C-SNARF-1 as a pHi fluoroprobe: discrepancies between conventional and intracellular data do not result from protein interactions

Yassine, Mohamed; Salmon, Jean‐Marie; Vigo, Jean; Viallet, Pierre

doi:10.1016/s1011-1344(96)07339-3

Cited by 20 publications

(20 citation statements)

References 27 publications

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“…However, it has also been suggested that SNARF-1 does not bind to bovine serum albumin but, rather, that a contaminant present in the commercially available SNARF-1 binds to bovine serum albumin (67). Furthermore, in the cytoplasm of cardiac myocytes, a major fraction of the fluoroprobes (0.5-0.9) appears to be bound to proteins (4).…”

Section: Discussionmentioning

confidence: 99%

Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Rojas¹,

Sennoune²,

Maiti³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3−-based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

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Section: Discussionmentioning

confidence: 99%

Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Rojas¹,

Sennoune²,

Maiti³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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“…These results differ from those previously reported (42), which show a significant alteration in emission spectra. More recently, however, it was determined that the SNARF variant used in that study (C-SNARF-1) contained a contaminant able to bind to BSA, which led to discrepancies between intracellular and cell-free spectral properties (55). C-SNARF-4 differs from the probe used in that study as it has a lower pKa value (6.4 compared to 7.5) due to a fluorine atom, and it lacks the acetoxymethylester substituent.…”

Section: Figmentioning

confidence: 93%

“…We have used C-SNARF-4 in combination with confocal scanning laser microscopy (CSLM) to study single-species Pseudomonas aeruginosa communities and the pH of their microenvironments. Since there have been recent reports suggesting C-SNARF emission characteristics can be influenced by the probe's interaction with various cell components (7,23,38,48,55), we wanted to determine the extent to which biofilm components (i.e., proteins, exopolymers, and bacterial cells) influence C-SNARF-4 emission properties. Accordingly, spectrofluorometric analyses were used to determine the effect of representative matrix components (alginate, bovine serum albumin, P. aeruginosa cells, and growth medium) on the spectral emission properties of the fluorophore.…”

mentioning

confidence: 99%

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

Hunter

Beveridge

2005

Appl Environ Microbiol

184

168

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An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.

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“…This seemed to be in contrast with reported changes in the pK a of cSNARF-1 inside cells (Owen 1992) that were attributed to the binding of the probe to cellular proteins (Seksek et al 1991). However, more-recent data revealed that a contaminant of the commercially distributed substance but not the molecule liberated from cSNARF-1 AM is able to bind to proteins (Yassine et al 1997).…”

Section: Calibration: Linking¯uorescence Signals To Ion Concentrationsmentioning

confidence: 99%

“…Compared with their parent compound¯uorescein the modern probes bear additional carboxyls that increase their polarity and intracellular retention. Today, the most widely applied probes for the mapping of cytosolic pH are cSNARF-1, a seminaphthorhoda¯uor dye that allows both excitation and emission ratioing Parton et al 1997;Yassine et al 1997) and Absorption optima are given; these are close to excitation optima but less sensitive to the environment of the molecule (Tsien 1989;Haugland 1996) BCECF, a carboxy¯uorescein derivative used for excitation ratioing (Haugland 1996). The SNAFL probes are seminaphtho¯uoresceins that, in contrast to cSNARF-1, display a stronger emission in their protonated than in their unprotonated form.…”

Section: Ph Indicatorsmentioning

confidence: 99%

Ion mapping in plant cells – methods and applications in signal transduction research

Roos

2000

Planta

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This review covers both methodical aspects and actual applications of ion imaging techniques in plant cell signal research. The methodological section explains the basic principles of fluorescence ion imaging, the impact of modern developments in fluorescence microscopy and introduces the most important fluorescence probes including aequorin and other photoproteins. It critically comments on loading strategies, intracellular compartmentation of probes and calibration procedures. The second part compiles actual research areas where the application of ion imaging procedures has gained substantial achievements and helped to establish new concepts of calcium- and pH-dependent signalling. Examples comprise the hormonal control of stomatal movements, effects of gibberellic and abscisic acids in aleurone cells, elicitation of phytoalexin production, cytosolic pH and cell development, and signatures of Ca2+ as a universal signal in plant cells.

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C-SNARF-1 as a pHi fluoroprobe: discrepancies between conventional and intracellular data do not result from protein interactions

Cited by 20 publications

References 27 publications

Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

Ion mapping in plant cells – methods and applications in signal transduction research

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C-SNARF-1 as a pHi fluoroprobe: discrepancies between conventional and intracellular data do not result from protein interactions

Cited by 20 publications

References 27 publications

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

Ion mapping in plant cells – methods and applications in signal transduction research

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Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Vacuolar-type H⁺-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells