title: 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 are available, 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 containing a variety of fluorophores and zinc-binding units. The new sensors 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Zastrow
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Radford
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Chyan
authors.affiliation: Departments of Otolaryngology and Neurobiology, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Anderson
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Daniel Y
authors.initials: DY
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Andrei
authors.initials: A
authors.lastname: Loas
authors.affiliation: Departments of Otolaryngology and Neurobiology, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States.
authors.forename: Thanos
authors.initials: T
authors.lastname: Tzounopoulos
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
keywords: ZnT3; auditory synapses; fluorescence; metalloneurochemistry; zinc sensors