Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes

Abstract

Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor-acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a K of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells

“…Although higher ratios appear in cell nuclei, their significance is uncertain as the emission intensities in both channels are five to eight times lower compared with the cytoplasmic region. Similar distributions were observed with other lipophilic probes (10,19) and presumably indicate a partition equilibrium that disfavors the polar nuclear environment. Addition of the membrane-permeant high-affinity Cu(I) chelator PSP-2 (50 μM) resulted in complete reversal of the initial change to reach an intensity ratio around 0.66 ± 0.05, slightly below the basal value (n = 20; P = 0.0008).…”

“…3 B, Inset) and clean isosbestic points in both absorption and emission spectra indicate a well-defined 1:1 Cu(I)-binding equilibrium. With these results at hand, we further characterized the probe in aqueous buffer in the presence of model lipid bilayers to evaluate its suitability for cellular imaging (19). Since cellular lipid bilayers comprise a variable mixture of neutral and anionic head groups, we prepared liposomes using a 4:1 ratio of zwitterionic palmitoyl oleoyl phosphatidylcholine (POPC) to anionic palmitoyl oleoyl phosphatidylglycerol (POPG) in aqueous buffer (13).…”

“…S9), a time frame that is sufficient for restoration of thioldisulfide homeostasis after oxidative insult (28). Given that phosphines are frequently employed as disulfide-reducing agents, it is remarkable that the phosphine-based probe crisp-17 remains functional under conditions that rapidly liberate metals by oxidation of thiolate binding sites to disulfides (19). To confirm that the observed ratio changes are not due to a direct interaction of the probe with DTDP, we performed control experiments under acellular conditions.…”

“…To harness the advantages of the PSP ligand design strategy within a Cu(I)-responsive fluorescent probe, we now report crisp-17 ( Fig. 1), in which the electron-withdrawing PSP motif is incorporated into a push-pull fluorophore architecture (19) to yield an emission-ratiometric indicator suitable for two-photon excitation microscopy. Employed in live mammalian cells, crisp-17 allows visualization of base levels of labile Cu(I) as well as rapid dynamic changes evoked by treatment with the membranepermeant thiosemicarbazone Cu(II) complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP).…”

The redox biology of redox-inert zinc ions

“…In erythrocytes, a concentrations of 24 pM was measured [18]. With the advent of fluorescent chelating agents for zinc, a few hundred picomolar were determined in several cell lines [19,20]. Thus, zinc ions are buffered at a pZn of about 10.…”