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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

Chemical structures of Superior LysoProbes (I–IV).
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f1: Chemical structures of Superior LysoProbes (I–IV).

Mentions: In earlier work, we developed several fluorescent probes that aggregate in lysosomes, and they were employed to monitor intracellular pH and localize lysosomes in cultured cells28. Nonetheless, these acidotropic probes are comparable to LysoTrackers in that they label compartments based upon their pKa values, thereby decreasing their specificity for lysosomes. To enhance lysosome-targeting specificity, we synthesized a series of novel Superior LysoProbes. We have taken advantage of the fact that lysosomal membrane proteins are heavily glycosylated with numerous N-linked glycans. The latter contain mannose, fucose, galactose, N-acetyl-glucosamine, and sialic acid monosaccharides, which protect lysosomal membrane proteins from degradation by lysosomal proteases such as cathepsins. We hypothesized that conjugation of the parent fluorophore with an N-linked glycan conjugate would aid targeting of fluorescent probes to lysosomes. To examine this hypothesis, we have synthesized a series of new fluorescent probes I–IV (chemical structure shown in Fig. 1) and documented selectivity for lysosomes and the capacity of these probes to label living cells at nanomolar concentrations over an extensive time course. For synthesis of Superior LysoProbes, various N-linked glycan moieties were introduced via “click” chemistry29 (Schemes S1 & S2). The spirocyclic structures of the rhodamine lactam-type derivatives were confirmed by NMR. When the spirocyclic compounds exist in the lactam (cyclic amide) form, they lack measurable absorbance and fluorescence in the visible spectrum, yet this returns when converted to the amide form. The binding activity and spirocyclic structure of Superior LysoProbes was confirmed using UV-Vis titration.


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)

Chemical structures of Superior LysoProbes (I–IV).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Chemical structures of Superior LysoProbes (I–IV).
Mentions: In earlier work, we developed several fluorescent probes that aggregate in lysosomes, and they were employed to monitor intracellular pH and localize lysosomes in cultured cells28. Nonetheless, these acidotropic probes are comparable to LysoTrackers in that they label compartments based upon their pKa values, thereby decreasing their specificity for lysosomes. To enhance lysosome-targeting specificity, we synthesized a series of novel Superior LysoProbes. We have taken advantage of the fact that lysosomal membrane proteins are heavily glycosylated with numerous N-linked glycans. The latter contain mannose, fucose, galactose, N-acetyl-glucosamine, and sialic acid monosaccharides, which protect lysosomal membrane proteins from degradation by lysosomal proteases such as cathepsins. We hypothesized that conjugation of the parent fluorophore with an N-linked glycan conjugate would aid targeting of fluorescent probes to lysosomes. To examine this hypothesis, we have synthesized a series of new fluorescent probes I–IV (chemical structure shown in Fig. 1) and documented selectivity for lysosomes and the capacity of these probes to label living cells at nanomolar concentrations over an extensive time course. For synthesis of Superior LysoProbes, various N-linked glycan moieties were introduced via “click” chemistry29 (Schemes S1 & S2). The spirocyclic structures of the rhodamine lactam-type derivatives were confirmed by NMR. When the spirocyclic compounds exist in the lactam (cyclic amide) form, they lack measurable absorbance and fluorescence in the visible spectrum, yet this returns when converted to the amide form. The binding activity and spirocyclic structure of Superior LysoProbes was confirmed using UV-Vis titration.

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