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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

(A) Detection of extensive microtubule disruption and lysosome perturbation in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). RBE cells were labeled with Tublin-GFP (green fluorescence), Superior LysoProbe (IV) (10 μM, red fluorescence) and Hoechst 33342 (1 μg/ml, blue fluorescence); (B) The dissolution of cytoskeletal F-actin structural characteristics was observed in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). F-actin exhibits green fluorescence and nuclei exhibit blue fluorescence associated with Hoechst 33342 staining. RBE cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective; (C) immunohistochemical images of α-Tublin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL).
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f10: (A) Detection of extensive microtubule disruption and lysosome perturbation in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). RBE cells were labeled with Tublin-GFP (green fluorescence), Superior LysoProbe (IV) (10 μM, red fluorescence) and Hoechst 33342 (1 μg/ml, blue fluorescence); (B) The dissolution of cytoskeletal F-actin structural characteristics was observed in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). F-actin exhibits green fluorescence and nuclei exhibit blue fluorescence associated with Hoechst 33342 staining. RBE cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective; (C) immunohistochemical images of α-Tublin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL).

Mentions: The previous findings of translocated organelles suggested that the cellular cytoskeleton might play a role in lobaplatin-induced apoptosis. To examine this possibility, we next investigated the relationship(s) between lysosomes and the cytoskeleton, since it was reasonable to assume that lysosomal relocation might be correlated with cytoskeleton integrity. Moreover, it is probable that lysosomal intracellular distribution would correlate with the extent of microtubule extension into the cytoplasm. In our studies evaluating these parameters, control cells demonstrated visible lysosomes distributed in the cell periphery superimposed on the microtubule (Fig. 10). Similar results were found at low concentrations of lobaplatin (2.5 μg/mL), but at higher lobaplatin concentrations the microtubules were interrupted and condensed to the perinuclear region. At these concentrations of lobaplatin, the lysosomes aggregated near the nucleus where the microtubule-organizing center was disrupted (Fig. 10A). Moreover, we found that the absolute levels of cytoskeletal F-actin protein was significantly diminished in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL) (Fig. 10B), a finding further confirmed by immune-histochemical assays of α-tubulin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL) (Fig. 10C). Although we suspect that the translocation of lysosomes may occur along microtubules, the present assays only indirectly suggest an association between lysosomes and cytoskeletal elements, a process that is under further investigation in our laboratory.


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)

(A) Detection of extensive microtubule disruption and lysosome perturbation in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). RBE cells were labeled with Tublin-GFP (green fluorescence), Superior LysoProbe (IV) (10 μM, red fluorescence) and Hoechst 33342 (1 μg/ml, blue fluorescence); (B) The dissolution of cytoskeletal F-actin structural characteristics was observed in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). F-actin exhibits green fluorescence and nuclei exhibit blue fluorescence associated with Hoechst 33342 staining. RBE cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective; (C) immunohistochemical images of α-Tublin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f10: (A) Detection of extensive microtubule disruption and lysosome perturbation in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). RBE cells were labeled with Tublin-GFP (green fluorescence), Superior LysoProbe (IV) (10 μM, red fluorescence) and Hoechst 33342 (1 μg/ml, blue fluorescence); (B) The dissolution of cytoskeletal F-actin structural characteristics was observed in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL). F-actin exhibits green fluorescence and nuclei exhibit blue fluorescence associated with Hoechst 33342 staining. RBE cells were imaged on an inverted laser scanning fluorescent microscope (Olympus) using a 60 × oil immersion objective; (C) immunohistochemical images of α-Tublin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL).
Mentions: The previous findings of translocated organelles suggested that the cellular cytoskeleton might play a role in lobaplatin-induced apoptosis. To examine this possibility, we next investigated the relationship(s) between lysosomes and the cytoskeleton, since it was reasonable to assume that lysosomal relocation might be correlated with cytoskeleton integrity. Moreover, it is probable that lysosomal intracellular distribution would correlate with the extent of microtubule extension into the cytoplasm. In our studies evaluating these parameters, control cells demonstrated visible lysosomes distributed in the cell periphery superimposed on the microtubule (Fig. 10). Similar results were found at low concentrations of lobaplatin (2.5 μg/mL), but at higher lobaplatin concentrations the microtubules were interrupted and condensed to the perinuclear region. At these concentrations of lobaplatin, the lysosomes aggregated near the nucleus where the microtubule-organizing center was disrupted (Fig. 10A). Moreover, we found that the absolute levels of cytoskeletal F-actin protein was significantly diminished in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL) (Fig. 10B), a finding further confirmed by immune-histochemical assays of α-tubulin in RBE cells exposed to lobaplatin (2.5 ~ 25 μg/mL) (Fig. 10C). Although we suspect that the translocation of lysosomes may occur along microtubules, the present assays only indirectly suggest an association between lysosomes and cytoskeletal elements, a process that is under further investigation in our laboratory.

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