Organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-Tag protein labeling

Abstract

Hydrogen peroxide (H(2)O(2)) is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of its production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring local, subcellular changes in H(2)O(2) levels by fluorescence imaging. Specifically, we have exploited the versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface or interior of living cells with the use of boronate-capped dyes to selectively visualize H(2)O(2). The resulting SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to SNAP-tag fusion proteins. Spectroscopic measurements of the SNAP-PG constructs confirm their ability to detect H(2)O(2) with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence turn-on in response to changes in local H(2)O(2) levels. This work provides a general molecular imaging platform for assaying H(2)O(2) chemistry in living cells with subcellular resolution.

Figure 1

Design strategy for organelle-specific hydrogen peroxide reporters using the SNAP tag methodology.

Figure 2

Fluorescence responses of 1 μM AGT-PG, a conjugate formed from the reaction of AGT with SPG2, to 100, 250, 500, and 1000 μM H₂O₂ in 20 mM HEPES, pH 7 at 25 °C. The plot shows emission responses at 0, 10, 20, 30, 40, 50, 60, and 120 min after H₂O₂ addition. The reactions are not complete at these early time points. The collected emission was integrated between 500–650 nm (λ_exc = 488 nm).

Figure 3

Absorption spectra of 1 μM AGT-PG in 20 mM HEPES, pH 7 upon addition of 1 mM H₂O₂. Spectra were acquired at one hour intervals for 8 hours. An isosbestic point was observed at 460 nm.

Figure 4

Fluorescence responses of 1 μM AGT-PG to various concentrations of added H₂O₂. Spectra were acquired in 20 mM HEPES, pH 7 at 25 °C after incubation of the probe with H₂O₂ for 30 min. Reactions are not complete at this early time point. The collected emission was integrated between 500–650 nm (λ_exc = 488 nm).

Figure 5

Targeted localization of STG1 and STG2 in living HEK293T cells by conjugation to SNAP-AGT fusion proteins. Cells were incubated with 5 μM STG1 or STG2 for 30 min, and washed with fresh DMEM + 10% FBS for 30 min (2 × 1 mL) before image acquisition. Rows (a) and (b) show HEK 293T cells transiently expressing SNAP-NK1R. Rows (c) and (d) display HEK 293T cells transiently expressing pSNAP for non-specific intracellular tagging, and row (e) presents HEK 293T cells transiently expressing SNAP-H2B. For each series: (1) emission from labeling with STG1 or STG2, (2) nuclear staining with Hoechst 33342, and (3) DIC image. Scale bar = 20 μm.

Figure 6

Targeted labeling of endoplasmic reticulum and mitochondria organelles with STG2. Row (a) shows HEK 293T cells expressing SNAP-KDEL in the ER lumen, and row (b) depicts HEK 293T cells expressing SNAP-Cox8A for mitochondrial tagging. For each series: (1) emission from labeling with STG2, (2) emission from (a) mCherry-KDEL or (b) mCherry-Cox8A, (3) overlay of STG2 and mCherrry, (4) nuclear staining with Hoechst 33342, and (5) DIC image. Scale bar = 20 μm.

Figure 7

Fluorescence detection of H₂O₂ on the surface of or within living HEK 293T cells. Row (a) shows cells transiently expressing SNAP-NK1R tagged with SPG1, row (b) displays cells transiently expressing pSNAP tagged with SPG2, and row (c) presents cells transiently expressing SNAP-H2B tagged with SPG2. H₂O₂ was added from 50 mM solution in Mili-Q water. Time-lapse image acquisition was assisted by a motorized stage equipped with incubator and humidifier maintaining 37 °C and 5% CO₂ atmosphere. For each series: (1–4) pseudocolor mode of fluorescent emission at 0, 10, 20, and 30 min after addition of 100 μM H₂O₂, (5, 6) fluorescent emission before (5) and after (6) treatment of cells with 100 μM H₂O₂ for 30 min (λ_exc = 488 nm, λ_em = 500–550 nm), (7) nuclear staining with Hoechst 33342, (8) DIC image. Scale bar = 20 μm.

Figure 8

Fluorescence detection of H₂O₂ in living HEK 293T cells transiently expressing SNAP-KDEL and mCherry-KDEL. Panels (a) and (b) show fluorescent emission from cells labeled with SPG2 before (a) and after (b) treatment with 100 μM H₂O₂ for 30 min (λ_exc = 488 nm, λ_em = 500–550 nm), panel (c) presents emission from mCherry-KDEL, panel (d) displays the overlay of (b) and (c), panel (e) shows nuclear staining with Hoechst 33342, and panels (f–i) display pseudocolor fluorescent emission at 0, 10, 20, and 30 min after addition of 100 μM H₂O₂. Panel (j) presents the DIC image of cells in (i). Scale bar = 20 μm.

Figure 9

Fluorescence detection of H₂O₂ in living HEK 293T cells transiently expressing SNAP-Cox8A and mCherry-Cox8A. Panels (a) and (b) show fluorescent emission from cells labeled with SPG2 before (a) and after (b) treatment with 100 μM H₂O₂ for 30 min (λ_exc = 488 nm, λ_em = 500–550 nm), panel (c) presents emission from mCherry-Cox8A, panel (d) displays the overlay of (b) and (c), panel (e) shows nuclear staining with Hoechst 33342, and panels (f-i) display pseudocolor fluorescent emission at 0, 10, 20, and 30 min after addition of 100 μM H₂O₂. Panel (j) presents the DIC image of cells in (i). Scale bar = 20 μm.

Figure 10

Relative fluorescence emission from time-lapse confocal images of SPG1 or SPG2 labeled HEK 293T cells in response to added 100 μM H₂O₂ at 0, 10, 20, 30 min. Error bars represent standard error measurement (s.e.m).

Scheme 1

Synthesis of SNAP-tag substrates

Scheme 2

Synthesis of SNAP-Peroxy-Green-1 (SPG1) and SNAP-Peroxy-Green-2 (SPG2)

Scheme 3

Synthesis of SNAP-Tokyo Green-1 (STG1) and SNAP-Tokyo Green-2 (STG2)