Highly stable and sensitive fluorescent probes (LysoProbes) for lysosomal labeling and tracking

Abstract

We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis.

Figure 1. Chemical structures of LysoProbes I-VI.

Figure 2. Intracellular distribution of LysoProbe II compared to MitoTracker and LysoTracker Green.

LysoProbe II (30 μM, B) was incubated with cells in DMEM media without FBS and counterstained with MitoTracker (80 nM, A), Hoechst 33342 (1 μg/mL, C); and overlay (D); (E–H): HeLa cells incubated with LysoProbe II (10 μM, E), followed by counterstaining with LysoTracker (2 μM, F), Hoechst 33342 (1 μg/mL, G); and overlay (H); Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Figure 3. Chemical structures of rhodamine-lactose conjugates (I and II) without glycan moieties.

Figure 4. Confocal laser-scanning fluorescent images of LysoProbe I in HeLa cells.

LysoProbe I (2 μM, green, C) was incubated with cells in non-FBS DMEM media for 15 min., and then counterstained with LysoTracker (2 μM, no fluorescence, B), Hoechst 33342 (1 μg/mL, blue, A); DIC image (D), overlay (A+B+C) with DIC (E), overlay (A+B+C) without DIC image (F). All images were acquired using a 60 × objective lens following 24 h incubation.

Figure 5. Characterization of intracellular pH using LysoProbe II (10 μM).

After washing three times with corresponding pH buffers, the cells pre-loaded with LysoProbe II were incubated with nigericin and subsequently imaged in pH 4.4 (A), pH 5.0 (B), pH 5.5 (C) and pH 6.5 (D) buffers. The cells were imaged for fluorescence on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Figure 6. Confocal laser-scanning fluorescence imaging of freshly frozen colon tumor tissue-slices from a patient following incubation of LysoProbe I/II (20 μM).

LysoProbe fluorescence images are displayed in green (A, D), with nuclei counterstained by Hoechst 33342 and displayed in blue (B, E), and overlay images (C, F). Tissues were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Figure 7. RBE cells transfected with GFP-LC3 and subsequently loaded with LysoProbe II (10 μM).

RBE cells treated with CQ in the presence of standard serum: (A) CQ (20 μM) for 1 h; (B) CQ (20 μM) for 24 h; (C) CQ (20 μM) + BafA1 (10 nM) for 24 h. Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Figure 8. Autophagosomes and swollen lysosomes fail to fuse in CQ-treated RBE cells (loaded with LysoProbe II, 10 μM):

(A) the control cells in medium with standard serum, and (B) after nutrient (serum) deprivation; (C) after CQ treatment (40 μM, 6 h) in the absence of standard serum; (D) after CQ treatment (40 μM, 16 h) in the absence of standard serum; (E) as positive control, RBE cells expressing GFP-LC3 were treated with rapamycin (1 μM, 16 h) in the absence of standard serum.Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Figure 9. Visualization of lysosomal alterations using LysoProbe II in RBE cells undergoing apoptosis:

(A–C): cells treated with CQ in standard serum for 16 h: (A) CQ (50 μM); (B) CQ (100 μM); (C) CQ (200 μM); (D) CQ (300 μM); (E): after treatment with CQ (50 μM), in the absence of standard serum, for 16 h. Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.