Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells

Abstract

Chelatable, mobile forms of divalent zinc, Zn(II), play essential signaling roles in mammalian biology. A complex network of zinc import and transport proteins has evolved to control zinc concentration and distribution on a subcellular level. Understanding the action of mobile zinc requires tools that can detect changes in Zn(II) concentrations at discrete cellular locales. We present here a zinc-responsive, reaction-based, targetable probe based on the diacetyled form of Zinpyr-1. The compound, (6-amidoethyl)triphenylphosphonium Zinpyr-1 diacetate (DA-ZP1-TPP), is essentially nonfluorescent in the metal-free state; however, exposure to Zn(II) triggers metal-mediated hydrolysis of the acetyl groups to afford a large, rapid, and zinc-induced fluorescence response. DA-ZP1-TPP is insensitive to intracellular esterases over a 2-h period and is impervious to proton-induced turn-on. A TPP unit is appended for targeting mitochondria, as demonstrated by live cell fluorescence imaging studies. The practical utility of DA-ZP1-TPP is demonstrated by experiments revealing that, in contrast to healthy epithelial prostate cells, tumorigenic cells are unable to accumulate mobile zinc within their mitochondria.

Keywords: fluorescence microscopy; prostate cancer; reaction-based probe; zinc biology.

Fig. 1.

Schematic representation of zinc-induced fluorescence from DA-ZP1-TPP. (A) ZP1-TPP is rendered nonfluorescent by addition of acetyl groups on the phenolic oxygen atoms of the xanthene ring. (B) Coordination of Zn(II) enhances fluorescence intensity by promoting ester hydrolysis and alleviating PeT originating from the two DPA arms. (C) Fluorescence enhancement is partially reversed by removing Zn(II), which restores the fluorescence-quenching ability of the DPA groups.

Fig. 2.

Fluorescence imaging of ZP1-TPP in live HeLa cells. HeLa cells were pretreated with a 5-µM solution of ZP1-TPP in dye- and serum-free DMEM for 30 min (37 °C, 5% CO₂). Initial fluorescence images reveal a distinct punctate pattern that does not respond significantly to medium supplemented with 50 µM ZnPT or 100 µM TPEN. (Scale bar: 25 µm.)

Fig. 3.

Zn(II)-dependent fluorescence signal enhancement of DA-ZP1-TPP. Normalized fluorescence spectra of a 1 µM solution of DA-ZP1-TPP in buffer (50 mM PIPES, 1 mM EDTA, 2 mM CaCl₂, and 100 mM KCl; pH 7) on increasing concentrations of free zinc ions. (Inset) Zinc-binding isotherm normalized to the integrated fluorescence signal intensity under zinc-saturating conditions.

Fig. 4.

Normalized fluorescence signal for DA-ZP1-TPP as a function of increasing pH in the presence of 125 µM ZnCl₂ (black) or 250 µM EDTA (gray). Data were acquired in 17.5 mM (0.1% vol/vol) acetic acid in Milli-Q water starting at pH 3.5. The pH of the solution was increased by sequential addition of aqueous KOH. The total volume of KOH added was less than 5% of the total volume. λ_ex = 475 nm.

Fig. 5.

Metal ion selectivity of DA-ZP1-TPP. Average normalized fluorescence intensities for a 1.1 μM solution of DA-ZP1-TPP in buffer (50 mM PIPES, 100 mM KCl; pH 7) at 25 °C, after addition of 50 µM–2 mM concentrations of various metal ions (white bars), followed by addition of 50 µM ZnCl₂ (black bars). The gray column represents the average sensor intensity after addition of 100 μM EDTA. Data were normalized to initial measured emission intensity.

Fig. 6.

Kinetic traces for the deacetylation of DA-ZP1-TPP. The half-life for deacetylation of a 2.75 µM solution of DA-ZP1-TPP at 37 °C in buffer (50 mM PIPES, 100 mM KCl; pH 7), was measured in the presence of 125 µM ZnCl₂ (black circles), 250 µM EDTA (triangle), or 1.25 U of porcine liver esterase (white circles). The change in absorbance at 520 nm (EDTA and esterase) or 510 nm (ZnCl₂) was used to monitor the reaction.

Fig. 7.

Fluorescence imaging of live HeLa cells. (A) DIC image. (B) Initial fluorescence image, showing a minimal fluorescence signal, which was stable for a period of 2 h (F). (C) Addition of 50 µM ZnPT. (D) Signal from MitoTracker Red. (E) Overlay of C and D. Pearson’s r = 0.64 ± 0.1 (n = 18). (G) Quantification of the change in fluorescence signal intensity of DA-ZP1-TPP (black bars) compared with ZP1-TPP (gray bars) under similar conditions. (Scale bar: 37 µm.)

Fig. 8.

Decreased mitochondrial zinc uptake in cancerous cell lines. RWPE-1, RWPE-2, and PC-3 prostate cell lines were incubated for 24 h in medium supplemented with (+) and without (−) 50 µM ZnCl₂. RWPE-1 is the only cell line demonstrating a statistically significant (P < 0.001) increase in mitochondrial zinc uptake when incubated in zinc-enriched media.