A microtubule-localizing activity-based sensing fluorescent probe for imaging hydrogen peroxide in living cells

Abstract

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species (ROS) in living systems with broad roles spanning both oxidative stress and redox signaling. Indeed, owing to its potent redox activity, regulating local sites of H₂O₂ generation and trafficking is critical to determining downstream physiological and/or pathological consequences. We now report the design, synthesis, and biological evaluation of Microtubule Peroxy Yellow 1 (MT-PY1), an activity-based sensing fluorescent probe bearing a microtubule-targeting moiety for detection of H₂O₂ in living cells. MT-PY1 utilizes a boronate trigger to show a selective and robust turn-on response to H₂O₂ in aqueous solution and in living cells. Live-cell microscopy experiments establish that the probe co-localizes with microtubules and retains its localization after responding to changes in levels of H₂O₂, including detection of endogenous H₂O₂ fluxes produced upon growth factor stimulation. This work adds to the arsenal of activity-based sensing probes for biological analytes that enable selective molecular imaging with subcellular resolution.

Keywords: Activity-based sensing; Fluorescent probe; Hydrogen peroxide; Molecular imaging; Reactive oxygen species.

Figure 1.

Structures of (a) microtubule-targeting molecule docetaxel; (b) mitochondria-localizing H₂O₂ probe Mito-PY1, and (c) microtubule-localizing H₂O₂ probe MT-PY1.

Figure 2.

In vitro characterization of MT-PY1. (a) Fluorescence emission spectrum of 5 μM MT-PY1 in 25 mM HEPES buffer pH 7.4 (bottom), and its turn-on response after treatment with 100 μM H₂O₂ at 37 °C for 5, 15, 30, 45 and 60 min. (b) Fluorescence responses of 5 μM MT-PY1 to 100 μM of various reactive oxygen and nitrogen species at 37 °C after 5, 15, 30, 45 and 60 min of incubation.

Figure 3.

Confocal microscopy images of MT-PY1 localization in HeLa cells. Cells were incubated with 1 μM MT-PY1 for 15 min at 37 °C prior to imaging. (a) A HeLa cell in metaphase. (b) A HeLa cell in interphase. Cell nucleus is stained by Hoechst 33342 in (a) and (b). (c) HeLa cells expressing mCherry-tubulin stained with MT-PY1. Enlarged image is shown in Supplementary Figure S2. Scale-bar: 10 μm.

Figure 4.

Fluorescence responses of MT-PY1 to exogenous addition of H₂O₂ in living cells. HeLa cells were incubated with 1 μM MT-PY1 for 0.5 h at 37 °C, followed by treatment with (a) vehicle control or (b) 100 μM H₂O₂ at 37 °C for 0.5 h; quantification is shown in (c). Scale-bar: 20 μm.

Figure 5.

Fluorescence imaging of endogenous H₂O₂ generated by the EGF signaling pathway in live A431 cells. A431 cells incubated with 1 μM MT-PY1 for 0.5 h at 37 °C were treated with (a) vehicle control, (b) 100 ng/mL EGF, (c) 100 ng/mL EGF and 500 μM L-NAME or (d) 100 ng/mL EGF and 50 μM PD15305 for 30 min at 37 °C and imaged. (e) Zoomed-in images of A431 stained with MT-PY1 showing the high spatial resolution of this fluorescence probe. (f) Quantification of fluorescence intensity of a-d shown as mean ± s.d. Scale-bar: 50 μm.

Scheme 1.

Synthesis of MT-PY1 and its fluorescence turn-on reaction upon activity-based sensing of H₂O₂.