Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues

Abstract

Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors have been developed, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes consisting of a variety of fluorophores and zinc-binding units. The results provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the μM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (~50–100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.

AMPA receptor inhibition by synaptically released zinc

“…To determine whether zinc modulation of PF EPSCs is input-specific in fusiform cells and whether zinc modulates PF EPSCs in another synapse with different AMPAR composition, we took advantage of this anatomical and functional segregation of synaptic inputs in the DCN. First, we demonstrated that the DCN molecular layer is zinc-rich by using a cell-permeable, acetylated zinc fluorescent sensor diacetylated Zinpyr-1 (DA-ZP1) (34). Consistent with previous anatomical studies (32), our imaging experiments revealed a zinc-specific fluorescent signal that is ZnT3 dependent and specific to the molecular layer of the DCN (Fig.…”

Tuning Mg(II) Selectivity: Comparative Analysis of the Photophysical Properties of Four Fluorescent Probes with an Alkynyl‐Naphthalene Fluorophore

“…[1] The application of fluorescent sensing is widespread, with avariety of fluorescent sensors providing detailed information on the roles of biologically active cations [2][3][4][5] and anions [6] in vitro and in cellulo.T he use of ratiometric probes is highly advantageous, as it allows quantitative informationt ob eo btained independent of environmental factors, such as uneven cell loading or photobleaching. [1] The application of fluorescent sensing is widespread, with avariety of fluorescent sensors providing detailed information on the roles of biologically active cations [2][3][4][5] and anions [6] in vitro and in cellulo.T he use of ratiometric probes is highly advantageous, as it allows quantitative informationt ob eo btained independent of environmental factors, such as uneven cell loading or photobleaching.…”

Purine‐Based Fluorescent Sensors for Imaging Zinc Ions in HeLa Cells

“…So there is redshift of 49 nm in the absorption spectrum, while for the emission spectrum, it is 17 nm. With other metal ions, such as Na + , K + , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Ag + , Cd 2+ , and Hg 2+ , there is no significant enhancement in the emission intensity (Figure ), and so, the observed enhancement in fluorescence could be due to photoinduced‐electron‐transfer (PET) quenching between the chelating unit and the fluorophore , , . As the concentration of zinc ions is increased, there is enhancement in the fluorescence of HL1 , as can be seen in Figures and S8; this could be due to an equilibrium between the free and bound zinc ions with the ligand.…”