Fluorescence Lifetime Characterization of Novel Low-pH Probes

Abstract

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH.

The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

“…Using this model, we designed a vital staining strategy incorporating the zinc-indicator zinquin (Zalewski et al, 1993(Zalewski et al, , 1994Snitsarev et al, 2001) and the pH-indicator LysoSensor Green DND-189 (Lin et al, 2001). Both dyes possess fluorescent properties that depend on the biochemical environment where they partition.…”

“…To explore this hypothesis, we determined, in cultured hippocampal neurons, both the vesicular ionic zinc content, by using the zinc-specific, fluoroprobe zinquin (Zalewski et al, 1993(Zalewski et al, , 1994Snitsarev et al, 2001) and the vesicular pH with LysoSensor Green DND-189. This dye stains plasma membrane and acidic compartments and possesses a pK a identical to the SV intravesicular pH (pH 5.2) (Lin et al, 2001).…”

“…To explore this hypothesis, we determined, in cultured hippocampal neurons, both the vesicular ionic zinc content, by using the zinc-specific, fluoroprobe zinquin (Zalewski et al, 1993(Zalewski et al, , 1994Snitsarev et al, 2001) and the vesicular pH with LysoSensor Green DND-189. This dye stains plasma membrane and acidic compartments and possesses a pK a identical to the SV intravesicular pH (pH 5.2) (Lin et al, 2001).Both zinquin and LysoSensor Green stained processes and boutons of isolated hippocampal neurons (Figure 8, A and B). Zinquin-and Lysosensor-positive compartments correspond to vesicles that undergo regulated secretion, as revealed by their redistribution from hippocampal neuronal processes after ␣-latrotoxin treatment ( Figure 8A).…”

Glucose Acutely Decreases pH of Secretory Granules in Mouse Pancreatic Islets

“…By acting on a sulfonylurea-like protein in the granule membrane, ATP (formed from glucose metabolism) could promote Cl Ϫ influx into the granule, thereby providing counterions for H ϩ pumping by the v-H ϩ -ATPase (16). In the present study, we have used Lysosensor DND-160, a fluorescent probe that, unlike Lysosensor DND-189 used previously, permits ratioing measurement of pH and therefore, decreases potential problems linked to differences in loading or redistribution between cellular compartments (26). This probe has previously been instrumental to measure stimulus-induced pH changes in lysosomes (27), in intracellular canaliculi of parietal gastric cells (28,29), and in eosinophil granules (30).…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…The applications of FLIM in the biosciences are manifold and reach from monitoring cellular parameters (Ca 2+ 55-57 and Na + concentrations 58 , pH [59][60][61][62][63][64] , pO 2 and ROS concentrations [65][66][67] , pCO 2 68 ) and the distances between proteins on the nm-scale by means of FRET 5,35,42,[46][47][48][68][69][70][71][72][73][74][75][76][77][78][79][80][81] to observing central cellular processes in real time, e.g. interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] .…”