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Lysosomal targeting with stable and sensitive fluorescent probes (Superior LysoProbes): applications for lysosome labeling and tracking during apoptosis.

Chen X, Bi Y, Wang T, Li P, Yan X, Hou S, Bammert CE, Ju J, Gibson KM, Pavan WJ, Bi L - Sci Rep (2015)

Bottom Line: The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy.Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis.Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Michigan Technological University, Houghton, MI 49931.

ABSTRACT
Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.

No MeSH data available.


Related in: MedlinePlus

Confocal laser-scanning fluorescent images of RBE cells treated with lobaplatin (5 μg/mL) with and without CQ (20 μM) for 24 h.GFP-LC3 (green fluorescence) transfected RBE cells were labeled with Superior LysoProbe (IV) (1 μM, red fluorescence), and Hoechst 33342 (1 μg/ml, blue fluorescence). Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.
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f9: Confocal laser-scanning fluorescent images of RBE cells treated with lobaplatin (5 μg/mL) with and without CQ (20 μM) for 24 h.GFP-LC3 (green fluorescence) transfected RBE cells were labeled with Superior LysoProbe (IV) (1 μM, red fluorescence), and Hoechst 33342 (1 μg/ml, blue fluorescence). Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.

Mentions: Autophagy may represent a cell-survival pathway in chemotherapy. To assess parameters of autophagy, RBE cells were transfected with the autophagosome marker LC33839 To monitor lysosomes, the GFP-LC3 transfected RBE cells were labeled with Superior LysoProbe IV (1 μM). Untreated cells demonstrated a primarily diffuse cytoplasmic staining of GFP-LC3 with very few punctuate autophagosomes and lysosomes (images not shown). An induction of autophagosome formation was observed in RBE cells following autophagic stimuli (starvation in serum-free medium) (Fig. 9, row 1). In contrast, treatment of RBE cells with CQ (20 μM, 24 h) resulted in a massive induction of GFP-LC3-positive autophagosomes. Further, CQ-treated cells displayed a marked increase in the number and size of lysosomes (Fig. 9, row 2), which retained Superior-LysoProbe fluorescence, indicating that the lysosomes did not lose their internal acidic nature. Yellow regions in the merged images indicate autophagolysosome formation, a result of combining GFP-LC3 (green fluorescence) and Superior LysoProbe (red fluorescence) colocalization. After lobaplatin treatment alone (5 μg/mL, 24 hr), a significant increase in the percentage of cells that were highly labeled with Superior LysoProbe (Fig. 9, row 3) was observed as compared to control untreated cells, suggesting that lobaplatin treatment alone resulted in an increase in lysosomal activity. However, after the combinatorial treatment with CQ (20 μM) + lobaplatin (5 μg/mL), many vesicles retained GFP staining and Superior-LysoProbe fluorescence, but an absence of yellow combinatorial fluorescence was revealed in the merged image (Fig. 9, row 4), indicating that combined treatment induced a marked accumulation of vesicles that included autophagosomes and lysosomes, but a blockade of the fusion of autophagosomes with lysosomes. We speculate that this result was the result of lysosomal dysfunctional. It has been reported elsewhere that CQ blocks autophagy via inhibition of lysosomal proteases and autophagosome-lysosomal fusion, consistent with our results suggesting that sensitization to lobaplatin treatment was induced by CQ treatment.


Lysosomal targeting with stable and sensitive fluorescent probes (Superior LysoProbes): applications for lysosome labeling and tracking during apoptosis.

Chen X, Bi Y, Wang T, Li P, Yan X, Hou S, Bammert CE, Ju J, Gibson KM, Pavan WJ, Bi L - Sci Rep (2015)

Confocal laser-scanning fluorescent images of RBE cells treated with lobaplatin (5 μg/mL) with and without CQ (20 μM) for 24 h.GFP-LC3 (green fluorescence) transfected RBE cells were labeled with Superior LysoProbe (IV) (1 μM, red fluorescence), and Hoechst 33342 (1 μg/ml, blue fluorescence). Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355733&req=5

f9: Confocal laser-scanning fluorescent images of RBE cells treated with lobaplatin (5 μg/mL) with and without CQ (20 μM) for 24 h.GFP-LC3 (green fluorescence) transfected RBE cells were labeled with Superior LysoProbe (IV) (1 μM, red fluorescence), and Hoechst 33342 (1 μg/ml, blue fluorescence). Cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective lens.
Mentions: Autophagy may represent a cell-survival pathway in chemotherapy. To assess parameters of autophagy, RBE cells were transfected with the autophagosome marker LC33839 To monitor lysosomes, the GFP-LC3 transfected RBE cells were labeled with Superior LysoProbe IV (1 μM). Untreated cells demonstrated a primarily diffuse cytoplasmic staining of GFP-LC3 with very few punctuate autophagosomes and lysosomes (images not shown). An induction of autophagosome formation was observed in RBE cells following autophagic stimuli (starvation in serum-free medium) (Fig. 9, row 1). In contrast, treatment of RBE cells with CQ (20 μM, 24 h) resulted in a massive induction of GFP-LC3-positive autophagosomes. Further, CQ-treated cells displayed a marked increase in the number and size of lysosomes (Fig. 9, row 2), which retained Superior-LysoProbe fluorescence, indicating that the lysosomes did not lose their internal acidic nature. Yellow regions in the merged images indicate autophagolysosome formation, a result of combining GFP-LC3 (green fluorescence) and Superior LysoProbe (red fluorescence) colocalization. After lobaplatin treatment alone (5 μg/mL, 24 hr), a significant increase in the percentage of cells that were highly labeled with Superior LysoProbe (Fig. 9, row 3) was observed as compared to control untreated cells, suggesting that lobaplatin treatment alone resulted in an increase in lysosomal activity. However, after the combinatorial treatment with CQ (20 μM) + lobaplatin (5 μg/mL), many vesicles retained GFP staining and Superior-LysoProbe fluorescence, but an absence of yellow combinatorial fluorescence was revealed in the merged image (Fig. 9, row 4), indicating that combined treatment induced a marked accumulation of vesicles that included autophagosomes and lysosomes, but a blockade of the fusion of autophagosomes with lysosomes. We speculate that this result was the result of lysosomal dysfunctional. It has been reported elsewhere that CQ blocks autophagy via inhibition of lysosomal proteases and autophagosome-lysosomal fusion, consistent with our results suggesting that sensitization to lobaplatin treatment was induced by CQ treatment.

Bottom Line: The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy.Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis.Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Michigan Technological University, Houghton, MI 49931.

ABSTRACT
Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.

No MeSH data available.


Related in: MedlinePlus