Using LysoSensor Yellow/Blue DND-160 to sense acidic pH under high hydrostatic pressures

DePedro, Hector Michael; Urayama, Paul

doi:10.1016/j.ab.2008.10.007

Cited by 38 publications

(32 citation statements)

References 14 publications

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“…As shown in Figure 2D, vacuolin-1 treatment raised lysosomal pH in HeLa cells. We further quantified lysosomal pH using quantitative ratiometric LysoSensor Yellow/Blue DND-160 38 -stained cells, and found that lysosomal pH in vacuolin-1-treated HeLa cells was increased from pH 4.7 in control cells to pH 5.2 (Fig. 2E).…”

Section: Vacuolin-1 Inhibited Autophagosome-lysosome Fusion In Hela Cmentioning

confidence: 99%

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

et al. 2014

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-l-phenylalanine 2-naphthylamide; LAMP1, lysosomal-associated membrane protein 1; Leup, leupeptin; MAP1LC3, microtubule-associated protein 1 light chain 3;MTOR, mechanistic target of rapamycin; RFP, red fluorescent protein; tfLC3, tandem fluorescence-tagged LC3.Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca 2C content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. IntroductionAmong 3 types of autophagy, including microautophagy, chaperone-mediated autophagy, and macroautophagy, in mammals, macroautophagy (hereafter referred as autophagy) is the most common type. Autophagy is an evolutionarily conserved catabolic degradation cellular process in which misfolded proteins or damaged organelles are first sequestered by a doublemembrane vesicle, called an autophagosome. Autophagosomes then fuse with lysosomes to form autolysosomes, inside which the sequestered contents are digested by lysosomal enzymes and recycled to maintain cellular homeostasis. Autophagy can also be markedly induced by a wide variety of stresses, e.g., nutrient starvation, infection, and aging, for cell survival. Dysfunctional autophagy has been associated with wide ranges of human diseases, e.g., cancer and neurodegenerative diseases. [1][2][3][4][5][6] Basal autophagy activity is essential for cell homeostasis, and it is tightly controlled by a complicated interplay among several key machineries, including ULK1 or ULK2 complexes and the class III phosphatidylinositol-3 kinase complexes. The MTOR (mechanistic target of rapamycin) Ser/Thr kinase suppresses autophagy by inhibiting the ULK1/2 comple...

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Section: Vacuolin-1 Inhibited Autophagosome-lysosome Fusion In Hela Cmentioning

confidence: 99%

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

et al. 2014

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“…Once incorporated into the biofilm matrix, the pH values can be measured based on fluorescence intensity ratio of the dual-wavelength fluorophore. 18 Lysosensor yellow/blue exhibits a dual-emission spectral peak (fluorescence emission maxima 452 and 521 nm) that is pH dependent. 18 The pH measurements are based on the principle that protonation of the fluorophore shifts its emission spectra, such that increasing ion concentrations increase fluorescence intensity at one wavelength, while decreasing it at the other.…”

Section: Methodsmentioning

confidence: 99%

“…18 Lysosensor yellow/blue exhibits a dual-emission spectral peak (fluorescence emission maxima 452 and 521 nm) that is pH dependent. 18 The pH measurements are based on the principle that protonation of the fluorophore shifts its emission spectra, such that increasing ion concentrations increase fluorescence intensity at one wavelength, while decreasing it at the other. 19 The fluorescence intensity of both emission wavelengths and the ratio of fluorescent intensity (I450/I520) within each biofilm confocal image were measured using Image J 1.44 and its calculation tools (for example, channel 1 divided by channel 2 as detailed in http://rsbweb.nih.gov/ij/download.html).…”

Section: Methodsmentioning

confidence: 99%

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Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface

et al. 2017

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To investigate how the biofilm three-dimensional (3D) architecture influences in situ pH distribution patterns on the enamel surface. Biofilms were formed on human tooth enamel in the presence of 1% sucrose or 0.5% glucose plus 0.5% fructose. At specific time points, biofilms were exposed to a neutral pH buffer to mimic the buffering of saliva and subsequently pulsed with 1% glucose to induce re-acidification. Simultaneous 3D pH mapping and architecture of intact biofilms was performed using two-photon confocal microscopy. The enamel surface and mineral content characteristics were examined successively via optical profilometry and microradiography analyses. Sucrose-mediated biofilm formation created spatial heterogeneities manifested by complex networks of bacterial clusters (microcolonies). Acidic regions (pH<5.5) were found only in the interior of microcolonies, which impedes rapid neutralization (taking more than 120 min for neutralization). Glucose exposure rapidly re-created the acidic niches, indicating formation of diffusion barriers associated with microcolonies structure. Enamel demineralization (white spots), rougher surface, deeper lesion and more mineral loss appeared to be associated with the localization of these bacterial clusters at the biofilm-enamel interface. Similar 3D architecture was observed in plaque-biofilms formed in vivo in the presence of sucrose. The formation of complex 3D architectures creates spatially heterogeneous acidic microenvironments in close proximity of enamel surface, which might correlate with the localized pattern of the onset of carious lesions (white spot like) on teeth.

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“…According to the principle of Le Chatelier and Braun, the equilibrium will shift to minimize the effect of pressure, thus favoring the ionization of weak acids in water. The pressure dependence of pH has been determined for a large number of weak acids and bases (8)(9)(10)(11)(12)(13)(14)(15)(16).…”

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confidence: 99%

Effect of Pressure-Induced Changes in the Ionization Equilibria of Buffers on Inactivation of Escherichia coli and Staphylococcus aureus by High Hydrostatic Pressure

Gayán

Condón

Álvarez

et al. 2013

Appl Environ Microbiol

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bSurvival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (⌬V°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ⌬V°values are approximately 5.0 to 7.0 cm 3 mol ؊1 , than in phosphate or dimethyl glutarate (DMG), whose ⌬V°values are about ؊25 cm 3 mol ؊1 . However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na 2 SO 4 and CaCl 2 ) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na 2 SO 4 than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes.

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Using LysoSensor Yellow/Blue DND-160 to sense acidic pH under high hydrostatic pressures

Cited by 38 publications

References 14 publications

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface

Effect of Pressure-Induced Changes in the Ionization Equilibria of Buffers on Inactivation of Escherichia coli and Staphylococcus aureus by High Hydrostatic Pressure

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