1p-Ato1p and Ato3p-Ato3p, but not Ato2p-Ato2p.  相似文献   

8.
Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers. Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.  相似文献   

9.
Flow cytometry was used to measure cytoplasmic pH (pHi) of B16 melanoma cells taken from tumor-bearing animals. We used a ratiometric method to allow measurements on an individual cell basis which were independent of cellular content of the pH indicator BCECF. In order to "freeze" any intercell variance which may have existed within the tumor mass, tumors were mechanically disaggregated in bicarbonate-free medium containing 0.5 mM amiloride at 4 degrees C and loaded with BCECF in choline chloride-based Earle's solution at 37 degrees C. Studies using cells grown in vitro showed that this protocol prevented acid load recovery during the 30-min period typically required between tumor excision and pHi measurement. A calibration curve was obtained by resuspending BCECF-stained cells in a range of buffers containing the proton ionophore nigericin. The range of values for individual cells was estimated by comparing the coefficient of variation of the test sample with that obtained when nigericin was used to reduce all cells to the pHi of the calibration buffer. The average value for mean tumor cell pH was 7.32 +/- 0.05 SD. Pretreatment of animals with intraperitoneal glucose for one hour resulted in an average for mean pHi of 7.17 +/- 0.17 SD. Mean coefficient of variation was 8.7%, and in the presence of nigericin, 8.1%. These values indicate a variance in measured pHi of approximately +/- 0.4 pH units, but most of this results from experimental error rather than true intercell pHi variance. The method used here is capable of detecting reduction in mean tumour pHi caused by ip glucose, but incapable of precise estimation of individual cell values. Despite these uncertainties, the results suggest that the range of pHi within B 16 tumors is small.  相似文献   

10.
It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.  相似文献   

11.
Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.  相似文献   

12.
It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.  相似文献   

13.
热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系   总被引:3,自引:0,他引:3  
应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pHI的影响,实验结果表明:细胞外pH对pHI略有影响,而生长碳源对pHI的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明:细胞比生长速率、CO2比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pHI的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pHI值维持在5.72~6.15范围内。  相似文献   

14.
On solid substrate, growing yeast colonies alternately acidify and alkalinize the medium. Using morphological, cytochemical, genetic, and DNA microarray approaches, we characterized six temporal steps in the "acid-to-alkali" colony transition. This transition is connected with the production of volatile ammonia acting as starvation signal between colonies. We present evidence that the three membrane proteins Ato1p, Ato2p, and Ato3p, members of the YaaH family, are involved in ammonia production in Saccharomyces cerevisiae colonies. The acid-to-alkali transition is connected with decrease of mitochondrial oxidative catabolism and by peroxisome activation, which in parallel with activation of biosynthetic pathways contribute to decrease the general stress level in colonies. These metabolic features characterize a novel survival strategy used by yeast under starvation conditions prevalent in nature.  相似文献   

15.
16.
Yuanyuan Li 《Luminescence》2017,32(8):1385-1390
A ratiometric fluorescent chemosensor 2‐(2′‐hydroxy‐3′‐formyl‐5′‐methoxyphenyl)benzothiazole ( 1 ) was developed for the detection of cysteine (Cys). In aqueous solution at neutral pH, 1 exhibited a ratiometric fluorescent response to Cys with a remarkable red‐to‐green shift in the emission wavelength. This fluorescence change was attributed to the cyclization reaction between the formyl group in 1 and the amino and sulfhydryl group in Cys in a stoichiometry of 1: 1 according to the proposed mechanism. At neutral pH, 1 displayed a significant fluorescence ratio signal enhancement with the addition of Cys. Furthermore, 1 showed good selectivity toward Cys. The detection limit and linear range were 5.6 and 0–100 μmol/L, respectively, which demonstrated that 1 could recognize relatively low concentrations of Cys and is a good candidate for applications in detecting Cys.  相似文献   

17.
Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery.  相似文献   

18.
Measuring yeast biomass is important in the processes of microbial fermentations. It has been demonstrated that synchronous light scattering (SLS) signals could be applied for the quantification of model bioparticles such as Saccharomyces cerevisiae. In this study, an improved synchronous light scattering method was developed for yeast biomass estimation. The settlement of yeast cells during SLS signals measuring process was studied, and hydrolysis anionic polyacrylamide was added into yeast suspensions to increase the stability of the cells in liquid environment. By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ?λ = 0), the SLS intensity was found to be proportional to the yeast concentration in the range from 0 to 4.9 × 106 cell/mL (R 2 = 0.9907), the detection limit is 8.1 × 103 cell/mL. The developed method exhibited good stability and sensitivity in the recovery test and growth curve drawing process, demonstrating the potential of the method in practical application of biomass estimation.  相似文献   

19.
Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.  相似文献   

20.
Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.  相似文献   

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